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Proteomic analysis of rod photoreceptor outer segment and characterization of an aminophospholipid transporter-like… Kwok, Michael Chung Ming. 2007

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PROTEOMIC ANALYSIS OF ROD PHOTORECEPTOR OUTER SEGMENT AND CHARACTERIZATION OF AN AMINOPHOSPHOLIPID TRANSPORTER-LIKE ATPASE by MICHAEL CHUNG MING KWOK B.Sc. (Honours), The University of British Columbia, 2005 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Biochemistry and Molecular Biology) THE UNIVERSITY OF BRITISH COLUMBIA September 2007 © Michael Chung Ming Kwok, 2007 ABSTRACT The photoreceptor outer segment is a specialized compartment of rod and cone photoreceptor cells which functions in vertebrate phototransduction, the process which converts light into electrical signals as the initial step in vision. Although the molecular mechanisms underlying phototransduction and light adaptation have been well examined, other cellular and molecular processes in the photoreceptor outer segment are not clearly understood. These include the mechanism of outer segment renewal, in particular the morphogenesis and shedding of disk membranes at opposite ends of the outer segment. In this study, a mass spectrometry-based proteomic approach was used to identify the complete proteome of the rod photoreceptor outer segment. Rod outer segments were isolated from bovine retina and separated into different subcellular preparations, followed by analysis on a tandem mass spectrometer. A total of 529 proteins were identified, including a large number of proteins that have not been previously reported to be present in the outer segments including a subset of proteins implicated in vesicle trafficking and membrane fusion. Using western blotting and immunofluorescence techniques, it was confirmed that the small GTPase Rab 11 and the Rab accessory protein Rab-GDI, as well as the SNARE protein VAMP 2/3 and SNARE-associated protein Munc 18-1 are true components of the rod outer segment. In addition, several uncharacterized proteins were identified one of which is aminophospholipid transporter-like ATPase IB (ATP8A2). The gene was cloned, expressed in cultured cells, and immunoaffinity purified. ATPase activity assay using radiolabeled [a- P] ATP showed that the purified protein was highly activated in the presence of brain polar lipid extract. Immunocytochemical studies showed a ii colocalization of the expressed protein with the ER-resident protein calnexin when expressed in HEK293 cells. These results, along with the proteomic data suggest that ATPase IB is a membrane protein in the rod outer segment that may play a role in transporting aminophospholipid across the membrane bilayer using ATP as an energy source. This study serves as a basis for understanding the role of this previously uncharacterized protein in the rod photoreceptor and determining whether mutations in the ATP8A2 gene may cause retinal disorders. T A B L E O F C O N T E N T S Abstract ii Table of Contents iv List of Tables vii List of Figures viii List of Abbreviations x Acknowledgements xii CHAPTER I Introduction 1 1.1 The Eye and the Retina 1 1.2 Photoreceptor cells 4 1.3 Rod Outer Segment 4 1.4 Phototransduction 6 1.5 Other Cellular Processes in the Rod Outer Segment 8 1.6 Retinal Degenerative Diseases 10 1.7 Thesis Investigation 11 CHAPTER II Proteomic Analysis of Rod Outer Segment 14 2.1 Introduction 14 2.1.1 Mass Spectrometry based Proteomics 14 2.1.2 Vesicle Trafficking and Membrane Fusion 15 2.2 Materials and Methods 19 2.2.1 Materials 19 2.2.2 Subcellular Fractionation of ROS 21 2.2.3 SDS-PAGE and Western Blot analysis 22 2.2.4 In-solution and In-gel Digestion of Proteins for Proteomic Analysis 22 iv 2.2.5 LC-MS/MS Analysis 23 2.2.6 Protein Identification 24 2.2.7 Immunofluorescence Microscopy 24 2.3 Results 25 2.3.1 Subfractionation of Bovine ROS 25 2.3.2 Proteomic Analysis of ROS 28 2.3.3 Rod and Cone Phototransduction Proteins 30 2.3.4 Metabolic, Structural, Transport, and Housekeeping Proteins ... 32 2.3.5 Vesicle Trafficking Proteins 33 2.3.6 Immunolocalizati on of Rab and SNARE Proteins 35 2.4 Discussion 42 2.4.1 Proteome of Bovine Photoreceptor Outer Segment 42 2.4.2 Rab and SNARE Proteins in ROS 43 CHAPTER III Cloning and Characterization of a Potential Aminophospholipid-Transporting ATPase IB (ATP8A2) 47 3.1 Introduction 47 3.1.1 Lipids and Biological Membrane Asymmetry 47 3.1.2 Lipid Transporters 49 3.1.3 P-type ATPases 51 3.1.4 Lipid Composition in ROS 52 3.2 Materials and Methods 53 3.2.1 DNA Sequencing and Bioinformatic Predictions 53 3.2.2 Cloning of lD4-tagged Human ATPase IB 53 3.2.3 Cell Culture and Transfection 54 3.2.4 Solubilization of Protein in HEK293 56 3.2.5 Immunoaffinity Purification 56 3.2.6 Immunofluorescence Microscopy 56 3.2.7 ATPase Activity Assay 57 3.3 Results 58 3.3.1 Gene Expression by RT-PCR 58 v 3.3.2 Cloning of human ATP8A2 58 3.3.3 DNA Sequencing and Sequence Alignments 60 3.3.4 Domain Analysis, Topology, and Glycosylation Sites Predictions 60 3.3.5 Expression and Solubilization by Different Detergents 62 3.3.6 Purification on lD4-Immunoaffinity Column 67 3.3.7 ATPase Activity Assay 67 3.3.8 Localization of Human ATPase IB in HEK293T Cells 67 3.3.9 Attempt to Generation of Monoclonal Antibody and Immunofluorescence Microscopy 71 3.4 Discussion 73 CHAPTER IV Summary and Future Studies 77 4.1 Summary 77 4.2 Future Studies 78 4.3 References 82 Appendix I Proteins Identified from each of the Five ROS Preparations 92 Appendix II Functions of ROS Proteins sorted into Various Categories 106 vi LIST OF T A B L E S Table 1.1 Table of Selected Genes Causing Human Retinal Diseases 12 Table 2.1 Selected Proteins involved in Phototransduction and Visual Cycle in Rods and Cones Present in the Proteomic Dataset 31 Table 2.2 Selected Proteins involved in Vesicle Trafficking and Other Functions Present in the Proteomic Dataset 34 vii LIST OF FIGURES Figure 1.1 The Anatomy of the Human Eye 2 Figure 1.2 Organization of the Retinal Cell Layers 3 Figure 1.3 Vertebrate Photoreceptor Cells 5 Figure 1.4 Schematic of the Phagocytosis of Rod Outer Segments by the Retinal Pigment Epithelium 7 Figure 1.5 The Phototransduction Cascade of Vertebrate Rod Photoreceptor 9 Figure 2.1 A General Overview of a Proteomic Analysis by Tandem Mass Spectrometry 16 Figure 2.2 SNARE and SNARE related protein Muncl8 in Membrane Fusion . . . 17 Figure 2.3 Rab proteins in the Trafficking and Fusion of Vesicles with Target Membrane 20 Figure 2.4 Subcellular Fractionation of Bovine Rod Outer Segment 26 Figure 2.5 Rod Outer Segment Subcellular Fractions analyzed by SDS-PAGE . . . 27 Figure 2.6 Summary of the Proteomic Analysis of Rod Outer Segment Preparations 29 Figure 2.7 Localization of Synaptophysin, Na+/K+ ATPase and ABCA4 36 in Retina Figure 2.8 Localization of Rab 11, Rab 14 and Rab-GDI in Retina 38 Figure 2.9 Localization of NSF, Syntaxin 3, Munc 18-1, and VAMP 2/3 in Retina 40 Figure 2.10 Distribution of Proteins in the Disk and Plasma Membrane 41 Figure 3.1 Regulation of Transbilayer Lipid Distribution in Cellular Membranes.. 50 Figure 3.2 Cloning of the Full Length Human ATP8A2 with 1D4 Affinity Tag . . . 55 3 Express ATP8A2 g ne in Various Tssues of Mouse 9 viii Figure 3.4 Complete Amino Acid Sequence of the Full Length ATP IB- 1D4 Clone 61 Figure 3.5 Hydropathy Profile for Human ATPase IB (ATP8A2) 63 Figure 3.6 Predicted Topological Model for Human ATPase IB using TopPred . . . 64 Figure 3.7 Predicted Topological Model for Human ATPase IB using TMHMM . . 65 Figure 3.8 Solubilization of ATP IB-1D4 Protein Expressed in HEK293 Cells using Various Detergents 66 Figure 3.9 Immunoaffinity Purification of ATP IB-1D4 Protein Expressed in HEK293T Cells 68 Figure 3.10 ATPase Activity of CHAPS-Solubilized ATP IB-1D4 is Stimulated by the Presence of Lipids 69 Figure 3.11 Immunofluorescence of ATPase IB-1D4, Peripherin, and Rhodopsin Expressed in HEK293T Cells 70 Figure 3.12 Localization of ATPase IB in Human Retina using Serum of Mouse Immunized with the C-Terminal GST-fusion Construct 72 ix LIST OF A B B R E V I A T I O N S ABC ATP-binding cassette ADP Adenosine diphosphate ATP Adenosine triphosphate cGMP cyclic-guanosinemonophosphate CHAPS 3 -[(3 -cholamidopropyl)dimethylammonio] -1 -propanesulfonate CNG Cyclic-nucleotide gated DHA Docosahexaenoic acid DM «-dodecyl-P-D-maltoside DMEM Dulbecco's modified Eagle medium DTT Dithiothreitol EDTA Ethylenediaminetetraacetic acid ESI Electrospray ionization ER Endoplasmic reticulum GDI GDP-dissociation inhibitor GTP Guanosine triphosphate HEK Human embryonic kidney HEPES 2-[4-(2-hydroxyethyl)-1 -piperazinyl]ethanesulfonic acid HPLC High performance liquid chromatography kDa Kilo Daltons mAb Monoclonal antibody MS/MS Tandem mass spectrometry NSF N-ethylmaleimide-sensitive factor pAb Polyclonal antibody PAGE Polyacrylamide gel electrophoresis PBS Phosphate buffered saline PC Phosphatidylcholine PDE Phosphodiesterase PE Phosphatidylethanolamine X PS Phosphatidylserine PVDF Polyvinylidene fluoride RDR Retina deficient in ROS PJS Rod inner segment ROS Rod outer segment RP Retinitis pigmentosa RPE Retinal pigment epithelium RT-PCR Reverse transcriptase-polymerase chain reaction SDS Sodium dodecyl sulfate SNAP Synaptosome-associated protein SNARE Soluble NSF attachment receptor Tris Tris(hydroxymethyl)aminomethane VAMP Vesicle-associated membrane protein ACKNOWLEDGEMENTS I would like to thank my supervisor, Dr. Robert Molday for giving me the opportunity to work on this interesting project and for his continuous guidance and tremendous insights. His passion and knowledge have truly motivated me in becoming a better scientist. I am really thankful to Dr. Leonard Foster for his patience in teaching me everything I know about mass spectrometry and help with the proteomic analysis. I am also grateful to Dr. Franck Duong for being my thesis committee member and his valuable advice. I would like to thank Laurie Molday for teaching me many of the techniques needed to complete this project as well as general help within the lab. Dr. Frank Dyka for sharing his expertise in molecular biology and cell culture techniques. Theresa Hii for antibody generation and screening, Dr. Juha Holopainen for assistance with the immunofluorescence microscopy studies, Dr. Seifollah Azadi for the results of RT-PCR screens, and Ming Zhong for teaching me the ATPase assay. I would also like to acknowledge other past and present members of the lab who have made working enjoyable: Dr. Jinhi Ann, Dr. Celene Grayson, Dr. Emmanuelle Reboul, Dr. Winco Wu, Karen Chang, Jonathan Coleman, Gurp Johal, Anke Roden, Sonke Stocker, and Julie Wong. Lastly, I would like to thank my family for their support and encouragement through the years. xii C H A P T E R I Introduction 1.1 The Eye and the Retina Light enters the vertebrate eye through the pupil and the iris regulates the amount of light entering the eye by dilation and contraction. The light is then focused by the cornea and lens onto the retina that is located at the posterior end of the eye. The eye is protected by a tough but flexible fibrous tissue called the sclera, and the choroid is a vascular layer that provides oxygen and nutrients for the retina (Figure 1.1). The retina is a highly complex, layered structure in the central nervous system that contains closely packed neural cells. It is made up of one type of glia (Miiller cells) and six types of neurons: rod and cone photoreceptors, horizontal, amacrine, bipolar, and ganglion cells (Adler, 1993). The nuclear layers are composed of the nuclei of the photoreceptor cells (outer nuclear layer; ONL) and the secondary neurons (inner nuclear layer; INL), while the plexiform layers contain synaptic junctions between the axons and dendrites of the neurons (Figure 1.2). Light rays pass through the whole thickness of the retina before reaching the sensory rod and cone photoreceptors where the signal is converted to neural activity and transmitted through the network of secondary neurons. The signal is finally relayed to the ganglion cells those axons compose the optic nerve and the signal is conveyed to the visual cortex of the brain. 1 Figure 1.1 The Anatomy of the Human Eye Light first travels through the cornea, where it is refracted and directed through the pupil, an opening in the center of the iris that regulates the amount of light entering the eye. The light is further refracted and focused by the lens and vitreous onto the retina, where it is converted to electrical impulses and transmitted through the optic nerve to the brain. The choroid is a layer of blood vessels that supplies oxygen and nutrients to the retina, and sclera is the opaque protective tissue that coats the eye. Figure modified from Kolb et al. (http://webvision.med.utah.edu/index.html) 2 Retinal Pigment Epithelium RPE OS IS ONL OPL ) INL IPL GCL Photoreceptors MUller Cells Horizontal Cells Bipolar Cells Amacrine Cells Ganglion Cells Figure 1.2 Organization of the Retinal Cell Layers The light-sensitive rod and cone photoreceptor cells are in close association with the retinal pigment epithelium (RPE) which is involved in the phagocytosis of the outer segments, the retinoid cycle, as well as the transport of nutrients to the photoreceptors. Electrical signals from the photoreceptors are integrated and processed by the horizontal, bipolar, and amacrine cells to the ganglion cells whose axons form the optic nerve. Muller cells are glial cells that provide support and help maintain homeostasis within the retina. RPE, retinal pigment epithelium layer; OS, outer segment; IS, inner segment; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer. Figure courtesy of Dr. R. Molday. 3 1.2 Photoreceptor Cells - There are two types of photoreceptors in vertebrates, the rods and cones, named by their shapes when they were first viewed in the microscope. Cones are the colour receptors of the eye and are responsible for acute vision in bright light. Rods function primarily in dim light, as they are very sensitive to light at low levels and a single photon can initiate a response in rods. Both photoreceptor cell types are highly polarized and have a similar cellular organization, consisting of five principle regions: an outer segment; a thin connecting cilium that serves as a passage for the transport of molecules between the outer and inner segments; an inner segment which contains the biosynthetic and metabolic machinery of the cell, including the endoplasmic reticulum (ER), Golgi apparatus, and mitochondria; a cell body containing the nucleus; and a synaptic region containing synaptic vesicles and ribbon synapse for the release of neurotransmitters to the aforementioned secondary neurons of the retina (Figure 1.3 A). 1.3 Rod Outer Segment The outer segments of vertebrate photoreceptor cells are a highly specialized compartment within which phototransduction takes place. The rod outer segment (ROS) consists of an ordered stack of tightly packed disks surrounded by a separate plasma membrane (Figure 1.3B). The disk membrane is filled with the visual pigment rhodopsin, which constitutes around 85% of all membrane proteins and responds to light and initiates the phototransduction process (Papermaster and Dreyer, 1974). The outer segment lacks the machinery required for protein synthesis and degration, yet it undergoes constant renewal. 4 Retinal Pigment Epithelium B ROS plasma membrane l€~ Disks Rim i egion IXL of Disks Cytoplasmic (Intradlskal) space Connecting Cilium Mitochondria » Golgi complex " Endoplasmic i etic ukim Rod Outer Segment Rod } Inner Segment nucleus ?• Cell Body Synaptic vesicles Synaptic terminal Figure 1.3 Vertebrate Photoreceptor Cells (A) Structural similarities and differences between the cone and rod photoreceptors. Both types of photoreceptors consist of an outer segment, a connecting cilium, an inner segment, a cell body, and a synaptic region. However, the shape, size, and arrangement of the outer segment clearly distinguish the two. (B) Diagram of vertebrate rod photoreceptor showing the basic cellular and subcellular features. Figure modified from Molday and Molday, 1987. 5 The disks undergo a continual renewal process in which packets of aged disk membranes are shed from the distal end of the outer segment while new disks are added at the base of the outer segment. Outer segment proteins such as rhodopsin are synthesized in the inner segments and transported through the modified connecting cilium to the base of the outer segments where they are incorporated into the new membranous disks. The old disks are removed daily by phagocytosis mediated by the adjacent retinal pigment epithelial (RPE) cells and the whole ROS is completely renewed over a period of around ten days (Figure 1.4) (Young 1967; Young and Bok, 1969). Since rods are about 20 times more abundant than cones and ROS can be easily purified from the rest of the retina (Molday and Molday, 1987), the focus of this study is on the rod photoreceptor outer segment. 1.4 Phototransduction Vision begins when a molecule of rhodopsin is excited by light and its 11-cw-retinal chromophore is isomerized into the a\\-trans configuration. This triggers rhodopsin to adopt an active conformation called metarhodopsin II (R*) and activates the G protein, transducin, by facilitating the exchange of GTP for GDP on its a-subunit. The transducin a-subunit stimulates its effector, cGMP phosphodiesterase (PDE), by binding to the y-subunit of PDE, which had inhibitory constraint on the catalytic a and p-subunits of PDE. The activated PDE hydrolyzes cGMP to 5'-GMP and leads to a rapid reduction in intracellular cGMP. This results in membrane hyperpolarization, which is governed by the closure of the cGMP-gated cationic channels in the plasma membrane (Figure 1.5) (Fesenko et al., 1985). The photoreceptor cells synapse with interconnecting neurons and the signal is relayed by 6 Retinal Pigment Epithelium (RPE) ROS Phagosomes Rod Outer Segments (ROS) Figure 1.4 Schematic of the Phagocytosis of Rod Outer Segments by the Retinal Pigment Epithelium Aged photoreceptor disks are displaced up the outer segment and the phagocytosis of ROS by RPE is initiated by the binding of shed disks at the apical membrane of RPE leading to the ingestion of the bound ROS. The ingested ROS phagosomes are eventually degraded by lysosomal enzymes. Figure modified from Kolb et al. (http://webvision.med.utah.edu/index.html). 7 suppressing the release of neurotransmitter glutamate. The electrical signal eventually reaches the ganglion cells which integrate the output from many cells and send the resulting signal through the optic nerve to the visual cortex of the brain. The recovery of the photoresponse is also rapid and requires efficient termination of each step of the phototransduction cascade. Photoexcited metarhodopsin R* is phosphorylated by rhodopsin kinase on multiple sites. This enables arrestin to bind to rhodopsin thereby blocking transducin activation (Mendez et al., 2000). The rate of rhodopsin phosphorylation is regulated by recoverin. The GTP-bound transducin a-subunit is deactivated by GTP hydrolysis accelerated by a protein complex containing regulator of G protein signaling protein 9 (RGS9), G protein beta 5, and membrane anchor R9AP (He et al., 1998; Makino et al., 1999; Hu and Wensel, 2002). The restoration of intracellular cGMP concentration is accomplished through a Ca2+ feedback mechanism. The activation of phototransduction cascade leads to a decline in intracellular Ca2+ as its entry through the cyclic nucleotide-gated (CNG) channels is reduced while its extrusion through the Na+/Ca2+-K+ exchanger is unaffected by light (Nakatani and Yau, 1988). The Ca2+ signal is sensed by guanylyl cyclase-activiating proteins (GCAPs). The reduction in Ca2+ results in the activation of guanylyl cyclase leading to the production of cGMP from GTP (Gorczyca et al., 1995). 1.5 Other cellular processes in the ROS Aside from phototransduction, the rod outer segment contains a set of proteins that has been suggested to carry out other essential cellular processes. A brain-type creatine 8 liv Disk MemlHone Pl.ism.i Membrane Figure 1.5 The Phototransduction Cascade of Vertebrate Rod Photoreceptor Phototransduction is initiated when rhodopsin captures a photon, isomerizes its retinal chromophore from the 1 l-cis to a\\-trans configuration and changes conformation. The activated rhodopsin (Rh ) activates the G-protein transducin by the exchange of GTP for GDP which in turn remove the inhibition on phosphodiesterase (PDE) by binding to its inhibitory y-subunit. This results in the hydrolysis of cGMP and subsequent closure of cGMP-gated ion channels on the plasma membrane. The decrease in the influx of Na+ and Ca leads to the hyperpolarization of the photoreceptors. The signal is then transmitted to the secondary neurons of the retina. The figure also shows the conversion of GTP to cGMP by guanylyl cyclase during dark recovery. The increase in cGMP level reopens the cGMP-gated channels and returns the photoreceptor to its dark adapted state. Figure modified from Fain, 2006. 9 kinase has been found in the ROS that transfers high-energy phosphate groups from phosphocreatine to ADP (Hemmer et al., 1993). The phosphocreatine shuttle is believed to be the major contributor that connects oxidative phosphorylation in the inner segment to ATP consumption in the outer segment. In addition, a subset of glycolytic enzymes and a GLUT-1 glucose transporter were discovered to be present in the rod outer segment by biochemical methods (Hsu and Molday, 1991). Glucose metabolism in the ROS has been thoroughly studied and found to be a significant contributor to the production of ATP and NADPH required for the energy-consuming processes in the ROS (Hsu and Molday, 1994). These include the regeneration of cGMP from ATP and GTP during phototransduction and the NADPH-dependent chromophore regeneration (Schnetkamp and Daemen, 1981). The recycling of retinoid is also a vital process in the photoreceptor cell that complements phototransduction. Retinol dehydrogenase (RDH8) and the photoreceptor specific ABC transporter ABCA4 are two proteins in the ROS that are responsible for the effective reduction of all-trans retinal to all-trans retinol following photoexcitation of rhodopsin (Ming et al., 1997; Weng et al., 1999; Rattner et al., 2000). The all-trans retinol is translocated from the ROS to the RPE where it is converted back to 11-cis retinal through a series of enzymatic reactions. 1.6 Retinal degenerative diseases Retinal degeneration comprises a large group of diseases that result in the death of photoreceptor cells and lead to visual impairment or blindness. Photoreceptor cell death caused by most retinal degenerative diseases is generally believed to be mediated solely by the apoptotic pathway (Chang et al., 1993), but it is suggested recently that autophagy and 10 complement-activated lysis may also play a role (Lohr et al., 2006). Retinitis pigmentosa (RP) and macular degeneration are two of the most common diseases and there are very few treatments available. Over the last two decades great progress in the field of molecular genetics has led to the identification of genes involved in inherited retinal degenerative diseases. As of July 2007, over 130 genes have been linked to various retinal degenerative diseases including retinitis pigmentosa, macular degeneration, and congenital stationary night blindness (RetNet, http://www.sph.uth.tmc.edu/Retnet/). Many of these genes are found to be specifically or highly expressed in photoreceptors. These include genes that encode proteins required for phototransduction, the visual cycle, or structural support of photoreceptors (Blackshaw et al., 2001). A list of selected genes is given in Table 1.1. However, there are over 50 disease-linked genetic loci that have been mapped but the gene responsible has not been determined (RetNet, http://www.sph.uth.tmc.edu/Retnet/). Population surveys of the genes known to be responsible for autosomal dominant retinitis pigmentosa indicated that they only account for 25% to 50% of all cases (Sullivan et al., 2006). Therefore, it suggests that mutations in genes encoding other unidentified ROS proteins may be responsible for the retinal degenerative diseases in which the defective gene is unknown. 1.7 Thesis investigation The outer segment of rod photoreceptor cell plays an essential role in phototransduction and vision, yet a complete catalog of ROS proteins has never been generated. The objective of this thesis is to identify all, or at least most proteins in bovine rod photoreceptor outer segments by using liquid chromatography coupled to tandem mass 11 Gene Protein Human retinal disease Rod Outer Segment Proteins Phototransduction and Visual Cycle: ABCA4 (ABCR) Rod ABC transporter AMD; Stargardt's disease CNGA1.B1 cGMP-gated channel a- and Autosomal recessive RP P-subunits GNAT1 Transducin a-subunit Autosomal dominant CSNB PDE6A PDE a-subunit Autosomal recessive RP PDE6B PDE P-subunit Autosomal recessive RP; Autosomal dominant CSNB RETGC1 (GUCY2D) Guanylyl cyclase 1 Autosomal recessive LCA RHO Rhodopsin Autosomal dominant RP; Autosomal recessive RP; Autosomal dominant CSNB RHOK Rhodopsin kinase Oguchi disease SAG Arrestin Autosomal recessive RP; Oguchi disease ROS Structure: PROML1 Prominin Autosomal recessive RP RDS Peripherin Autosomal dominant RP; Dominant MD; Digenic RP with ROM1 ROM1 Rom-1 Autosomal dominant RP; Digenic RP with RDS Other Retinal Proteins RP1 Rpl Autosomal dominant RP; Autosomal recessive RP RP2 Xrp2 X-linked RP RPGR Retinitis pigmentosa GTPase X-linked dominant RP regulator X-linked recessive RP XLRS1 Retinoschisin X-linked retinoschisis Table 1.1 Table of Selected Genes Causing Human Retinal Diseases Genes encoding proteins that function in phototransduction, visual cycle, or maintenance of ROS structure are often linked to inheritied retinal degenerative diseases. AMD, age-related macular degeneration; CSNB, congenital stationary night blindness; LCA, Leber's congenital amaurosis; RP, retinitis pigmentosa RetNet (http://www.sph.uth.tmc.edu/Retnet/) 12 spectrometry (LC-MS/MS). It is anticipated that the outcome of these studies will provide insight into cellular processes in the ROS that are, as yet, not well defined. These include the renewal process of the ROS and the formation and maintenance of its highly organized structure. In addition, the results should prove to be a valuable resource to the research community studying the structure and function of previously uncharacterized ROS proteins. Chapter two of this thesis describes the design and results of the proteomic analysis. It was shown by proteomics that a subset of SNARE and Rab proteins are present in the rod outer segment preparations. Immunofluorescence microscopy was used to confirm the presence of Muncl8, VAMP-2/3, Rab 11, and Rab-GDI in the rod outer segment. Chapter three of this thesis describes the cloning and characterization of a potential aminophospholipid-transporter IB (ATP8A2) that was identified in the proteomic study. This protein has never been studied and very little is known about its structure, function, or localization. The full length human ATP IB was successfully cloned, expressed, and purified from cultured cells. It was shown that the purified protein possesses ATP hydrolytic activity which is enhanced in the presence of lipids. This provides support for its proposed role as an ATP-dependent lipid transporter. Lastly, chapter four summarizes the research findings and suggests future directions for the studies. All studies were carried out by me except for the confocal microscopy studies and associated immunoblots (Figures 2.7-2.9) (Dr. J. Holopainen), and the RT-PCR experiment (Figure 3.3) (Dr. S. Azadi). 13 C H A P T E R II Proteomic Analysis of Rod Outer Segment 2.1 Introduction 2.1.1 Mass Spectrometry based Proteomics Proteomics is defined as the study of all proteins, including all isoforms and modifications, of a given cell or organism at a specified time (de Hoog and Mann, 2004). Proteins encoded by genes carry out the majority of biological functions within cells, and to understand how cells work, it is necessary to identify what proteins are present, how they interact and when they are expressed. Mass spectrometry has proven to be an indispensable tool in the field of proteomics. A mass spectrometer consists of an ion source, a mass analyzer, and a detector. Electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI) are two most used ionization methods for the analysis of peptides and proteins (Aebersold and Mann, 2003). Two mass analyzers can be used in tandem to provide information about the peptide sequence, in what is known as tandem mass spectrometry (MS/MS). ESI-MS/MS systems are often coupled to a HPLC (LC-MS/MS), where the peptides are separated on a chromatographic column and then eluted directly into the electrospray ion source. The first analyzer selects a precursor ion, which is then fragmented in the collision cell with an inert gas, breaking bonds along the polypeptide backbone. The second mass analyzer measures the resulting fragments and records the MS/MS spectra of the selected precursor ion. The 14 amino acid sequence of the precursor peptide ion and the identity of the original protein can be deduced by searching the MS/MS spectra against protein sequence databases (Figure 2.1). 2.1.2 Vesicle Trafficking and Membrane Fusion Vesicle trafficking is an essential cellular process in the secretory and endocytic pathways that is involved in the delivery of newly synthesized proteins and lipids from the endoplasmic reticulum to their destined intracellular compartment, as well as the regulated secretion of neurotransmitters at neuronal synapses. The mechanisms underlying membrane trafficking can be summarized in four essential steps: vesicle formation, intracellular transport, vesicle tethering, and membrane fusion. Although the precise mechanism mediating membrane fusion is still unknown, the basic molecular components have been identified. SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins and the syntaxin-associated protein Muncl8 have emerged as key components of the membrane fusion machinery (reviewed by Jahn and Scheller, 2006). It was discovered that SNARE proteins reconstituted into proteoliposomes are capable of inducing membrane fusion without the need for other factors (Weber et al., 1998). The SNARE proteins have been proposed to account for the specificity of membrane fusion initially and to directly execute fusion by forming a tight SNARE complex that brings the vesicle and target membrane in close proximity (McNew et al., 2000). However, it was later determined that the specificity of membrane trafficking is most likely defined at the vesicle targeting and tethering steps (Zerial and McBride, 2001). Syntaxin, SNAP-25 (25 kDa synaptosome-associated protein), and VAMP (vesicle-associated membrane protein, also called synaptobrevin) were the first SNARE proteins to be discovered (Figure 2.2). They were 15 — as • • . . . P e p t i d e s i ,1 Sample SDS-PAGE fractionation 536 T a n d e m m a s s s p e c t r u m 1 1 0 3 722 8 5 1 11 F \ Y L w 1204 1495 1 567 Y 1 K S e q u e n c e d a t a b a s e M V K W P D V Q K E K C G M N E R Y R 0 f" F Y V F H V. T I V A L P P ^ T T S V K . S e q u e n c e a n d p r o t e i n i d e n t i f i e d Figure 2.1 A General Overview of a Proteomic Analysis by Tandem Mass Spectrometry Complex samples can be separated into simpler fractions, and the proteins can be either digested in-solution or in-gel after separation by SDS-PAGE with a specific protease. The resulting peptides are separated by HPLC and eluted directly into the tandem mass spectrometer where they are ionized and fragmented. Sequence information is collected and the tandem mass spectra of the peptides are searched against predicted mass spectra of proteins in a sequence database. Figure modified from Gygi and Aebersold, 2000 16 Figure 2.2 SNARE and SNARE related protein Muncl8 in Membrane Fusion In the current model of vesicle fusion, t-SNARE proteins syntaxin and SNAP-25 form a SNARE complex with v-SNARE synaptobrevin (VAMP) that brings together the lipid bilayers of the vesicle and target membrane to induce membrane fusion. Muncl8 has recently been shown to bind to the assembled SNARE complexes as well as the closed conformation of syntaxin and is believed to play a central role in membrane fusion. Figure modified from Dulubova et al, 2007. 17 classified into v-SNAREs and t-SNAREs according to their vesicle or target membrane localization, and they have also been reclassified as R-SNAREs (arginine-containing SNAREs) or Q-SNAREs (glutamine-containing SNAREs) based on the identity of a highly conserved residue (Fasshauer et al., 1998). Different combinations of SNARE complexes have been observed from a limited number of SNARE proteins. The precise function of Muncl8-1 is still unclear, but recent studies have shown that it binds directly to the neuronal SNARE complex (Dulubova et al., 2007). Other components involved include NSF (N-ethylmaleimide-sensitive factor), which is an ATPase associated with the disassembly of the SNARE complex (Sollner et al., 1993). Studies on NSF indicated that NSF is not needed for fusion but functions as an activation or priming factor (Mayer et al., 1996). Rab proteins belong to the Ras superfamily of GTPases and more than 60 Rab proteins have been identified in mammalian cells (Schultz et al., 2000). It is now widely accepted that Rab proteins have different subcellular localizations and regulate distinct steps in membrane trafficking through their downstream effector molecules (Stenmark and Olkkonen, 2001). Experimental evidence has suggested that Rab proteins are central regulators of all steps of vesicle transport (reviewed by Grosshans et al., 2006). Like other members of the Ras-like superfamily of proteins, Rabs cycle between a GDP-bound ('off) and a GTP-bound ('on') conformations, and thus are able to function as molecular switches. Conversion between the two conformations is catalyzed by GDP/GTP exchange factors (GEF) and GTPase-activating proteins (GAP). Specific effector molecules are recruited and bind exclusively to each GTP-bound Rab to transduce the signal in the trafficking mechanism. Rab effectors have been identified to be a very diverse group of proteins that include proteins involved in membrane tethering, enzymes, or cytoskeleton-associated proteins, making it possible that a single Rab GTPase may be regulating several molecular events in the same 18 compartment (Stenmark and Olkkonen, 2001). Rab proteins are also geranylgeranylated and have a continuous cycle of association and dissociation from membranes (Anant et al., 1998). The membrane-bound Rab is activated by a GEF (GDP/GTP exchange factor) through the exchange of GDP for GTP and interacts with downstream effectors. Activation of the Rab is terminated upon GTP hydrolysis catalyzed by a GAP, and the Rab protein is released from the membrane by Rab GDP-dissociation inhibitor (GDI) (Ullrich et al., 1993) (Figure 2.3). 2.2 Materials and Methods 2.2.1 Materials Monoclonal antibodies (mAb) to ABCA4 (Rim 3F4), the cGMP-gated channel (PMc 2G11), and the rod Na+/Ca2+-K+ exchanger (PMe 2D9) have been previously described (Cook et al, 1989; Illing et al., 1997; Kim et al., 1998). Polyclonal antibody (pAb) to syntaxin 3 was a generous gift of Dr. Vesa Olkkonen, National Public Health Institute, Finland. Other antibodies were obtained from the following sources: synaptophysin mAb (Santa Cruz Biotechnology, Santa Cruz, CA); Rabl 1 pAb (Sigma-Aldrich) to the N-terminal segment of Rabl 1 (crossreacts with Rabl la and Rabl lb); Rabl la pAb (Zymed Laboratories, South San Francisco, CA) to the C-terminal region of human Rabl la; Muncl8 (syntaxin binding protein 1) pAb and RabGDI (a-subunit) mAb (Synaptic Systems, Goettingen, Germany); VAMP2 pAb that crossreacts with VAMP3 (Stressgen, Victoria, Canada); NSF pAb and mAb (Abeam, Cambridge, MA); and Na+-K+ ATPase a3 mAb (Affinity BioReagents, Golden, CO). All antibodies labeled a single band (or a doublet in the case of 19 S N A R E Figure 2.3 Rab proteins in the Trafficking and Fusion of Vesicles with Target Membrane Rab proteins are key regulators in the targeting and fusion of vesicles. They can be anchored to the membrane or present in the cytoplasm. They cycle between GTP- and GDP-bound forms. The interaction between the active GTP-bound Rab with its effector tethers the vesicle to its appropriate target membrane and allows SNARE-mediated membrane fusion. Rab-GDI binds the membrane-associated GDP-bound Rab and inhibits the exchange of GDP for GTP. The GDI-bound Rab protein is released from the membrane and is recycled back to its original membrane. Figure modified from Olkkonen and Ikonen, 2000. 20 syntaxin 3) of the predicted protein molecular weight in retinal extracts as analyzed by Western blotting. An exception was the Rab 11 pAb which labeled a 35 kDa protein in addition to the Rab 11 protein as documented in the technical specifications for this antibody. 2.2.2 Subcellular Fractionation of ROS ROS were isolated on a continuous sucrose gradient from fresh or previously frozen, dark-adapted bovine retinas as previously described (Molday and Molday, 1987). The remaining retinal extract designated as retina deficient in ROS (RDR) was stored frozen until required. ROS were separated into a ROS membrane and soluble (cytoplasmic) fraction by hypotonic lysis of ROS followed by centrifugation as previously described (Molday and Molday, 1987). The soluble fraction was concentrated on a Nanosep 10K spin column (Pall Corporation, East Hills, NY) to 1 mg/ml protein. The ROS membrane pellet was washed twice in Tris-EDTA buffer (10 mM TrisHCl, pH 7.4, 1 mM EDTA) and stored in the same buffer. ROS membranes were further separated into highly pure disks and enriched plasma membrane by a modification of the immunogold density perturbation method (Molday and Molday, 1987). Briefly, the extracellular surface of ROS was labeled for 2 h at 4°C with the PMe 2D9 mAb conjugated to lOnm gold-dextran particles (Kim et al., 1998). The labeled ROS were washed three times in Tris-EDTA buffer by centrifugation at 12,000 rpm for 10 min and resuspended in 2.5 mM Tris, pH 7.4 containing 1 mM EDTA and 1 mM DTT. Dissociation of disk membranes from the plasma membrane was allowed to occur overnight at 4°C. The sample was subsequently vortexed, pelleted and resuspended in 7% sucrose/20mM Tris, pH 7.4. The sample was centrifuged on a 20-40% continuous sucrose gradient at 22,500 rpm for 2 h. Unlabeled disk membranes were collected as a band near the 21 top of the gradient, while the enriched labeled plasma membrane was collected near the bottom of the tube. 2.2.3 SDS-PAGE and Western Blot Analysis For western blotting, samples were denatured in 10 mM Tris-HCl, pH 6.8, 4% SDS, 20% sucrose, and 4% (3-mercaptoethanol and separated on 8% or 11.5% SDS polyacrylamide gels. Proteins were transferred onto Immobilon-FL membranes (Millipore, Bedford, MA). Blots were blocked in 0.5% skim milk in phosphate-buffered saline (PBS) for 30 min and labeled with the primary antibody in 0.5% skimmed milk in PBS containing 0.05% Tween 20 (PBS-T) at the recommended dilution. The blots were washed in PBS-T and labeled with a secondary anti-mouse or anti-rabbit antibody conjugated to Alexa Fluor 680 (1:40,000) (Molecular Probes, Eugene, OR) or LI-COR IRDye 800 (1:10,000) (Rockland, Gilvertsville, PA) for analysis on a LI-COR Odyssey imager (Lincoln, NE). For MS analysis, samples (50 pg protein) were denatured in lithium dodecyl sulfate denaturing buffer containing 4% P-mercaptoethanol and separated on 4-12% Tris/Glycine NuPAGE gels according to the manufacturer's instructions (Invitrogen, Carlsbad, CA). 2.2.4 In-solution and In-gel Digestion of Proteins for Proteomic Analysis For in-solution digests, protein samples were prepared as previously described (Foster et al., 2003). Briefly, samples (5pg protein) were treated with 6M urea, 2M thiourea in lOmM HEPES, pH 8.0, reduced with lpg DTT per 50pg protein for 30 min, alkylated with 5pg iodoacetamide per 50pg protein for 30 min, digested with lpg endopeptidase LysC per 50pg protein for 3 h, and diluted 4x with 50mM ammonium bicarbonate. The proteins were 22 subsequently digested overnight at 37°C with 1 jag porcine modified trypsin (Promega, Nepean, ON) per 50pg protein. For in-gel digests, each lane of a NuPAGE gel was cut into 15 pieces and digested as described (Shevchenko et al., 1996). Peptide mixtures were desalted and concentrated using stop and go extraction (STAGE) tips (Rappsilber et al., 2003). 2.2.5 LC-MS/MS Analysis Peptides were resolved by reverse phase chromatography on 15 cm long, 75 pm inner diameter fused silica emitters with 8 pm diameter opening (Polymicro, Phoenix, AZ) packed with 3 pm diameter ReproSil-Pur Cig beads (Dr. Maisch, Ammerbuch-Entringen, Germany) with an Agilent 1100 Series nanoflow HPLC coupled online to LTQ-FT and LTQ-Orbitrap systems (ThermoFisher, Bremen, Germany) using nanospray ionization sources (Proxeon Biosystems, Odense, Denmark). Running buffer A consisted of 0.5% acetic acid and running buffer B consisted of 0.5% acetic acid and 80% acetonitrile. Gradients were run from 6% B to 30% B over 60 min, 30% B to 80% B for 10 min, held at 80% B for 5 min, and then dropped to 6% B for 15 min to recondition the column. The LTQ-FT was set to acquire a full range scan at 25,000 resolution in the FT, from which the three most intense multiply-charged ions per cycle were isolated for fragmentation in the LTQ. Selected ion monitoring scans in the FT were also carried out on each of the three precursor ions as described (Olsen and Mann, 2004). The LTQ-Orbitrap was set to acquire a full-range scan at 60,000 resolution from 350 to 1500 Th in the Orbitrap and to simultaneously fragment the top five peptide ions in each cycle in the LTQ. 23 2.2.6 Protein Identification Fragment peak lists were generated by ExtractMSN software (ThermoFisher) and monoisotopic peak and charge state assignments were checked by DTA Supercharge, part of the MSQuant suite of software (http://msquant.sourceforge.net). Fragment spectra were searched against the bovine IPI database (v3.03, 46,730 sequences, ftp://ftp.ebi.ac.uk/pub/ databases/IPI/old/BOVIN/) using Mascot Server v2.1 with the following parameters: Trypsin specificity allowing up to one missed cleavage, cysteine carbamidomethylation, ESI-TRAP fragmentation, 5 ppm mass tolerance for precursor ion mass, and 0.8 Da mass tolerance for fragment ions. Sequences of human keratins, trypsin and LysC were added to the bovine IPI database prior to searching. Since the complete bovine genome was not available and spectra not matching any proteins in the bovine database were subsequently searched against the mammalian taxa of the Mass Spectrometry protein sequence DataBase (MSDB, 306,702 sequences, http://csc-fserve.hh.med.ic.ac.uk/ msdb.html) using the same limits as for the bovine search. A non-redundant list of proteins and sequence coverage data was then compiled as described (Foster et al., 2006). Acceptance criteria for protein identifications were set to require two or more peptides of seven or more amino acids and each with scores greater than 24, corresponding to a false positive rate of less than 1% as calculated by reversed database searching. 2.2.7 Immunofluorescence Microscopy Cryosections of rat or mouse eyes fixed with 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.0 (PB) for 4 h and subsequently permeabilized and blocked in PB 24 containing 0.2% Triton X-100 and 10% normal goat serum for 20 min. The sections were then labeled overnight with the primary antibody diluted in PB containing 0.1 % Triton X-100 and 2.5%) normal goat serum as per manufacturers' specifications. Sections were rinsed in PB and labeled for 1 h with the secondary antibody conjugated with Cy3, and counterstained with DAPI nuclear stain for analysis with a Zeiss LSM510 Meta confocal microscope equipped with a Zeiss LSM5 Image Browser. 2.3 Results 2.3.1 Subfractionation of Bovine ROS In order to simplify the mixture of proteins in the rod photoreceptor outer segment, purified bovine ROS were separated into a membrane and soluble fraction (Figure 2.4). The soluble fraction contains proteins that do not require detergent for solubilization including cytoplasmic proteins and proteins that are weakly associated with either integral membrane proteins or the lipid bilayer. ROS membranes were further separated into a disk membrane fraction and an enriched plasma membrane fraction using an immunogold density perturbation procedure (Molday and Molday 1987). Gold dextran particles were conjugated to the monoclonal antibody 2D9 generated against the plasma membrane specific Na+/Ca2+-K+ exchanger protein and used to separate the plasma membrane from the disk membranes by sucrose density gradient centrifugation (Figure 2.4). The proteins of each subcellular fraction were separated by SDS-PAGE (Figure 2.5). The purity of the membrane fractions was confirmed by the dominance of the rhodopsin monomer (~36kDa), and to a lesser extent the dimer (~72kDa) as previously reported (Papermaster and Dreyer, 1974). Other major bands in the membrane fractions included the ABCA4 transporter (250kDa) (Ming et al., 25 Bovine Retina ^ Sucrose Gradient Purified ROS Hypotonic Lysis in 20mM Tris Centrifugation ROS Membranes Soluble Proteins ^ Labeled with 2D9-go!d ^ Hypotonic separation of disks and P M Sucrose Gradient Disk Membrane Plasma Membrane Figure 2.4 Subcellular Fractionation of Bovine Rod Outer Segment Flow diagram showing the main steps in the purification and subcellular fractionation of rod outer segment from bovine retina. Samples highlighted in boxes represent the five fractions used for subsequent proteomic analysis. 26 kDa 250-150-100-75-50-37-25-< R O S S o l M e m D i s k P M ^mnp ^BP* j^jp^ ^jp^ ;^ |||^ p Rho(2) Rho Figure 2.5 Rod Outer Segment Subcellular Fractions analyzed by SDS-PAGE Coomassie blue-stained SDS gel of the various ROS fractions used in the proteomic studies. From left to right are: molecular weight markers; ROS; ROS soluble fraction; ROS membrane fraction; disk fraction; and enriched plasma membrane fraction. Thirty pg of proteins was loaded on each lane of the 8% SDS gel. All fractions were dominated with rhodopin, Rho and its dimer, Rho(2) except the soluble fraction. 27 1997), and the P-subunit of the cGMP-gated channel (~270kDa), which is present in the ROS, ROS membrane, and plasma membrane fractions, but absent in the disk membrane fraction (Korschen et al., 1995). The soluble fraction also agreed with previous studies showing pairs of double bands at 37-39 kDa and 89-91 kDa corresponding to the a- and (3-subunits of transducin and phosphodiesterase (Molday, 1998). A series of weaker bands were also visible between 20-150 kDa in the soluble fraction indicating the presence of a complex mixture of proteins. 2.3.2 Proteomic Analysis of ROS In-gel and in-solution tryptic digestions were carried out on the five fractions: total ROS, ROS membranes, soluble, disk membranes, and plasma membranes, and the peptides were analyzed by LC-MS/MS. Database searching of the fragmentation spectra resulted in the identification of a combined total of 529 proteins from 3958 peptides (Appendix I). The selection criteria required each protein to be identified from two or more peptides each having a length of seven or more amino acids and Mascot score of 24 or higher. Reducing the criteria to one peptide and/or shorter peptide lengths resulted in a secondary dataset (data not shown) of over 500 additional proteins, many of which may represent false positives due to the reduced selection criteria (Cargile et al., 2004). The identified proteins were separated into six broad categories based on their principal known or predicted function (Figure 2.6; Appendix II). A seventh category "Uncharacterized" was added to include hypothetical proteins annotated in the genomic database for which no information on their putative function is available. 28 Total ROS Figure 2.6 Summary of the Proteomic Analysis of Rod Outer Segment Preparations Five ROS fractions were analyzed by LC-MS/MS. A total of 3958 peptides were detected resulting in the identification of 529 proteins. The proteins were classified into seven broad categories according to their primary known or predicted functions. 29 2.3.3 Rod and Cone Phototransduction Proteins Most of the proteins that are known to function in phototransduction and the visual cycle were identified in the proteomic approach (Table 2.1), with the exception of guanylate cyclase-activating protein 1 (GCAP 1) and the y subunit of phosphodiesterase. However, these proteins were present in the secondary dataset containing proteins identified from a single peptide or peptides of less than seven amino acids in length. This suggests that trypsin digestion of these relatively small proteins may not yield a sufficient number of long peptides to be included in the primary dataset. Conversely, the primary dataset includes RPE65 and cellular retinal binding protein (CRALBP), which are two abundant proteins that function in the visual cycle and retinoid binding. Since these proteins have been reported to reside in RPE and/or Mueller cells, they most likely represent minor contaminants of the ROS preparations (Bunt-Milam and Saari, 1983; Hamel et al., 1993). In addition to ROS proteins, most of the cone specific phototransduction proteins were also detected, including the red and blue opsin, cone transducin, cone arrestin, cone opsin kinase, cone cGMP-gated channel and the cone Na+/Ca2+-K+ exchanger (Table 2.1). The ability to detect the relatively scarce cone proteins truly demonstrated the extraordinary sensitivity of the LC-MS/MS system. 30 Protein Unique Sequence Peptides Coverage (%) Phototransduction, Rods Calmodulin 1 4 17 cGMP-gated cation channel alpha 1 23 34 cGMP-gated cation channel 240kDa protein 38 28 Guanine nucleotide-binding protein, beta-5 subunit 17 43 Phosducin 8 42 Protein phosphatase 2 regulatory subunit A isoform alpha 2 5 Recoverin 17 78 Regulator of G-protein signaling 9 40 66 Regulator of G protein signaling 9-binding protein (R9AP) 9 39 Retinal guanylyl cyclase 1 35 38 Retinal guanylyl cyclase 2 15 17 Rhodopsin 9 23 Rhodopsin kinase 25 52 Rod cGMP-specific 3',5'-cyclic phosphodiesterase alpha subunit 53 55 Rod cGMP-specific 3',5'-cyclic phosphodiesterase beta subunit 57 60 Sodium/potassium/calcium exchanger 1 14 11 Splice isoform A of S-arrestin 38 76 Splice isoform B of S-arrestin 35 73 Transducin, alpha-1 subunit 32 70 Transducin, beta-1 subunit 19 65 Transducin, gamma-Tl subunit 7 77 Phototransduction, Cones Arrestin 3 (X-arrestin) 6 19 Blue-sensitive opsin 2 7 cGMP-gated cation channel alpha 3 5 6 Cone cGMP-specific 3',5'-cyclic phosphodiesterase alpha subunit 27 28 Red opsin 7 13 Retina G protein-coupled receptor kinase 7 5 11 Sodium/potassium/calcium exchanger 2 3 7 Transducin alpha-2 subunit 18 49 Transducin gamma-T2 subunit 3 45 Visual / Retinoid Cycle A B C A 4 104 43 All-trans retinol dehydrogenase 7 26 Cellular retinaldehyde-binding protein 14 47 Interphotoreceptor retinoid-binding protein (IRBP) 46 45 Retinal pigment epithelium-specific 65 kDa protein (RPE65) 3 7 Table 2.1 Selected Proteins involved in Phototransduction and Visual Cycle in Rods and Cones Present in the Proteomic Dataset. The number of unique peptides used for the identification and their sequence coverage percentages are given. 31 2.3.4 Metabolic, Structural, Transport, and Housekeeping Proteins A significant number of proteins that are involved in metabolic pathways, structural support, molecule transport, and basic cellular functions were detected (Table 2.2). Many of these proteins are involved in glycolysis, the hexose monophosphate shunt, and the creatine phosphate shuttle pathways, thus confirming earlier studies (Hsu and Molday, 1990; Hsu and Molday, 1991; Hemmer et al., 1993). Peripherin/rds (Arikawa et al., 1992), rom-1 (Moritz and Molday, 1996), prominin-1 (Maw et al., 2000), and RP1 (Liu et al., 2003) are proteins implicated in ROS morphogenesis and structure were also detected. Other cytoskeletal and membrane associated proteins such as tubulin are believed to play an important role in the overall structure of the ROS as well (Matesic et al., 1992). Kinesin, which has been reported to be in close association with microtubules in ROS and believed to transport molecules longitudinally along microtubules was among the proteins identified (Eckmiller and Toman, 1998). Crbl, which is implicated in photoreceptor morphogenesis and is found in the cone outer segments but not in ROS, was also detected (Pellikka et al., 2002). This again demonstrated the ability of our proteomic analysis to detect cone-specific proteins despite their scarcity in the preparations. A large proportion of proteins were classified as housekeeping proteins. These proteins are found in most cells and perform basic cellular functions, including chaperone proteins, signal transduction proteins, ribosomal proteins, and nucleotide processing proteins etc. Some of these proteins have been reported in the outer segment of the photoreceptors, such as protein kinase C (Udovichenko et al., 1996), heat shock protein 27 (Strunnikova et al., 2001), 5'-nucleotidase (Takazawa, 1998) and 2',3'-cyclic nucleotide 3'-phosphodiesterase (Kohsaka et al., 1983). The presence of semenogelin I and II which have unknown functions 32 in the outer segment and the retina were also confirmed in this study (Bonilha et al., 2006). Additional studies, such as immunofluorescence microscopy, are required to determine if other proteins in our dataset are real resident proteins of outer segments or contaminants from the other cellular compartments. 2.3.5 Vesicle Trafficking Proteins Proteins implicated to play a role in vesicle trafficking and membrane fusion comprised a major class of proteins identified (Table 2.2; Appendix II). Twenty-seven of the fifty-seven proteins in this category are members of the Rab family of small GTPase proteins known to regulate vesicular transport (Grosshans et al., 2006). In addition, the a- and P-subunits of the Rab GDP dissociation inhibitor (Rab-GDI) were also identified. Most SNARE proteins implicated in membrane fusion were detected, including syntaxin 3, VAMP 2 or 3 (VAMP 2/3, or synaptobrevin 2/3), NSF, and the syntaxin binding protein Muncl8-1 (Jahn and Scheller, 2006). SNAP-25, another key SNARE protein, was present in the secondary dataset containing proteins identified from a single peptide or peptides of less than seven amino acids in length. Synaptophysin, an abundant synaptic protein was not detected by LC-MS/MS suggesting that synaptosomes were not a major contaminant of our ROS preparations (Brandstatter et al., 1996). 33 Protein Unique Sequence Peptides Coverage (%) Structure Actin, cytoplasmic 2 14 47 Peripherin 15 36 Prominin-1 12 30 Retinitis pigmentosa 1 protein 15 9 Rom-1 11 35 Tubulin, alpha 1 22 55 Tubulin, beta 2 22 53 Metabolism / Transport Brain creatine kinase 16 50 Glucose transporter I (GLUT-1) 6 10 Glyceraldehyde-3-phosphate dehydrogenase 19 67 Hexokinase 1 24 27 Phosphoglycerate kinase 1 22 63 Sodium/potassium-transporting ATPase alpha 3 34 37 Transketolase 7 14 Vesicle Trafficking N-ethylmaleimide-sensitive factor (NSF) 17 19 Rab 11A/B 14 70 Rab 14 10 29 Rab GDP-dissociation inhibitor alpha 10 29 Syntaxin 3 5 20 Syntaxin-binding protein 1 (Muncl8-1) 22 41 Vesicle associated membrane protein 2/3 (VAMP 2/3) 3 46 Table 2.2 Selected Proteins involved in Vesicle Trafficking and Other Functions Present in the Proteomic Dataset. The number of unique peptides used for the identification and their sequence coverage percentages are given. 34 2.3.6 Immunolocalization of Rab and SNARE Proteins The localization of a subset of Rab and SNARE proteins in the retina were reported to be in the inner segments and the synaptic regions of photoreceptors and secondary neurons, but their presence in the outer segments has not been reported (Deretic 1997; Von Kriegstein et al., 1999). As a result, it was important to verify that the Rab and SNARE proteins identified in our proteomic analysis are true components of the photoreceptor outer segments. Both western blotting of isolated ROS and retina deficient in ROS (RDR) and immunofluoresence labeling of mouse and rat retinal crysections were carried out. As a control experiment, the distribution of ABCA4, Na+/K+ ATPase (a-3 subunit), and synaptophysin known to be localized to the outer segment, inner segment, and plexiform (synaptic) layers of the retina were examined (Brandstatter et al., 1996; Illing et al., 1997; Wetzel et al., 1999). ABCA4 and Na+/K+ ATPase were detected by LC-MS/MS in our ROS preparations, whereas synaptophysin was not detected. As shown by western blotting in Figure 2.7A, ABCA4 was almost exclusively found in the isolated ROS fraction, whereas Na+/K+ ATPase and synaptophysin were predominantly found in the RDR fraction. This distribution was in agreement with the literature as well as the immunofluorescence labeling (Figure 2.7B and 2.7C), showing the preferential distribution of ABCA4 in the outer segment of the retina, Na+/K+ ATPase in the inner segment layer, and synaptophysin in the outer and inner plexiform layers. 35 A Synaptophysin B Na/K ATPase ABCA4 RDR ROS RDR ROS RDR ROS RPE Figure 2.7 Localization of Synaptophysin, Na+/K+ ATPase and ABCA4 in Retina (A) Western blots of bovine retina deficient in ROS (RDR) and isolated ROS labeled for synaptophysin (38 kDa), Na+/K+ ATPase a-subunit (110 kDa), and ABCA4 (250 kDa). (B) Confocal scanning microscopy of rat retinal cryosections labeled with primary antibodies to synaptophysin, Na+/K+ ATPase, and ABCA4 followed by a secondary antibody tagged with the Cy3 fluorescent dye (green). The sections were counterstained with DAPI (blue) nuclear dye. (C) Magnification of Panel B showing a restricted region of the outer and inner segment of photoreceptors. Similar labeling results were obtained with mouse retina. RPE, retinal pigment epithelium layer; OS, outer segment; IS, inner segment; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer. Figure courtesy of Dr. J. Holopainen. 36 Next, we examined proteins that have never been reported to reside in the photoreceptor outer segments. Western blotting showed that Rabl 1 and Rab 14, as well as the Rab-GDI a, were present in both the ROS and RDR fractions (Figure 2.8A). Localization of these proteins to the outer segments and other retinal layers were confirmed by immunofluorescence microscopy (Figure 2.8B, 2.8C). Since both Rabl la and Rabl lb were identified from the proteomic study, we have used two separate antibodies to examine their localization: a polyclonal antibody that crossreacts with both Rabl la and Rabl lb isoforms as well as a 35 kDa protein of unknown function; and a polyclonal antibody specific for the C-terminal region of Rabl la. As shown in Fig 2.8B, the second antibody labeled the outer segments and the outer nuclear layer, while the first Rabl la-specific antibody only labeled the outer segments. These results confirmed the presence of Rabl la in the outer segments, where it is inconclusive for Rabl lb as an antibody specific for Rabl lb would be required. Rab-GDI immunoactivity was also observed in the photoreceptor cells, with moderate staining in the inner segment and intense staining in the outer segment. In contrast, Rab 14 immunoactivity was relatively weak in the outer segment compared to the inner segment. 37 Figure 2.8 Localization of Rab 11, Rab 14 and Rab-GDI in Retina (A) Western blots of bovine retina deficient in ROS (RDR) and isolated ROS labeled for Rab 11, Rab 14, and Rab-GDI a-subunit. (B) Confocal scanning microscopy of rat retinal cryosections labeled with primary antibodies to Rab 11 (C-terminal specific antibody specific for Rab 1 la), Rab 11 (N-terminal specific antibody crossreacts with Rab 1 la and 1 lb), Rab 14, and Rab-GDI a-subunit followed by a secondary antibody tagged with the Cy3 fluorescent dye (green). The sections were counterstained with DAPI (blue) nuclear dye. (C) Magnification of Panel B showing a restricted region of the outer and inner segment of photoreceptors. Similar labeling results were obtained with mouse retina. RPE, retinal pigment epithelium layer; OS, outer segment; IS, inner segment; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer. Figure courtesy of Dr. J. Holopainen. 38 The distribution of several SNARE proteins in the retina was also examined. All of Syntaxin 3, VAMP 2/3, Muncl8-1, and NSF were detected in both the ROS and RDR fractions by western blotting (Figure 2.9A), but only VAMP 2/3 and Muncl8-1 showed immunolabeling throughout the outer segment layer of rat and mouse retina by immunofluorescence microscopy (Figure 2.9B, 2.9C). Syntaxin 3 and NSF showed a striking punctuate pattern of labeling near the junction between the outer and inner segments and strong labeling in the distal region of the outer segment layer close to the RPE. The latter may reflect the labeling of aged disks within the outer segments or apical processes of RPE cells which penetrate into the outer segment layer. All of the SNARE proteins were also observed in the plexiform layers consistent with their role in neurotransmitter release at ribbon synapses (Von Kriegstein et al., 1999). The localization of Rabl 1, Rab-GDI, VAMP 2/3, and Muncl8-1 within ROS was further evaluated using isolated disk and plasma membrane preparations by Western blotting. As shown in Figure 2.10, the distribution of Muncl8-1 and VAMP 2/3 was similar to the cGMP-gated channel, a plasma membrane marker (Cook et al., 1989), with labeling primarily in the plasma membrane fraction. In contrast, Rabl 1 showed a labeling pattern similar to the disk membrane specific ABCA4 with labeling in both the disk and plasma membrane fractions (Ming et al., 1997). The labeling in the plasma membrane is primarily due to the presence of disk rim membrane which is not completely dissociated from the plasma membrane under the conditions used in the isolation procedure (Molday and Molday, 1987). These results suggest that Muncl8-1 and VAMP 2/3 are localized to the plasma membrane of ROS, while Rabl 1 could be disk membrane specific or present in both the disk and plasma membrane. 39 Figure 2.9 Localization of NSF, Syntaxin 3, Munc 18-1, and VAMP 2/3 in Retina (A) Western blots of bovine retina deficient in ROS (RDR) and isolated ROS labeled for NSF, syntaxin 3, Munc 18-1, and VAMP 2/3. (B) Confocal scanning microscopy of rat retinal cryosections labeled with primary antibodies to NSF, syntaxin 3, Munc 18-1, and VAMP 2/3 followed by a secondary antibody tagged with the Cy3 fluorescent dye (green). The sections were counterstained with DAPI (blue) nuclear dye. (C) Magnification of Panel B showing a restricted region of the outer and inner segment of photoreceptors. Similar labeling results were obtained with mouse retina. RPE, retinal pigment epithelium layer; OS, outer segment; IS, inner segment; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer. Figure courtesy of Dr. J. Holopainen. 40 ROS Disk PM ABCA4 CNG-cc Munc18 VAMP-2/3 Rab 11 Figure 2.10 Distr ibut ion of Proteins in the Disk and Plasma Membrane Western blots of total ROS, disks, and enriched plasma membrane were labeled for ABCA4 (a disk membrane marker); cGMP-gated channel (CNG-a; a plasma membrane marker), Munc 18-1, VAMP 2/3, and Rab 11. 41 2.4 Discussion 2.4.1 Proteome of Bovine Photoreceptor Outer Segment In this study we have used sensitive tandem mass spectroscopic technique together with subcellular fractionation procedures to identify proteins in bovine photoreceptor outer segments. Most previously identified ROS proteins known to function in phototransduction, retinoid cycling, metabolic pathways, maintenance of ROS structure, and protein associated with retinal degenerative diseases were detected. In addition, a significant number of cone-specific proteins that function in phototransduction in cone outer segments were detected (Table 2.1). Since cones constitute only about 3% of the photoreceptors in bovine retina and cone outer segments are considerably smaller than rod outer segments, cone phototransduction proteins are on average more than two orders of magnitude less abundant than rod phototransduction proteins. Therefore, it is believed that our approach would contain the majority of ROS proteins if these relatively scarce cone proteins were detected. A small number of proteins may have been missed due to the inability of trypsin digestion to produce multiple peptides of seven or more amino acids. This could be due to the intrinsic properties of those proteins, such as the amino acid composition, the size, the hydrophobicity, or post-translational modifications. In addition, the use of an incomplete bovine genomic database may also result in missed identifications, although the analysis of a mouse ROS preparation showed comparable results (data not shown). The 529 proteins identified in our dataset most likely represent an over estimation of the number of proteins in the photoreceptor outer segments. Although the subcellular fractionation methods used in the study are known to produce highly pure preparations based on biochemical and electron microscopic analyses (Molday and Molday, 1987), the high 42 sensitivity of the tandem MS system still resulted in the detection of some minor contaminating proteins from other cellular compartments. These include Na+/K+ ATPase (Figure 2.7), RPE65 (Hamel et al., 1993), CRALBP (Bunt-Milam and Saari, 1983), myosin VI (Kitamoto et al., 2005), and the plasma membrane Ca2+ ATPase (Krizaj et al., 2002). Although it is possible that these proteins are present in the outer segments in exceedingly low quantities that could not be detected by immunofluorescence in the previous studies, more likely they represent contaminants from the inner segments or from neighbouring RPE cells. Abundant proteins from the interphotoreceptor matrix such as serum albumin and interphotoreceptor retinoid binding protein (IRBP) were also found to be a source of contamination in the ROS preparations (Adler and Edwards, 2000). Therefore, it is important to validate the proteomic results with other biochemical and immunocytochemical techniques. It is also interesting to note that many of the photoreceptor cilium proteins were not detected, such as the intraflagellar transport proteins (Rosenbaum et al., 1999), retinitis pigmentosa GTPase regulator (RPGR) and RPGR-interacting protein (RPGRIP) (Hong et al., 2001; Hong et al., 2003), indicating that the connecting cilium was not a major contaminant of the ROS preparations. 2.4.2 Rab and SNARE Proteins in Rod Outer Segments Rab and SNARE proteins are known to function in membrane trafficking and fusion in cellular events such as neurotransmitter release, endocytosis, membrane protein trafficking, and protein secretion (Grosshans et al., 2006; Jahn and Scheller, 2006). Here we report the presence of VAMP2/3 and Muncl8-1 throughout the photoreceptor outer segment, while syntaxin 3 and NSF are present at the boundary of the inner and outer segments as well as the 43 distal region of outer segment. These results suggest that these SNARE proteins may mediate membrane fusion associated with the ROS membrane renewal process. ROS membrane renewal is a dynamic process involving vesicle trafficking and membrane fusion within the inner segment, protein transport through the connecting cilium, formation of new disk membranes at the base of the outer segment, and the removal of aged membranes at the distal end. Disk morphogenesis takes place at the base of the outer segment by evagination of the ciliary plasma membrane followed by the joining of adjacent membranes through the outgrowth of the specialized disk rim region to generate mature, closed disks in ROS (Steinberg et al., 1980). The aged packet of disks are intermittently removed at the distal end of the outer segment through a process of shedding and phagocytosis (Young and Bok, 1969). Molecular mechanisms underlying these processes are not understood, but membrane fusion must play a central role in both disk morphogenesis and outer segment shedding. The difference in the localization of the SNARE proteins examined may partly be explained by the differences in the onset of immunoreactivity observed for SNAP-25, syntaxin, and VAMP in developing and mature retina (Greenlee et al., 2001). The fact that the disk shedding process is not continuous suggests the possibility that the necessary proteins may translocate to the site of membrane fusion only when required, similar to the translocation of transducin, arrestin and recoverin between the outer and inner segments (Calvert et al., 2006). Previous studies have shown that partially assembled SNARE complexes do exist (Hua and Charlton, 1999). The Rab family of GTPases also plays an important role as regulators in vesicle trafficking and membrane fusion (Grosshans et al., 2006). Membrane proteins such as rhodopsin that are destined for the ROS are synthesized in the ER of the inner segments and targeted to the base of the connecting cilium on post Golgi vesicles, a process that was shown 44 to be regulated by Rab 6 and Rab 8 proteins (Deretic and Papermaster, 1993; Deretic 1997; Moritz et al., 2001). Several genetic diseases have been linked Rab or their interacting proteins (Olkkonen and Ikonen, 2000). Choroideremia is an X-linked disease caused by mutation in REP-1, a protein needed for the geranylgeranylation of Rabs, leading to the degeneration of RPE and the adjacent photoreceptor cell layers (Sebra et al., 1993). The presence of Rab 11 in the outer segment of photoreceptors identified in this study raises the possibility that it may play a key role in regulating the outer segment renewal process. The proteomic and immunofluorescence data suggest that it is likely the most abundant member of the Rab GTPase within the photoreceptor outer segments, as it has a strong immunoreactivity throughout the outer segments and is consistently identified from the most number of peptides in our ROS preparations (Figure 2.8; Table 2.2). It is possible that Rabl 1 interacts with Rab-GDI, which was also shown to be present throughout the outer segments (Figure 2.8), to be maintained in its inactive state over the length of the outer segment to prevent disk membranes from fusing with the plasma membrane. A signal may induce the switch of Rabl 1 to its active GTP bound state at the proximal and distal ends of the outer segment to initiation the membrane fusion events in disk morphogenesis and/or disk shedding. However, detailed studies are needed to examine the exact roles of Rab 11 and the SNARE proteins play in the photoreceptor outer segment. In summary, a total of 529 proteins were identified in bovine rod photoreceptor outer segment preparations and most of the proteins known to function in phototransduction, the visual cycle, maintenance of outer segment structure, metabolic pathways, and retinal degeneration were identified. In addition, a large number of proteins were detected that have not been previously shown to be present in the photoreceptor outer segments including Rab and SNARE proteins implicated in vesicle trafficking and membrane fusion. The dataset of 45 proteins generated in the study should serve as a valuable resource to further define the molecular and cellular basis of outer segment function, its morphogenesis and renewal. These results should also be useful in the identification of photoreceptor proteins linked to various inherited retinal degenerative diseases such as retinitis pigmentosa and macular degeneration. 46 CHAPTER III Cloning and Characterization of a Potential Aminophospholipid Transporter ATPase IB (ATP8A2> 3.1 Introduction 3.1.1 Lipids and Biological Membrane Asymmetry Biological membrane is a double layer of lipid that surrounds cells and cellular compartments, acting as an impermeable barrier to the passage of most polar molecules. Biological membrane is made up of three types of membrane lipids: glycerophospholipids, sphingolipids, and sterols. Phospholipids contain a polar head group that is joined to the hydrophobic moiety through a phosphodiester linkage. Phosphatidylcholine (PC), sphingomyelin, and glycosphingolipids are enriched primarily on the exoplasmic leaflet of the plasma membrane or the topologically equivalent luminal leaflet of internal organelles. On the contrary, the aminophospholipids are typically found on the cytoplasmic leaflet. Phosphatidylserine (PS) is present almost exclusively in the cytoplasmic leaflet, whereas phosphatidylethanolamine (PE) is found predominantly on the same leaflet. Other minor phospholipids, phosphatidic acid, phosphatidylinositol, are also enriched on the cytoplasmic side of the membrane (Gascard et al., 1991). According to the fluid mosaic model, phospholipids form a lipid bilayer in which the hydrophobic fatty acyl tails of the lipid face each other at the core of the bilayer and their polar head groups face outward. Membrane proteins are embedded within the bilayer held by hydrophobic interactions between their stretches of hydrophobic residues in an alpha-helical conformation and the fatty acyl chains of membrane lipids. Membrane lipids and membrane 47 proteins are distributed asymmetrically within the bilayer. The relative proportion of each varies with the type of membrane reflecting the diverse biological roles of membranes. Lipids are distributed asymmetrically across biological membrane bilayers (Bretscher, 1972). Loss of transmembrane phospholipid asymmetry leads to changes in the membrane's physical properties with consequent effects on cell-cell and lipid-protein interactions. Exposure of PS in the external monolayer of the plasma membrane induces apoptosis mediated by cell surface PS receptors on macrophages (Fadok et al., 1992). Other cytoplasmic or membrane proteins can also be affected by the loss of membrane transbilayer asymmetry. Protein kinase C is one of the important enzymes involved in signal transduction that interacts with the cytofacial surface of membranes and requires diacylglycerol and PS for activation (Nishizuka, 1986; Newton and Johnson, 1998). Lipid biosynthesis is intrinsically asymmetrical, as glycerophospholipids are synthesized on the cytoplasmic side of the endoplasmic reticulum (ER) (Bell et al., 1981). Once the lipid asymmetry has been established, it is maintained by a combination of slow transbilayer diffusion, protein-lipid interactions, and protein-mediated transport (reviewed by Daleke, 2003). Since it had been shown that transbilayer diffusion has a half time of hours to days (Kornberg and McConnell, 1971) and that protein-lipid interactions do not play a major role in the maintenance of lipid asymmetry (Gudi et al., 1990), it is believed that protein-mediated transport is the major contributor that gives rise to lipid bilayer asymmetry and its maintenance. 48 3.1.2 Lipid Transporters Lipid transporters are classified into three types according to their direction of transport and requirement for energy: scramblase, floppase and flippase (reviewed by Daleke 2007). Scramblases are ATP-independent nonspecific bidirectional transporters that 2"t-randomize phospholipid distribution across the lipid bilayer. Examples include a Ca -activated scramblase in the plasma membrane of erythrocytes (Williamson et al., 1992) and an ER scramblase (Buton et al., 1996). Floppases are ATP-dependent transporters that catalyze the inner (cytoplasmic) to outer (external or luminal face) transport of lipids, including most members of the ATP-binding cassette (ABC) superfamily. Examples include the well studied ABCA1 (ABC1) that transports cholesterol and PS out of cells (Hamon et al., 2000) and ABCB1 (MDR1) that has broad substrate specificity and is responsible for multidrug resistance (van Helvoort et al., 1996). Flippases are ATP-dependent transporters that catalyze the transport of lipids in the opposite direction from floppases, that is from the outer to inner monolayer (Figure 3.1). Flippase activity was first discovered in human erythrocytes (Seigneuret and Devaux, 1984), which consumes approximately one ATP per lipid transported (Beleznay et al., 1993). Aside from erythrocytes, aminophospholipid flippase activity has since been detected in a wide variety of cells including lymphocytes, fibroblasts, synaptosomes, and aortic endothelial cells (Daleke, 2003). ATP-dependent flippase activity was also observed in chromaffin granules, indicating that flippases are present in intracellular organelles as well (Zachowski et al., 1989). Although the identities of the aminophospholipid flippases remain elusive due to difficulties in purification and direct evidence for their lipid transport activity, it is widely believed that the members of the P4-ATPases family are lipid flippases. 49 Figure 3.1 Regulation of Transbilayer Lipid Distribution in Cellular Membranes The transbilayer movement of lipids within cellular membranes is catalyzed by three classes of lipid transporters which transport lipids in different directions and have different requirements for energy. The ATP-dependent ABC transporters transport lipids from the cytoplasmic monolayer to the exoplasmic (or lumenal) monolayer, while the P4-ATPases transport lipids in the opposite direction. Scramblases are ATP-independent and function to abolish transbilayer phospholipid asymmetry by catalyzing bidirectional transport of lipids. 50 3.1.3 P-type ATPases P-type adenosinetriphosphatase (P-type ATPase) is a large and varied family of ATP-driven transporters involved in many transport processes (reviewed by Kiihlbrandt, 2004). They are widely distributed, including the ubiquitously expressed Na+/K+ ATPase and Ca2+ ATPase. They have a characteristic DKTG[T,S][L,I,V,M][T,I] sequence motif in which the Asp(D) residue is reversibly phosphorylated by ATP as part of the transport cycle, hence the name "P-type". The majority of the P-type ATPases contain ten transmembrane helices, while some only have six or eight. Both the amino and carboxyl termini are on the cytoplasmic side of the membrane and the cytoplasmic domains between the second/third and third/fourth transmembrane helices are well conserved. All the P-type ATPases studied to date have molecular masses between 70-150 kDa and are all sensitive to inhibition by the phosphate analog vanadate. P-type ATPases have been divided into five major subtypes according to their substrate specificities: Type I includes heavy metal ion pumps; Type II includes Na+/K+ ATPases, Ca2+ ATPases, and H+/K+ ATPases; Type III includes H + and Mg2+ pumps; Type TV are phospholipid pumps; and Type V which have no assigned substrate specificity (Axelsen and Palmgren, 1998). Type IV P-type ATPases (P4-ATPases) are a class of P-type ATPases that was first discovered in 1996 to be involved in lipid transport and the maintenance of lipid-bilayer asymmetry (Tang et al., 1996). Since then over a hundred members of the P4-ATPase have been identified in eukaryotes including approximately a dozen human isoforms (Halleck et al., 1999). 51 3.1.4 Lipid composition in ROS ROS membranes are composed of approximately 50% protein and 50% lipid by weight, and phospholipids account for 90 to 95% of the total lipids (Fliesler and Anderson, 1983). The major phospholipids are PE and PC like in all eukaryotic cells with a relatively large proportion of PS that accounts for around 15% of all phospholipids (Giusto et al., 1986). One of the most prominent characteristic of ROS membranes is the unusually high content of long chain polyunsaturated acids, with docosahexaenoic acid (DHA) being the most abundant (Aveldano, 1987). The content of DHA within PC and PI also increases as disks age, which suggests that disk membranes undergo changes to prepare for eventual disk shedding and phagocytosis (Albert et al., 1998). The disk membrane contains significantly more DHA (22:6n-3) in its phospholipids than the plasma membrane, and DHA was shown to be essential in visual function (Fliesler and Anderson, 1983; Bush et al., 1994). ROS is also relatively deficient in cholesterol and glycosphingolipids compared to other eukaryotic membranes. Consistent with the preference of aminophospholipids to be on the cytoplasmic monolayer, it was estimated that at least 77-88% of PS and 73-87% of PE are in the outer monolayer (facing the cytoplasm) of ROS disk membranes. (Miljanich et al., 1981). The charge asymmetry caused by the cytoplasmically positive rhodopsin has been suggested to play a role in this asymmetry (Hubbell, 1990). 52 3.2 Materials and Methods 3.2.1 DNA Sequencing and Bioinformatic Predictions All clones were sequenced using BigDye® Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA) according to the manufacturer's instruction. Protein domain and pattern analysis was performed by InterProScan v. 4.3 (http://www.ebi.ac.uk/InterProScan) (Quevillon et al., 2005). Prediction of N-glycosylation was performed using NetNGlyc 1.0 (http://www.cbs.dtu.dk/services/NetNGlyc) and O-glycosylation was performed using NetOGlyc 3.1 (http://www.cbs.dhi.dk/services/NetOGlyc) (Julenius et al., 2005). Protein topology was predicted by TMHMM v.2.0 (http://www.cbs.dtu.dk/services/TMHMM) and TopPred (http://bioweb.pasteur.fr/seqanal/interfaces/toppred.html). Physical and chemical properties of the protein were predicted by ProtParam (http://www.expasy.ch/tools/protparam.html). All bioinformatics predictions were performed on the published sequence from National Center for Biotechnology Information (NCBI) accession number: AK127263. Sequence alignments were performed by ClustalW (http://www.ebi.ac.uk/Tools/clustalw/). 3.2.2 Cloning of lD4-tagged Human ATPase IB The first 1060bp on the 5' end of the gene (N-terminal fragment) was cloned from human retinal cDNA by PCR using sequence-specific primers containing a unique restriction site Kpn I at the 5'end based on the published sequence from NCBI accession number: AK127263: 53 Forward: 5'-GCGGGTACCATGCTGAACGGCGCAGGC-3' Reverse: 5'-GTGTCCATCTTCTTGATGTAC-3' The C-terminal fragment containing the last 2987bp of the gene (581-3567) was amplified by PCR from a clone obtained from the German Resource Centre for Genome Research, Clone: DKFZp686K0636Q using sequence-specific primers containing a unique restriction site Xba I and the lD4-tag epitope at the 3' end. Forward 5'-GAACCTCAGGCAATGTGTTATG-3' Reverse 5' -GCGTCTAGATTAGGCAGGCGCCACTTGGCTGGTCTCTGTTTTCTT CCTGGATTTCTTTTTG-3' The N and C terminal fragments were ligated together by taking advantage of the unique EcoR I restriction site GAATTC in the overlapping sequence (891-896) of the fragments (Figure 3.2) and cloned into mammalian expression vector pcDNA3 (Invitrogen, Carlsbad, CA). The full-length coding sequence in pcDNA3 was sequenced to verify correct sequence and the absence of random mutations. 3.2.3 Cell Culture and Transfection HEK293T cells were grown in Dulbecco's modified Eagle medium (DMEM) with L-glutamine, 10% bovine growth serum. Single transfections were carried out with lOpg DNA per plate for immunofluorescence microscopy and 20pg per plate for purification. 500pL BES-buffered saline (50mM N,N-bis(2-hydroxyethyl)-2-aminoethane, 280mM NaCl, 1.4mM Na2P04, pH 6.95) was added dropwise to 500pL DNA solution containing 250mM calcium chloride and incubated for 20 minutes at room temperature. 54 ATP8A2 1D4 ! j ! 581 1060 3567 bp | C-terminal fragment EcoR I German Resource Centre for Genome Research N-terminal fragment i Clone: DKFZp686K0636Q Figure 3.2 Cloning of the Full Length Human ATP8A2 with 1D4 Affinity Tag Diagram illustrating the cloning of the full length human ATP8A2-IDA from two fragments. The N-terminal fragment was cloned from human retinal cDNA by PCR using primers containing a Kpn I restriction site at the 5'end (1-1060). The C-terminal fragment (581-3567) was obtained from the German Resource Centre for Genomic Research (Clone:DKFZp686K0636Q) and amplified by PCR using a reverse primer containing the lD4-tag epitope and Xba I restriction site at the 3' end. The two fragments were cloned into pcDNA3 vector and ligated together by taking advantage of the unique EcoR I restriction site in the overlapping sequence in the fragments. 55 3.2.4 Solubilization of protein in HEK293 Transfected HEK293T cells were harvested by washing the cells twice in PBS (137mM NaCl, 2.7mM KC1, lOmM Na2HP04, 1.8mM KH2P04, pH 7.4) by low-speed centrifugation. The pellet was resuspended in 200 pL of PBS and added to an equal volume of PBS containing one of the following detergents: 2% TX-100; 2% NP40; 40mM CHAPS, 5% octoglucopyranoside; 1.6% «-dodecyl-P-D-maltoside (DM) and stirred at 4°C for 20 minutes. Unsolubilized materials were centrifuged at 50,000 rpm in a Beckman TLA-100.4 rotor for 15 minutes and the supernatant was retained for SDS gel electrophoresis. 3.2.5 Immunoaffinity Purification HEK293T cells solubilized in 20mM CHAPS were incubated with 50pL monoclonal antibody Rho lD4-conjugated Sepharose 2B beads at 4°C for 1 h. The unbound material was discarded by low speed centrifugation through an Ultrafree filter unit (Millipore, Bedford, MA) and the beads were washed three times with lOmM CHAPS in PBS (washing buffer). Bound proteins were eluted sequentially by incubation in lOOpL washing buffer containing 0.2mg/mL, 0.5mg/mL, LOmg/mL 1D4 synthetic peptide (pH 7.5), or 2% SDS for 15 minutes each at room temperature. The elutions were collected by low speed centrifugation. 3.2.6 Immunofluorescence Microscopy Transfected HEK293T cells were grown in DMEM with L-glutamine and 10% bovine growth serum on polylysine-coated coverslips at 37°C and fixed with 4% paraformaldehyde in phosphate buffer (PB; 0.1M phosphate, pH 7.4) for 25 minutes. 56 Coverslips were blocked and permeabilized in PB with 10% goat serum and 0.5% TX-100 for 30 minutes and labeled with the Rho 1D4 monoclonal (1:50 dilution) or Per 2B6 against peripherin (1:20 dilution) and a polyclonal antibody against the ER marker calnexin (Stressgen, Victoria, BC; 1:200 dilution). After washing with PB, the samples were labeled with goat anti-mouse immunoglobulin conjugated with Alexa 488 (Molecular Probes, Burlington, ON; 1:1000 dilution) and goat anti-rabbit immunoglobulin conjugated with Alexa 594 (Molecular Probes, Burlington, ON; 1:1000 dilution) for 1 h and counterstained with DAPI for detection of nuclei. Labeled cells were washed with PB and examined under a fluorescence microscope (Axioplan 2, Zeiss, Oberkochen, Germany) equipped with a digital image analysis system. 3.2.7 ATPase Activity Assay ATPase activity was measured as the percentage of [a-32P] ADP produced from the enzymatic hydrolysis of [oc-32P] ATP (NEN Life Science Products, Boston, MA). CHAPS-solubilized protein (7pL) was pipetted into 0.5mL microcentrifuge tube with 5mM MgCl2 to a final volume of 9pL in the absence or presence of 25pg of porcine brain polar lipid extract (Avanti Polar Lipids Inc, Alabaster, AL). The reaction was initiated with the addition of lpL of a 1 Ox ATP solution (Final concentration: 0.2pCi) and incubated at 37°C for 30 minutes. The assay was stopped with the addition of 4pL 10% SDS and the tube was centriftiged briefly. One pL of the reaction mixture was spotted onto a polyethyleneimine cellulose plate (Aldrich, Milwaukee, Wl) and separated by thin layer chromatography in 0.5M LiCl/ 1M formic acid. The plate was exposed to a storage phosphor screen overnight and scanned in a Typhoon 8600 imager (Molecular Dynamics, Sunnyvale, CA). Spots corresponding to ADP 57 and ATP were quantified using IPLab Gel Analysis software (Signal Analytics Corp., Vienna, VA). Each sample was assayed in triplicate and buffer blanks were included to determine nonenzymatic ATP hydrolysis. 3.3 Results 3.3.1 Gene Expression by RT-PCR The gene expression profile of mouse aminophospholipid transporter-like ATPase IB (ATP8AT) was examined in five tissues: heart, kidney, brain, spleen, and retina. It was found that the mouse ATP8A2 was expressed predominantly in the retina and minimally in the brain, while expression in the other three tissues tested were not detected (Figure 3.3). This expression profile agrees with the published profile from EST (expressed sequence tag) counts, in which the expression levels were the highest in the pituitary gland, lung, nerve, testis, brain, and eye (UniGene Accession Number: Hs.444957, NCBI). 3.3.2 Cloning of human ATP8A2 The cDNA sequence of the human ATP8A2 was obtained from a partial cDNA clone and a PCR fragment amplified from total retinal cDNA (Figure 3.2). The cloned human ATP8A2-1D4 construct contains 3594bp and the open reading frame encodes a protein of 1197 amino acids (nine of which is the 1D4 epitope). It has a predicted molecular weight of 134 kDa and a theoretical pi of 8.3 (ProtParam, Swiss Institute of Bioinformatics). It is 94% identical in sequence to the mouse ATP8A2 and 93% identical to the bovine ATP8A2. 58 400 bp - I Figure 3.3 Expression oiATP8A2 gene in Various Tissues of Mouse RT-PCR of a 400bp gene-specific sequence of ATP8A2 with cDNAs from various tissues of mouse: heart, kidney, brain, spleen, and retina. Figure courtesy of Dr. S. Azadi. 5 9 3.3.3 DNA Sequencing and Sequence Alignments The cloned human ATP8A2-ID4 was sequenced on both strands and aligned with two human sequences in the National Center for Biotechnology Information (NCBI) database (Accession numbers: AK127263.1 (Last updated: Sep 14, 2006) and NM_016529.4 (Last updated: June 27, 2007). The nucleic acid sequences were more than 99% identical to each other with a total of seven mismatched nucleotides between the three sequences. Translation of the sequences revealed that six of the seven mismatches resulted in the same amino acids, with the exception of position 573 where the cloned construct and NM_016529.4 have an arginine but the AK127263 has a lysine residue (Figure 3.4, highlighted in grey). It is also noted that the nine amino acid epitope of the 1D4 tag (TETSQVAPA) was incorporated into the C-terminal of the construct as intended. 3.3.4 Domain Analysis, Topology, and Glycosylation Sites Predictions A thorough bioinformatics analysis of the aminophospholipid transporter-like ATPase IB was performed using a variety of tools available on the internet. The human ATP8A2 had been mapped to chromosome 13 (Unigene, NCBI), and domain analysis using InterProScan (European Bioinformatics Institute) confirmed that the protein under study belongs to the phospholipid-translocating P-type (E1-E2 type) ATPase, flippase family. It contains a conserved signature P-type ATPase phosphorylation site on residues 428-434 and a P-type ATPase associated region between residues 126-384. Potential glycosylation sites were predicted using NetNGlyc 1.0 and NetOGlyc 3.1 (Center for Biological Sequence Analysis, Technical University of Denmark) for N-linked and O-linked glycosylation respectively. The protein contained three potential N-linked glycosylation sites at residues 298, 339, 848 60 AK127263 Clone AK127263 Clone AK127263 Clone AK127263 Clone AK127263 Clone AK127263 Clone AK127263 Clone AK127263 Clone AK127263 Clone AK127263 Clone AK127263 Clone AK127263 Clone AK127263 Clone AK127263 Clone AK127263 Clone AK127263 Clone AK127263 Clone AK127263 Clone AK127263 Clone AK127263 Clone MLNGAGLDKALKMSLPRRSRIRSSVGPVRSSLGYKKAEDEMSRATSVGDQLEAPARTIYL 60 MLNGAGLDKALKMSLPRRSRIRSSVGPVRSSLGYKKAEDEMSRATSVGDQLEAPARTIYL 60 ************************************************************ NQPHLNKFRDNQISTAKYSVLTFLPRFLYEQIRRAANAFFLFIALLQQIPDVSPTGRYTT 120 NQPHLNKFRDNQISTAKYSVLTFLPRFLYEQIRRAANAFFLFIALLQQIPDVSPTGRYTT 120 ************************************************************ LVPLIIILTIAGIKEIVEDFKRHKADNAVNKKKTIVLRNGMWHTIMWKEVAVGDIVKVVN 180 LVPLIIILTIAGIKEIVEDFKRHKADNAVNKKKTIVLRNGMWHTIMWKEVAVGDIVKVVN 180 ************************************************************ GQYLPADVVLLSSSEPQAMCYVETANLDGETNLKIRQGLSHTADMQTREVLMKLSGTIEC 24 0 GQYLPADVVLLSSSEPQAMCYVETANLDGETNLKIRQGLSHTADMQTREVLMKLSGTIEC 24 0 ************************************************************ EGPNRHLYDFTGNLNLDGKSLVALGPDQILLRGTQLRNTQWVFGIVVYTGHDTKLMQNST 300 EGPNRHLYDFTGNLNLDGKSLVALGPDQILLRGTQLRNTQWVFGIVVYTGHDTKLMQNST 300 * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * KAPLKRSNVEKVTNVQILVLFGILLVMALVSSAGALYWNRSHGEKNWYIKKMDTTSDNFG 360 KAPLKRSNVEKVTNVQILVLFGILLVMALVSSAGALYWNRSHGEKNWYIKKMDTTSDNFG 360 ************************************************************ YNLLTFIILYNNLIPISLLVTLEVVKYTQALFINWDTDMYYIGNDTPAMARTSNLNEELG 420 YNLLTFIILYNNLIPISLLVTLEVVKYTQALFINWDTDMYYIGNDTPAMARTSNLNEELG 420 ************************************************************ QVKYLFSDKTGTLTCNIMNFKKCSIAGVTYGHFPELAREPSSDDFCRMPPPCSDSCDFDD 4 80 QVKYLFSDKTGTLTCNIMNFKKCSIAGVTYGHFPELAREPSSDDFCRMPPPCSDSCDFDD 4 80 ************************************************************ PRLLKNIEDRHPTAPCIQEFLTLLAVCHTVVPEKDGDNIIYQASSPDEAALVKGAKKLGF 54 0 PRLLKNIEDRHPTAPCIQEFLTLLAVCHTVVPEKDGDNIIYQASSPDEAALVKGAKKLGF 540 ************************************************************ VFTARTPFSVIIEAMGQEQTFGILNVLEFSSDKKRMSVIVRTPSGRLRLYCKGADNVIFE 600 VFTARTPFSVIIEAMGQEQTFGILNVLEFSSDRKRMSVIVRTPSGRLRLYCKGADNVIFE 600 ******************************* *p* ************************** RLSKDSKYMEETLCHLEYFATEGLRTLCVAYADLSENEYEEWLKVYQEASTILKDRAQRL 660 RLSKDSKYMEETLCHLEYFATEGLRTLCVAYADLSENEYEEWLKVYQEASTILKDRAQRL 660 ************************************************************ EECYEIIEKNLLLLGATAIEDRLQAGVPETIATLLKAEIKIWVLTGDKQETAINIGYSCR 720 EECYEIIEKNLLLLGATAIEDRLQAGVPETIATLLKAEIKIWVLTGDKQETAINIGYSCR 720 ************************************************************ LVSQNMALILLKEDSLDATRAAITQHCTDLGNLLGKENDVALIIDGHTLKYALSFEVRRS 780 LVSQNMALILLKEDSLDATRAAITQHCTDLGNLLGKENDVALIIDGHTLKYALSFEVRRS 780 * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * FLDLALSCKAVICCRVSPLQKSEIVDVVKKRVKAITLAIGDGANDVGMIQTAHVGVGISG 840 FLDLALSCKAVICCRVSPLQKSEIVDVVKKRVKAITLAIGDGANDVGMIQTAHVGVGISG 840 ************************************************************ NEGMQATNNSDYAIAQFSYLEKLLLVHGAWSYNRVTKCILYCFYKNVVLYIIELWFAFVN 900 NEGMQATNNSDYAIAQFSYLEKLLLVHGAWSYNRVTKCILYCFYKNVVLYIIELWFAFVN 900 ************************************************************ GFSGQILFERWCIGLYNVIFTALPPFTLGIFERSCTQESMLRFPQLYKITQNGEGFNTKV 960 GFSGQILFERWCIGLYNVIFTALPPFTLGIFERSCTQESMLRFPQLYKITQNGEGFNTKV 960 ************************************************************ FWGHCINALVHSLILFWFPMKALEHDTVLTSGHATDYLFVGNIVYTYVVVTVCLKAGLET 1020 FWGHCINALVHSLILFWFPMKALEHDTVLTSGHATDYLFVGNIVYTYVVVTVCLKAGLET 1020 ************************************************************ TAWTKFSHLAVWGSMLTWLVFFGIYSTIWPTIPIAPDMRGQATMVLSSAHFWLGLFLVPT 1080 TAWTKFSHLAVWGSMLTWLVFFGIYSTIWPTIPIAPDMRGQATMVLSSAHFWLGLFLVPT 1080 ************************************************************ ACLIEDVAWRAAKHTCKKTLLEEVQELETKSRVLGKAVLRDSNGKRLNERDRLIKRLGRK 1140 ACLIEDVAWRAAKHTCKKTLLEEVQELETKSRVLGKAVLRDSNGKRLNERDRLIKRLGRK 114 0 ************************************************************ TPPTLFRGSSLQQGVPHGYAFSQEEHGAVSQEEVIRAYDTTKKKSRKK 1188 TPPTLFRGSSLQQGVPHGYAFSQEEHGAVSQEEVIRAYDTTKKKSRKKTETSQVAPA 1197 ************************************************ Figure 3.4 Complete Amino Acid Sequence of the Full Length ATP IB-1D4 Clone The ATP IB-1D4 clone was translated into its corresponding amino acids and aligned with the published sequence (NCBI accession number: AK127263). and one potential O-linked glycosylation site at residue 115. Two protein topology prediction programs were also used: TMHMM v. 2.0 (Center for Biological Sequence Analysis, Technical University of Denmark) predicted seven transmembrane helices, while TopPred (Pasteur Institute, France) predicted ten. The hydropathy profile of the protein generated by TopPred was given in Figure 3.5. Since most of the P-type ATPases contain ten transmembrane helices and none has been shown to contain seven, the TopPred model most likely reflects the actual topology of the protein. The predicted topological models and the relative positions of the potential glycosylation sites are illustrated in Figures 3.6 and 3.7. 3.3.5 Expression and Solubilization by Different Detergents Transfected HEK293T cells were harvested and solubilized with different detergents. The solubilized materials were separated by SDS-PAGE and the expression of the aminophospholipid transporter-like ATPase IB was detected by Western blots with anti-lD4 antibody. As shown in Figure 3.8, 1% NP-40 alternative, 20mM CHAPS, and 0.8% n-dodecyl-(3-D-maltopyranoside (DM) were able to solubilize the expressed protein that corresponds to the expected size of aminophospholipid transporter-like ATPase IB (~130kDa). Solubilization with TX-100 resulted in a series of smaller bands (Lane a), while 2.5% octyl p-D-glucopyranoside was unable to solubilize any detectable proteins (Lane d). Twenty mM CHAPS in PBS was used for the solubilization of the ATPase IB-1D4 in all subsequent experiments unless otherwise noted. These results demonstrated that our cloned human ATPase IB-1D4 construct can be expressed in HEK293T cells and detected by the monoclonal antibody 1D4. 62 a o t_ "O - 4 h - 5 Upper c u t o f f L outer c u t o f f h u d r b p h o b icLtu I J L 200 4:08. :600 8@B 1:000 p o s i t i o n -oT window Iri- sequence' Figure 3.5 Hydropathy Profile of Human ATPase IB (ATP8A2) Hydropathy plot predicted by TopPred program on the NCBI published sequence (accession number: AK127263) using the Goldman Engelman Steitz (GES) scale and default parameters. The hydrophobicity of the protein is plotted in blue with the amino terminus starting at residue zero on the x-axis. The lower and upper cutoff values are shown in green (0.5) and red (1.0) respectively. 63 Figure 3.6 Predicted Topological Model for Human ATPase IB using TopPred Topological model predicted by TopPred program on the NCBI published sequence (accession number: AK127263) using the default parameters contained ten transmembrane regions. The conserved P-type ATPase phosphorylation site was identified by InterProScan v.4.3 (residues 428-434) and potential glycosylation sites were predicted by NetNGlyc 1.0 and NetOGlyc 3.0 respectively. Potential glycosylation sites on the cytoplasmic space were not shown. 64 Figure 3.7 Predicted Topological Model for Human ATPase IB using TMHMM Topological model predicted by TMHMM program on the NCBI published sequence (accession number: AK127263) using the default parameters contained seven transmembrane regions. The conserved P-type ATPase phosphorylation site (residues 428-434) was identified by InterProScan v.4.3. Potential glycosylation sites were predicted by NetNGlyc 1.0 and NetOGlyc 3.0 respectively. Potential glycosylation sites on the cytoplasmic space were not shown. 65 a b c d e Figure 3.8 Solubilization of ATP IB-1D4 Protein Expressed in HEK293 Cells using Various Detergents Western blot of a) 1% Triton X-100 (TX-100); b) 1% NP40 alternative; c) 20mM CHAPS; d) 2.5% octylglucopyranoside (OG); e) 0.8% «-dodecyl-P-Z)-maltoside (DM) solubilized or f) unsolubilized HEK293T cells expressing ATP IB-1D4 construct labeled with monoclonal antibody Rho 1D4. 66 3.3.6 Purification on lD4-Immunoaffinity Column The expressed lD4-tagged protein could be efficiently purified from CHAPS solubilized HEK293T cells on a lD4-Sepharose 2B column. As shown in Figure 3.9, the majority of the bound protein was eluted in the first two elutions with 0.2mg/mL and 0.5mg/mL 1D4 peptide. The third elution with l.Omg/mL peptide and the final SDS elution did not result in a significant amount of protein, confirming that the 1D4 purification system used was highly efficient and specific. 3.3.7 ATPase Activity Assay The ability of the expressed protein to bind and hydrolyze ATP was tested by the radiolabeled [a-32P] ATP hydrolysis assay as previously described by Ann et al., 2000. Solubilized protein was incubated with and without a brain polar lipid extract and the results were shown in Figure 3.1 OA. In the absence of any lipids, the solubilized protein was able to hydrolyze 4.4% ± 0.1% (one standard deviation) of the ATP to ADP compared to 3.5% ± 0.1% by the buffer blank due to nonenzymatic ATP hydrolysis. The ATPase activity of the protein is much more evident in the presence of a brain polar lipid extract, resulted in the hydrolysis of 15.1% ± 1.8% of the ATP (Figure 3.10B). 3.3.8 Localization of Human ATPase IB in HEK293T cells The cellular distribution of the human aminophospholipid transporter-like ATPase IB-1D4 construct was compared to that of rhodopsin and peripherin in a mammalian expression system. Figure 3.11 showed that all three proteins have a distinct expression 67 f kDa 250-150-100-75-50 37 ~ i — f i i r % <<L <> %, ^  v<>- ^ a . WW °* <CL <CL Figure 3.9 Immunoaffinity Purification of ATP IB-1D4 Protein Expressed in HEK293T Cells CHAPS solubilized HEK293T cells expressing ATP IB-1D4 (lane a) was applied to a Rho lD4-Sepharose column and the unbound proteins (lane b) were removed by washing. The bound proteins were eluted successively using 0.2mg/mL 1D4 peptide (lane c), 0.5mg/mL 1D4 peptide (lane d), l.Omg/mL 1D4 peptide (lane e), and 2% SDS (lane f). The proteins were transferred to PVDF membranes following SDS-PAGE and labeled with the monoclonal antibody Rho 1D4. 68 A ADP 10 -11 Vi 13 14 \t 16 17 '<ti A T P I i M I I I I I B 1 2 3 4 5 6 7 8 9 1 I I I I I Blank - Lipid + Lipids Figure 3.10 ATPase Activity of CHAPS-Solubilized ATP IB-1D4 is Stimulated by the Presence of Lipids ATP IB-1D4 was solubilized and purified from HEK293T cells by immunoaffinity chromatography in the presence of CHAPS. The ATPase activity of the eluate was assayed in the absence and presence of 25pg brain polar lipid extract. (A) Phosphorimage of thin layer chromatography-separated [a- PJADP and [a- P]ATP. (B) Graphical representation of ATPase activity expressed as percentage of ATP hydrolyzed. The experiment was performed in triplicate and the results were averaged. Error bars represent ± standard deviation. 69 Calnexin Merge ATPase IB Peripherin Rhodopsin Figure 3.11 Immunofluorescence of ATPase IB-1D4, Peripherin, and Rhodopsin Expressed in HEK293T Cells Transfected cells were fixed and labeled with the Rho 1D4 monoclonal antibody (rhodopsin, ATPase IB-1D4) (green) or Per 2B6 monoclonal antibody (peripherin) (green) and the calnexin (ER marker) polyclonal antibody (red). The nuclei were visualized with DAPI (blue). The merged images of labeled cells are shown. 70 pattern in HEK293T cells. Permeabilized HEK 293T cells expressing the human ATPase IB-1D4 construct was localized predominantly in the ER, as evidenced by the complete colocalization with the ER marker calnexin. Peripherin also showed extensive colocalization with the ER when labeled with the monoclonal antibody Per 2B6 (Molday et al., 1987), and additional labeling were observed in the cytoplasm and perimeter of the cells. In contrast, HEK 293T cells expressing bovine rhodopsin showed labeling at the perimeter of the cells as well as punctuate labeling within the cells when labeled with anti-rhodopsin monoclonal antibody 1D4 against the carboxyl terminus (Molday and MacKenzie, 1983). Minimal colocalization with the ER-marker calnexin was observed. 3.3.9 Attempt to Generation of Monoclonal Antibody and Immunofmoresence Microscopy To study the expression and cellular distribution of the aminophospholipid transporter-like ATPase IB within the retina, a monoclonal antibody against the endogenous protein would be required. Mice serum from mice injected with GST fusion proteins containing the N-terminal 90 amino acids and the C-terminal 76 amino acids of human ATP8A2 were tested by Western blotting against human ROS preparations. Out of the seven mice injected, only one showed immune response to the antigen. Two bands at ~250kDa and ~130kDa were labeled in human ROS, corresponding to the expected size of the aminophospholipid transporter-like ATPase IB and possibly the dimer or protein aggregate (Figure 3.12A). However, hybridoma cells fused with spleen cells of the positive mouse did not result in any cell lines that secrete an antibody specific for the aminophospholipid transporter-like ATPase IB. The inability to generate a monoclonal antibody was likely due 71 B ROS kDa 250 — 150 — 100 Mgi 75 g j j 37 « M mAb 3F4 (ABCA4) Mouse serum Figure 3.12 Localization of ATPase IB in Human Retina using Serum of Mouse Immunized with the C-Terminal GST-fusion Construct (A) Western blot of human ROS labeled with serum from mice immunized with ATPase IB C-terminal GST-fusion construct (B) Immunofluorescence microscopy of human retinal cryosections labeled with monoclonal antibody 3F4 against ABCA4 and mouse serum immunized with an ATPase IB C-terminal GST-fusion construct, followed by a secondary antibody tagged with the Cy3 fluorescent dye (green) and DAPI (blue) nuclear dye. 72 to the sequence similarity between the human and mouse homologues, thus the antigen was not immunogenic enough to provoke a strong immune response in the mice. The mouse serum from the only positive mouse was used for immunofluorescence labeling on human retina cryosection. As shown in Figure 3.12B, the mouse serum labeled practically all cell layers in the retina except the outer and inner nuclear layers, with the strongest immunoreactivity observed in the photoreceptor outer segment layer. Although the data suggests that the aminophospholipid transporter-like ATPase IB may be present in the outer segments, conclusions cannot be drawn from this study since the mouse serum used may contain multiple non-specific antibodies. Our laboratory is currently in the process of making longer GST-fusion constructs as well as constructs against the bovine sequence in an attempt to increase the immunogenicity of the antigens being injected. 3.4 D i s c u s s i o n In this study, a previously uncharacterized protein identified from the proteomic analysis of rod photoreceptor outer segment was examined. An aminiophospholipid transporter-like ATPase IB (ATP8A2) was consistently detectable from the ROS membranes, particularly in the disk membrane preparations. It was identified from a total of 24 unique peptides, which is more than a number of well-characterized photoreceptor outer segment proteins, such as the Na+/Ca2+-K+ exchanger 1 and the cGMP-gated cation channel alpha 1 subunit. Its presence in the highly pure disk preparation also led to the hypothesis that it is very likely to be a true component in the rod outer segments and may play an important role in the transport of aminophospholipids across disk membranes. 73 The human orthologue of the ATP8A2 gene was cloned and exhibited features similar to other members of the P4-ATPase family. These include similar hydropathy profiles, conserved phosphorylation motif DKTGTLT at residues 428-434, and the cytoplasmic orientation of both the amino and carboxyl termini. Sequence analysis predicted topological models consisting of seven (TMHMM) or ten (TopPred) transmembrane spanning regions. Although TMHMM is the more selective than TopPred in avoiding false positive predictions (Kali and Sonnhammer, 2002), it is believed that the TopPred model reflects the true conformation of the protein in biological membranes more closely as most members of the P-type ATPases studied to date contained ten transmembrane regions and have both the N- and C- termini on the cytoplasmic side. The expression of the aminophospholipid transporter-like ATPase IB in mammalian cells transfected with the lD4-tagged construct was examined by immunocytochemistry. The ATPase IB-1D4 protein was found predominantly in the ER of the HEK293T cells, and had a distinct expression pattern from two other ROS membrane proteins rhodopsin and peripherin. The expressed protein could be efficiently solubilized in a number of detergents and purified on a lD4-immunoaffinity column. We have also confirmed that the cloned protein is indeed an ATPase, and its ATP hydrolytic activity was increased over two-fold in the presence of a brain polar lipid extract. This is consistent with the proposed role of this protein as an ATPase that is activated upon binding to its phospholipid substrate. The specific activity of the protein could not be calculated due to the inability to measure the concentration of the purified proteins which would require a larger batch of samples. Nonetheless, the ATPase activity assay provided compelling evidence that the protein of interest hydrolyzes ATP and is activated in the presence of lipids. 74 Over the years a lot of experimental data have supported the proposed phospholipid transporting activity of the P4-ATPases, but it cannot be ruled out that they play an indirect role by regulating the activity or localization of the proteins directly responsible. Many of the early lipid translocation studies were based only on observations of human erythrocytes, which lack intracellular organelles and vesicular traffic. The closest member in the P 4 -ATPase family to the ATPase IB is the ATPase II (ATP8A1) and both the bovine and murine ATP8A1 homologues have been cloned and purified. (Tang et al., 1996; Paterson et al., 2006). It was determined that the ATPase activity of the murine ATPase II was activated maximally by PS and minimally by PE or phosphatidylglycerol (Paterson et al., 2006). However, its direct role in lipid transport remains to be established. Interestingly, the proposed phospholipid translocating activity of these putative flippases has also been linked to vesicle trafficking. Inactivation of yeast Drs2p, the founding member of the P4-ATPase family, resulted in a decrease in clathrin-coated vesicle budding from the trans-Golgi network (Gall et al., 2002). It was believed that the translocation of lipids may aid in the curvature of the membrane bilayer by generating an imbalance of phospholipids between the two leaflets (Graham, 2004). It is also possible that the imbalance of phospholipids aid in the recruitment of the vesicle budding machinery. It will be of particular interest if the aminophospholipid transporter-like ATPase IB is found to be a true resident protein in the rod outer segments, as phospholipids have been shown to play important roles in photoreceptor function. Transducin and phosphodiesterase have been found to bind to phospholipids in the disk membranes and the lipid-protein interactions are proposed to be important in visual transduction (Hessel et al., 2003). Once the lipid asymmetry has been established, other proteins may contribute to its preservation through binding to the phospholipids. Cytoskeletal proteins actin, spectrin, and band 4.1 75 have been shown to bind to PS in previous studies (Mombers et al, 1979; Sato and Ohnishi, 1983), all of which were detected in our proteomic study. ABCA4, the only member of the ABC transporter family that functions like a flippase (instead of floppase), is essential in the transport of TV-retinylidiene PE from the lumen of disk membranes to the cytosolic side during retinoid recycling (Beharry et al., 2004). It has been observed that the transmembrane movement of phospholipids in disks is partially inhibited by N-ethylmaleimide (Wu and Hubbell, 1993), consistent with the fact that all P4-ATPases are sensitive to N-ethylmaleimide. Localization of the ATPase IB protein to the disk membrane of ROS may also help to explain the existence of rim region within disks. 76 C H A P T E R IV Summary and Future Studies 4.1 Summary This study has provided a comprehensive proteomic analysis of the bovine rod photoreceptor outer segments by tandem mass spectrometry. Well-defined subcellular fractionation techniques were used to separate ROS into soluble and membrane proteins, as well as disk and plasma membrane proteins. The purity of the subcellular fractions was confirmed by SDS-PAGE and Western blotting before being subjected to in-gel and in-solution tryptic digestions. The resulting peptides were separated by high performance liquid chromatography and fragment spectra were generated by tandem mass spectrometry. A total of 529 proteins were identified from genomic database searching and almost all the previously known ROS proteins were detected in our preparations. These include proteins involved in phototransduction and the visual cycle, metabolic pathways, maintenance of ROS structure, and proteins linked to various retinal degenerative diseases. Immunofluorescence microscopy revealed that a small number of proteins detected were contaminants from the adjoining inner segment and retinal pigment epithelium, such as Na+/K+ ATPase. We have detected a subset of Rab and SNARE proteins that have not been reported in the photoreceptor outer segments. Immunofluorescence microscopy confirmed that a number of them are true components of the ROS, including the syntaxin-binding protein Muncl8-1, VAMP 2 or 3 (Synaptobrevin), Rab 11, and Rab-GDI. The function of these proteins implicated in vesicle trafficking and membrane fusion remained unknown in the ROS, but we proposed that they may be involved in ROS morphogenesis and renewal since both processes 77 would require fusion of membranes and regulated movement of intracellular components. Furthermore, a number of uncharacterized proteins were detected in the ROS preparations in which their functions remained to be identified. This dataset of ROS proteins should be a valuable resource for the vision research community to further define the molecular and cellular basis of outer segment morphogenesis, function, and renewal. It also represents an important first step towards the identification of photoreceptor proteins that may be linked to various inherited retinal degenerative diseases. One of the uncharacterized proteins identified from the proteomic study was the aminophospholipid transporter-like ATPase IB (ATP8A2). It belongs to the P4-ATPase family and has been proposed to transport phospholipids across the membrane bilayer. The human ATPase IB was cloned, sequenced, and found to be localized in the endoplasmic reticulum when expressed in HEK293T cells. Bioinformatic analysis predicted ten transmembrane regions for the protein with both the amino and carboxyl termini on the cytoplasmic side, consistent with other members of the P-type ATPases. The expressed ATPase IB-1D4 tagged protein was efficiently solubilized in detergent and purified on a lD4-immunoaffinity column. The ATP hydrolytic activity of the purified protein was examined using a radiolabeled [a-32P]ATP assay and found to be significantly enhanced in the presence of a polar lipid extract. This result is consistent with the proposed role of the ATPase IB as a phospholipid-translocating ATPase. 4.2 Future Studies The proteomic analysis of the rod photoreceptor outer segments has opened many doors for research on this physiologically important compartment that is vital for vision. 78 Prior to obtaining these results, it was thought that the protein composition of the bovine ROS was relatively simple, as rhodopsin comprises over 85% of all proteins with phototransduction, metabolic, and structural proteins accounting for the rest. However, our results suggested that the ROS may contain many proteins that have not been previously reported. This study has shown that immunofluorescence microscopy of retinal cryosections is an indispensable tool for verifying whether proteins are actual ROS resident proteins (eg: Rabl 1, Rab-GDI, Muncl8-1, etc) or contaminants from other cellular compartments (eg: Na+/K+ ATPase). Therefore, one must obtain antibodies specific for each of the proteins identified in order to confirm their localizations within the retina. We have shown that a subset of Rab and SNARE proteins are present in the photoreceptor outer segments, but their functions in ROS remain unclear. According to the current understanding of SNARE proteins, they are only functional as a complex of v- and t-SNAREs with other accessory proteins. Although immunofluorescence microscopy of syntaxin 3 and NSF suggested that they are only present in low levels in ROS, it is possible that these proteins are translocated to the outer segments through the connecting cilium under certain conditions. They may undergo light-dependent translocation into and out of ROS in a way similar to that of a number of phototransduction proteins. Immunohistochemical studies using animals sacrificed under different illumination conditions may help address this issue. It is also important to examine if other isoforms or related proteins may be present in ROS but not recognized by the antibodies used. For example, syntaxin 4 and syntaxin 7 were also detected in the proteomic study and antibodies specific for each protein are required to determine if they are present at a higher amount in ROS than syntaxin 3. The subset of uncharacterized proteins identified from the proteomic analysis is also of interest for further studies. In the second part of the study, the aminophospholipid transporter-like ATPase IB (ATP8A2) was cloned and its ATPase activity was shown to be activated by the presence of a brain polar lipid extract. It would be a logical next step to determine the lipid specificity of the ATPase IB by incubating the purified protein with each of the phospholipids and measure its ATPase activity. It is hypothesized that the presence of at least one of the aminophospholipids (PE and PS) would provide maximum activation. There are still controversies about the identities and actual biological function of the potential lipid flippases, since there is no established method for the direct measure of endogenous lipid transport. Reconstitution of the purified lipid flippase candidates into proteoliposomes with a variety of reporter lipids have been the method of choice for the study of phospholipid transporting activity. However, the use of labeled lipids has been criticized as steric hindrance of the fluorescent lipid analogues and the polarity of spin-labeled probes may affect the phospholipid transporting activity. The limited size of reconstituted proteoliposome may also affect lipid translocation activity due to its large surface tension (Devaux et al., 2006). Recently the changes in the shape of reconstituted giant unilamellar vesicles have been proposed as a new tool to detect flippase activity (Papadopulos et al., 2007). However, this still does not reflect the conditions of a biological membrane and the potential requirement of other accessory or regulatory proteins. The generation of a monoclonal antibody against ATPase IB is essential to examine the presence and localization of the protein within the retina and photoreceptor cell layer. The failure to obtain a specific antibody was likely due to the lack of immunogenicity of the antigen injected. GST-fusion proteins should be made from sequences that are dissimilar to that of the mouse ATPase IB homologue. Once an antibody is purified and tested, it could be used for immunolabeling studies as well as immunoprecipitation to identify potential protein interactors. Ultimately, it is desirable to investigate whether mutations in the ATP8A2 gene may be linked to any retinal degenerative disorders such as retinitis pigmentosa or macular degeneration by generation of a knockdown/knockout animal. 81 4.3 References Adler, R. 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(2001). 91 APPENDIX I - Protiens identified in each of the five ROS preparations Access ion # Name Peptides Detected in R O S Disk Mem P M Sol I P I 0 0 0 1 7 3 7 3 . 1 Rep l i ca t ion pro te in A 14 k D a subun i t 2 Y I P I 0 0 0 1 9 5 6 8 . 1 P ro th romb in p r e c u r s o r (F ragmen t ) 3 Y I P I 0 0 0 2 5 4 1 5 . 1 S e m e n o g e l i n - 2 p recu rso r 7 Y I P I 0 0 0 3 7 0 7 0 . 2 I so fo rm 2 of Hea t s h o c k c o g n a t e 71 k D a pro te in 2 Y Y Y Y I P I 0 0 2 2 0 8 3 4 . 7 A T P - d e p e n d e n t D N A he l i case 2 subun i t 2 2 Y I P I 0 0 2 9 0 2 7 9 . 1 I so fo rm Long of A d e n o s i n e k inase 2 Y Y I P I 0 0 2 9 5 3 8 6 . 6 C a r b o n y l r educ tase [ N A D P H ] 1 3 Y Bone m a r r o w m a c r o p h a g e c D N A , R I K E N fu l l - leng th I P I 0 0 3 3 1 2 8 6 . 2 en r i ched l ib rary , c l o n e : I 8 3 0 1 2 9 N 0 7 p r o d u c t : b e t a - 2 2 Y m i c rog lobu l i n , ful l inser t s e q u e n c e I P I 0 0 3 7 7 3 5 1 . 2 Apo l i pop ro te i n A - I V p recu rso r 4 Y I P I 0 0 4 0 7 2 7 3 . 2 P R E D I C T E D : s im i l a r to Ig k a p p a cha in V reg ion E V 1 5 p r e c u r s o r 2 Y I P I 0 0 4 1 2 5 9 2 . 4 c h r o m o s o m e 9 o p e n read ing f r a m e 77 i so fo rm 2 2 Y Y I P I 0 0 4 1 3 7 3 1 . 2 Pro te in p h o s p h a t a s e 3 2 Y Y Y I P I 0 0 4 1 4 6 8 4 . 6 I so fo rm 2 of S e m e n o g e l i n - 1 p recu rso r 6 Y I P I 0 0 4 7 9 1 8 6 . 4 p y r u v a t e k i nase 3 i so fo rm 1 6 Y Y Y Y Y I P I 0 0 5 5 5 6 2 8 . 1 Neura l cel l a d h e s i o n mo lecu le 1, 120 k D a i so fo rm 2 Y Y Y va r i an t ( F r a g m e n t ) I P I 0 0 6 4 2 9 7 1 . 2 e u k a r y o t i c t rans la t i on e longa t ion fac to r 1 de l ta 4 Y i so fo rm 1 I P I 0 0 6 5 6 3 2 9 . 1 K n g l p ro te in (K in inogen 1) 2 Y I P I 0 0 6 8 5 1 9 6 . 1 S o l u t e car r ie r f am i l y 3 (Ac t i va to rs of d ibas ic a n d 9 Y Y Y Y neut ra l a m i n o ac id t r anspo r t ) , m e m b e r 2 I P I 0 0 6 8 6 0 7 4 . 1 A T P a s e , H+ t r anspo r t i ng , l y s o s o m a l (Vacuo la r p ro ton 5 Y Y Y p u m p ) , a lpha po l ypep t i de , 7 0 k D , i so fo rm 1 I P I 0 0 6 8 6 0 9 2 . 1 P e r o x i r e d o x i n - 1 6 Y Y Y I P I 0 0 6 8 6 1 7 3 . 1 G lu ta th i one S - t r a n s f e r a s e P 10 Y Y Y I P I 0 0 6 8 6 3 3 1 . 1 Exc i ta to ry a m i n o ac id t r anspo r te r 1 3 Y Y Y I P I 0 0 6 8 6 5 4 6 . 1 Hypo the t i ca l p ro te in M G C 1 2 8 8 5 2 (s im i la r to 4 Y Y c h a p e r o n i n con ta in ing T C P 1 , subun i t 2) I P I 0 0 6 8 6 7 3 3 . 1 D P P 3 pro te in (d ipept idy l pep t i dase III) 4 Y Y I P I 0 0 6 8 6 8 0 3 . 1 Hypo the t i ca l p ro te in M G C 1 2 7 2 5 7 (S im i l a r to 6 Y Y r i bonuc lease U K 1 1 4 ) I P I 0 0 6 8 6 9 4 3 . 1 P R E D I C T E D : s im i l a r to C D W 9 2 an t i gen i so fo rm 2 , 4 Y par t ia l I P I 0 0 6 8 6 9 4 8 . 1 S e r p i n B6 3 Y I P I 0 0 6 8 6 9 8 1 . 1 A n n e x i n A 4 15 Y Y Y Y Hypo the t i ca l p ro te in ( P R E D I C T E D : s im i l a r to A l coho l I P I 0 0 6 8 6 9 8 4 . 3 d e h y d r o g e n a s e [ N A D P + ] ( A l d e h y d e reduc tase ) (A ldo - 4 Y Y ke to r educ tase fam i l y 1 m e m b e r A l ) ) I P I 0 0 6 8 7 0 3 3 . 1 P R E D I C T E D : s im i l a r to R A B 3 3 B , m e m b e r R A S 3 Y Y o n c o g e n e fam i l y I P I 0 0 6 8 7 1 3 0 . 2 P R E D I C T E D : s im i l a r to A P 2 a s s o c i a t e d k i n a s e 1 2 Y I P I 0 0 6 8 7 1 3 5 . 1 Euka ryo t i c t rans la t i on e longa t ion fac to r 1 beta 2 2 Y I P I 0 0 6 8 7 1 4 4 . 1 Hypo the t i ca l p ro te in 2 Y I P I 0 0 6 8 7 2 1 1 . 1 H e x o k i n a s e 1 24 Y Y Y Y Y I P I 0 0 6 8 7 2 4 6 . 2 V o l t a g e - d e p e n d e n t a n i o n - s e l e c t i v e channe l p ro te in 2 4 Y Y Y I P I 0 0 6 8 7 3 3 4 . 2 M G C 1 3 3 8 3 0 pro te in (s im i la r to inner m e m b r a n e 3 Y Y Y p ro te in , m i t o c h o n d r i a l , par t ia l ) I P I 0 0 6 8 7 5 3 9 . 1 P R E D I C T E D : s im i l a r to D i h y d r o p y r i m i d i n a s e - r e l a t e d 22 Y Y Y Y Y pro te in 3 I P I 0 0 6 8 7 5 6 0 . 1 P o l y ( R C ) b ind ing p ro te in 1 2 Y Y I P I 0 0 6 8 7 6 2 5 . 2 P R E D I C T E D : s im i l a r to h e t e r o g e n e o u s nuc lea r 2 Y r i bonuc leop ro te in AO 92 Access ion # Name Peptides Detected in R O S Disk Mem P M Sol I P I 0 0 6 8 7 8 5 9 . 1 I n te rpho to recep to r re t i no id -b ind ing pro te in p r e c u r s o r ( IRBP) 46 Y Y Y Y Y I P I 0 0 6 8 8 0 0 6 . 1 Proh ib i t in 2 Y Y I P I 0 0 6 8 8 0 5 4 . 1 81 k D a pro te in (s im i la r to Prote in k i nase C , 2 Y nu t y p e ( n P K C - n u ) (Pro te in k inase E P K 2 ) ) I P I 0 0 6 8 8 0 8 1 . 1 M i c r o t u b u l e - a s s o c i a t e d pro te in 4 7 Y Y I P I 0 0 6 8 8 5 2 2 . 1 V a c u o l a r A T P s y n t h a s e subun i t B, bra in i so fo rm 7 Y Y Y I P I 0 0 6 8 8 6 4 9 . 2 P R E D I C T E D : s im i l a r to A n n e x i n A 6 M G C 1 2 8 5 7 6 pro te in ( P R E D I C T E D : s im i l a r to 8 Y Y Y I P I 0 0 6 8 8 6 5 1 . 3 C a l c i u m / c a l m o d u l i n - d e p e n d e n t pro te in k i nase t ype II de l ta cha in ( C a M - k i n a s e II de l ta cha in ) ( C a M k i n a s e II de l ta subun i t ) ( C a M K - I I de l ta subun i t ) ) 4 Y Y Y I P I 0 0 6 8 8 8 1 6 . 1 C la th r in h e a v y cha in 11 Y Y Y I P I 0 0 6 8 8 8 8 7 . 1 Hypo the t i ca l p ro te in (s im i la r to a l pha i so fo rm of 2 Y Y regu la to ry subun i t A , p ro te in p h o s p h a t a s e 2) I P I 0 0 6 8 8 9 9 8 . 1 T r a n s f o r m i n g pro te in R h o A p r e c u r s o r 5 Y I P I 0 0 6 8 9 2 2 9 . 1 R a s - r e l a t e d C 3 bo tu l i num tox in subs t ra te 1 p r e c u r s o r 3 Y Y ( R a c l ) I P I 0 0 6 8 9 3 2 5 . 1 Pro te in d i s u l f i d e - i s o m e r a s e A 3 p recu rso r 6 Y Y Y I P I 0 0 6 8 9 3 6 2 . 1 T r a n s t h y r e t i n p recu rso r Hypo the t i ca l p ro te in M G C 1 2 8 0 9 6 (s im i la r to T-2 Y I P I 0 0 6 8 9 3 7 7 . 1 c o m p l e x pro te in 1, ze ta subun i t ( T C P - l - z e t a ) ( C C T -ze ta ) ( C C T - z e t a - 1 ) ) 9 Y Y I P I 0 0 6 8 9 4 3 0 . 1 P R E D I C T E D : s im i l a r to C G 3 1 3 2 - P A 7 Y Y Y I P I 0 0 6 8 9 4 3 6 . 2 P R E D I C T E D : s im i l a r to es te rase D / fo rmy lg l u ta th i one 4 Y Y h y d r o l a s e i so fo rm 1 I P I 0 0 6 8 9 4 4 0 . 2 N u c l e a s e sens i t i ve e l e m e n t - b i n d i n g pro te in 1 3 Y I P I 0 0 6 8 9 5 1 5 . 2 P R E D I C T E D : s im i la r to F r u c t o s e - b i s p h o s p h a t e a l d o l a s e 16 Y Y Y Y Y A ( M u s c l e - t y p e a ldo lase ) i so fo rm 1 I P I 0 0 6 8 9 6 3 8 . 1 Hypo the t i ca l p ro te in (Sp l i ce I so fo rm Long of 5 Y Y Y Lac tadhe r i n p recu rso r ) I P I 0 0 6 8 9 6 4 6 . 1 ce l lu la r re t ino ic ac id b ind ing pro te in 1 3 Y Y I P I 0 0 6 8 9 7 8 9 . 1 P R E D I C T E D : s im i l a r to ves i c le a m i n e t r anspo r t p ro te in 1 i so fo rm 1 5 Y Y Y Y I P I 0 0 6 8 9 8 5 7 . 1 P e r o x i r e d o x i n - 6 8 Y Y Y Y I P I 0 0 6 8 9 9 5 4 . 1 146 k D a pro te in ( S y n a p t o j a n i n - 1 ) 4 Y I P I 0 0 6 9 0 0 7 3 . 1 23 k D a pro te in ( 4 0 S r i bosoma l pro te in S 3 ) 3 Y I P I 0 0 6 9 0 1 4 1 . 1 S i m i l a r to H L A - B a s s o c i a t e d t ransc r ip t 1 3 Y I P I 0 0 6 9 0 3 6 7 . 1 Hypo the t i ca l p ro te in M G C 1 2 6 9 5 1 (Ras re la ted v - ra l 6 Y Y Y Y s i m i a n l e u k e m i a v i ra l o n c o g e n e h o m o l o g A) I P I 0 0 6 9 0 5 0 8 . 3 M G C 1 3 7 3 3 6 pro te in 3 Y I P I 0 0 6 9 0 6 2 3 . 1 119 k D a pro te in 2 Y I P I 0 0 6 9 0 7 5 1 . 2 P R E D I C T E D : s im i la r to a n k y r i n repea t d o m a i n 33 13 Y Y Y Y I P I 0 0 6 9 0 8 0 5 . 1 Rod c G M P - s p e c i f i c 3 ' , 5 ' - cyc l i c p h o s p h o d i e s t e r a s e 53 Y Y Y Y Y a l p h a - s u b u n i t I P I 0 0 6 9 0 8 7 0 . 1 Acon i t a te h y d r a t a s e , m i tochondr ia l p recu rso r 9 Y Y Y I P I 0 0 6 9 0 9 6 2 . 1 S y n t a x i n - b i n d i n g pro te in 1 ( M u n c - 1 8 - 1 ) 22 Y Y Y Y Y I P I 0 0 6 9 0 9 9 3 . 1 Hypo the t i ca l p ro te in (Pro te in C 1 1 0 R F 6 7 homo log ) L O C 5 1 3 4 1 0 pro te in ( P R E D I C T E D : s im i l a r to 2 Y I P I 0 0 6 9 1 0 6 8 . 3 h e t e r o g e n e o u s nuc lea r r i bonuc leopro te in A B i so fo rm a , par t ia l ) 2 Y I P I 0 0 6 9 1 2 0 6 . 1 P R E D I C T E D : s im i l a r to R A B 3 9 B , m e m b e r R A S o n c o g e n e fam i l y 3 Y I P I 0 0 6 9 1 2 7 1 . 1 2 0 3 k D a pro te in (K ines in fami l y m e m b e r I B ) 6 Y I P I 0 0 6 9 1 4 0 7 . 1 Pro te in k i nase C beta t ype 4 Y Y I P I 0 0 6 9 1 4 4 0 . 2 P R E D I C T E D : s im i l a r to d i m e r i c d ihyd rod io l d e h y d r o g e n a s e 3 Y Y I P I 0 0 6 9 1 6 4 1 . 2 s im i l a r to A D P - r i b o s y l a t i o n fac to r - l i ke 1 0 C 3 Y Y 93 Access ion # Name Peptides Detected in R O S Disk Mem P M Sol I P I 0 0 6 9 1 7 4 7 . 3 B e t a - s y n u c l e i n 3 Y Y I P I 0 0 6 9 1 8 2 6 . 2 P R E D I C T E D : s im i l a r to R a s - r e l a t e d pro te in R a b - 1 8 i so fo rm 4 7 Y Y Y I P I 0 0 6 9 1 8 2 7 . 1 S p l i c e I so fo rm L iver of A T P s y n t h a s e g a m m a c h a i n , 3 Y Y m i tochond r i a l p recu rso r P R E D I C T E D : s im i l a r to 60 k D a hea t shock p ro te in , I P I 0 0 6 9 1 8 5 0 . 1 m i tochondr ia l p recu rso r ( H s p 6 0 ) (60 k D a c h a p e r o n i n ) 4 Y ( C P N 6 0 ) (Hea t shock pro te in 60 ) ( H S P - 6 0 ) (M i tochondr ia l ma t r i x p ro te in P I ) i so fo rm 1 I P I 0 0 6 9 2 0 3 4 . 1 C r e a t i n e k i n a s e , ub iqu i tous m i tochondr ia l p recu rso r 5 Y Y Y Y Y I P I 0 0 6 9 2 0 7 9 . 1 M i c r o t u b u l e - a s s o c i a t e d pro te in 4 i so fo rm 5 ( F r a g m e n t ) 3 Y I P I 0 0 6 9 2 1 3 2 . 2 P R E D I C T E D : s im i l a r to p lecks t r in h o m o l o g y d o m a i n 5 Y Y Y c o n t a i n i n g , f am i l y B (evec t ins ) m e m b e r 1 i so fo rm 1 M G C 1 2 8 2 9 7 prote in ( P R E D I C T E D : s im i l a r to H e t e r o g e n e o u s nuc lea r r i bonuc leopro te in A l (He l i x -I P I 0 0 6 9 2 2 3 5 . 1 des tab i l i z i ng pro te in) (S i ng l e - s t r and b ind ing pro te in) ( h n R N P co re pro te in A l ) ( H D P - l ) ( T o p o i s o m e r a s e -inh ib i to r s u p p r e s s e d ) ) 4 Y I P I 0 0 6 9 2 2 4 7 . 1 Heat s h o c k 7 0 k D a pro te in 9 B 13 Y Y Y I P I 0 0 6 9 2 2 9 5 . 1 P la te le t -ac t i va t ing fac to r a c e t y l h y d r o l a s e IB beta 2 Y Y Y subun i t I P I 0 0 6 9 2 5 0 3 . 1 P R E D I C T E D : s im i l a r to R a s - r e l a t e d pro te in R a b - 3 2 4 Y Y I P I 0 0 6 9 2 7 3 3 . 1 P R E D I C T E D : s im i l a r to t r a n s m e m b r a n e pro te in 3 0 A , par t ia l 5 Y Y I P I 0 0 6 9 2 8 1 9 . 1 Inos i to l m o n o p h o s p h a t a s e 2 Y Y I P I 0 0 6 9 2 8 6 5 . 1 E n d o p l a s m i n p recu rso r 6 Y Y I P I 0 0 6 9 2 9 1 1 . 2 P R E D I C T E D : s im i la r to ac id ic ( leuc ine- r i ch ) 5 Y Y Y nuc lea r phosphop ro te i n 32 fam i l y , m e m b e r A I P I 0 0 6 9 3 0 1 8 . 1 G l y c y l - t r a n s y n t h e t a s e (F ragmen t ) 3 Y I P I 0 0 6 9 3 1 0 6 . 1 1 4 - 3 - 3 pro te in g a m m a 5 Y Y Y I P I 0 0 6 9 3 2 6 4 . 1 Pho to recep to r ou te r s e g m e n t a l l - t rans ret inol 7 Y Y Y Y d e h y d r o g e n a s e I P I 0 0 6 9 3 5 0 6 . 1 P R E D I C T E D : s im i l a r to a m y o t r o p h i c latera l sc le ros i s 2 2 Y Y ( juven i le ) c h r o m o s o m e reg ion , c a n d i d a t e 4 I P I 0 0 6 9 3 6 3 6 . 2 P R E D I C T E D : s im i l a r to R a s - r e l a t e d prote in R a b - 8 B 6 Y I P I 0 0 6 9 3 8 2 3 . 1 P R E D I C T E D : s im i l a r to S y n t a x i n - b i n d i n g pro te in 3 7 Y Y I P I 0 0 6 9 3 8 2 4 . 1 P R E D I C T E D : s im i l a r to R a s - r e l a t e d prote in R a b - 5 A i so fo rm 1 5 Y Y Y I P I 0 0 6 9 3 9 0 0 . 2 Tol l in te rac t ing prote in 4 Y Y Y Y I P I 0 0 6 9 4 0 4 0 . 2 P R E D I C T E D : s im i l a r to p r o g r a m m e d cel l dea th 6 7 Y Y in te rac t ing pro te in i so fo rm 1 I P I 0 0 6 9 4 0 8 2 . 1 Hypo the t i ca l p ro te in M G C 1 2 7 3 2 5 ( P R E D I C T E D : s im i l a r 3 Y to C N D P d ipep t i dase 2 (me ta l l opep t i dase M 2 0 fam i l y ) I P I 0 0 6 9 4 1 0 6 . 1 G u a n i n e nuc leo t i de -b ind ing pro te in G ( I ) / G ( S ) / G ( 0 ) g a m m a - 7 subun i t p recu rso r 3 Y I P I 0 0 6 9 4 1 0 7 . 1 Prof i l in -1 2 Y Y I P I 0 0 6 9 4 1 4 2 . 2 M a c r o p h a g e m ig ra t i on inh ib i to ry fac to r 2 Y Y Y I P I 0 0 6 9 4 1 9 8 . 1 R a s - r e l a t e d pro te in R a b - 5 C 5 Y Y Y I P I 0 0 6 9 4 2 0 2 . 1 L y s o p h o s p h o l i p a s e I 2 Y Y I P I 0 0 6 9 4 2 4 3 . 2 P R E D I C T E D : s im i l a r to 4 0 S r i bosoma l pro te in S 7 3 Y I P I 0 0 6 9 4 2 9 5 . 1 A T P s y n t h a s e a lpha cha in hear t i s o f o r m , m i tochondr ia l p r e c u r s o r 25 Y Y Y Y I P I 0 0 6 9 4 3 8 3 . 2 P R E D I C T E D : s im i la r to H y p o x a n t h i n e - g u a n i n e p h o s p h o r i b o s y l t r a n s f e r a s e 3 Y Y I P I 0 0 6 9 4 4 7 2 . 2 P R E D I C T E D : s im i l a r to Monog l yce r i de l ipase 3 Y I P I 0 0 6 9 4 4 8 2 . 1 Regu la to r of G -p ro te i n s igna l ing 9 ( R G S 9 ) 40 Y Y Y Y I P I 0 0 6 9 4 6 1 2 . 1 A s p a r t a t e a m i n o t r a n s f e r a s e , c y t o p l a s m i c 2 0 Y Y I P I 0 0 6 9 4 6 4 1 . 1 Ezr in 6 Y Y Y I P I 0 0 6 9 4 7 6 0 . 2 s im i l a r to V e s i c l e - a s s o c i a t e d m e m b r a n e pro te in 5 2 Y 94 Access ion # Name Peptides Detected in R O S Disk Mem P M Sol I P I 0 0 6 9 5 0 7 8 . 1 Hypo the t i ca l p ro te in M G C 1 2 6 9 2 9 (S im i l a r to 3 Y Y P r o t e a s o m e subun i t a l pha t ype 7) I P I 0 0 6 9 5 1 8 4 . 1 M i tochondr ia f i ss ion 1 prote in 2 Y Y Y I P I 0 0 6 9 5 2 0 8 . 1 Pro te in k i n a s e C a lpha t ype 7 Y I P I 0 0 6 9 5 4 4 8 . 1 1 4 - 3 - 3 pro te in e ta 8 Y Y Y Y I P I 0 0 6 9 5 7 5 9 . 1 G u a n i n e nuc leo t i de -b ind ing prote in G(T ) g a m m a - T l 7 Y Y Y Y Y subun i t p r e c u r s o r I P I 0 0 6 9 5 7 7 6 . 1 R a s - r e l a t e d pro te in R a p - l b p recu rso r 8 Y I P I 0 0 6 9 5 8 8 1 . 2 P R E D I C T E D : s im i l a r to R A B 2 , m e m b e r R A S o n c o g e n e 5 Y fam i l y I P I 0 0 6 9 6 0 1 1 . 1 M i t o g e n - a c t i v a t e d p ro te in -b ind ing p ro te in - in te rac t i ng 2 Y Y pro te in I P I 0 0 6 9 6 0 2 1 . 1 Sp l i ce I so fo rm C S P 1 of DnaJ h o m o l o g sub fam i l y C m e m b e r 5 2 Y Y I P I 0 0 6 9 6 0 3 0 . 1 P R E D I C T E D : s im i la r to R a s - r e l a t e d prote in R a b - 2 3 2 Y Y I P I 0 0 6 9 6 2 0 3 . 1 c A M P - d e p e n d e n t p ro te in k i n a s e , a l pha -ca ta l y t i c 3 Y subun i t I P I 0 0 6 9 6 2 5 7 . 1 P R E D I C T E D : s im i l a r to g a m m a - a m i n o b u t y r i c ac id 2 Y Y Y ( G A B A - A ) t r anspo r te r 4 , par t ia l I P I 0 0 6 9 6 2 8 6 . 1 G l y c e r a l d e h y d e - 3 - p h o s p h a t e d e h y d r o g e n a s e l i ke -17 3 Y pro te in ( F r a g m e n t ) I P I 0 0 6 9 6 3 2 5 . 1 Hypo the t i ca l p ro te in ( P R E D I C T E D : s im i l a r to bas ig in 6 Y Y Y Y i so fo rm 2 i so fo rm 1 ) I P I 0 0 6 9 6 3 9 3 . 1 Ret ina l g u a n y l y l c y c l a s e 2 p recu rso r 15 Y Y Y Y I P I 0 0 6 9 6 4 3 5 . 1 1 4 - 3 - 3 pro te in eps i l on 13 Y Y Y Y I P I 0 0 6 9 6 5 3 9 . 1 c A M P - d e p e n d e n t prote in k i n a s e , b e t a - 2 - c a t a l y t i c 3 Y subun i t I P I 0 0 6 9 6 6 4 8 . 1 P R E D I C T E D : s im i l a r to B a r d e t - B i e d l s y n d r o m e 7 17 Y Y Y Y Y pro te in i so fo rm a , par t ia l I P I 0 0 6 9 6 6 5 3 . 2 P R E D I C T E D : s im i l a r to C 0 3 A 3 . 3 P R E D I C T E D : s im i l a r to G lu ta th i one S - t r a n s f e r a s e Mu 1 2 Y Y I P I 0 0 6 9 6 7 6 6 . 1 ( G S T M 1 - 1 ) ( G S T c l a s s - m u l ) ( G S T M l a - l a ) ( G S T M l b -l b ) (HB subun i t 4) ( G T H 4 ) i so fo rm 1 9 Y Y Y Y I P I 0 0 6 9 6 7 9 3 . 2 P R E D I C T E D : s im i l a r to Th io redox in - l i ke pro te in 1 2 Y I P I 0 0 6 9 6 8 2 4 . 1 T h i o r e d o x i n - d e p e n d e n t pe rox ide r e d u c t a s e , 2 Y Y m i tochond r i a l p recu rso r I P I 0 0 6 9 6 9 2 2 . 1 Red ops in 7 Y Y Y Y I P I 0 0 6 9 7 1 0 7 . 1 Be ta tubu l in 22 Y Y Y I P I 0 0 6 9 7 1 2 9 . 1 10 k D a pro te in ( A c y l - C o A - b i n d i n g pro te in) 2 Y I P I 0 0 6 9 7 2 9 0 . 1 1 4 - 3 - 3 pro te in b e t a / a l p h a 9 Y Y Y Y I P I 0 0 6 9 7 4 8 6 . 2 A d e n y l a t e k i n a s e 1 2 Y Y I P I 0 0 6 9 7 4 8 8 . 1 Hypo the t i ca l pro te in M G C 1 2 8 1 5 8 (S im i l a r to 2 Y Op t i neu r i n ) I P I 0 0 6 9 7 5 7 3 . 2 P R E D I C T E D : s im i l a r to pa ra thy ro id h o r m o n e - 4 Y Y Y r e s p o n s i v e B l g e n e i so fo rm 2 ( B B S 9 ) I P I 0 0 6 9 7 6 9 1 . 1 P R E D I C T E D : s im i l a r to B a n d 4 .1 - l i ke pro te in 2 13 Y Y Y Y ( G e n e r a l l y e x p r e s s e d pro te in 4 .1 ) ( 4 . 1 G ) i so fo rm 1 I P I 0 0 6 9 7 8 2 7 . 1 - U b i q u i n o l - c y t o c h r o m e - c reduc tase 4 Y Y c o m p l e x co re pro te in 2 , m i tochondr ia l p recu rso r I P I 0 0 6 9 7 8 5 1 . 1 K e r a t i n , t y p e II cy toske le ta l 5 5 Y Y I P I 0 0 6 9 8 0 4 3 . 2 P R E D I C T E D : s im i l a r to N A D - d e p e n d e n t d e a c e t y l a s e 2 Y Y s i r t u in -2 I P I 0 0 6 9 8 0 6 9 . 1 Neu roca l c i n de l ta 5 Y Y I P I 0 0 6 9 8 1 0 2 . 1 S e r i n e / t h r e o n i n e k i nase recep to r assoc ia ted p ro te in 3 Y I P I 0 0 6 9 8 3 3 8 . 3 D i h y d r o p y r i m i d i n a s e - r e l a t e d pro te in 2 21 Y Y Y I P I 0 0 6 9 8 5 2 9 . 1 Bre fe ld in A - i nh ib i t ed g u a n i n e n u c l e o t i d e - e x c h a n g e pro te in 1 ( A R F G E P 1) 2 Y I P I 0 0 6 9 8 5 6 8 . 2 s im i l a r to T B C 1 d o m a i n f am i l y , m e m b e r 10A 3 Y Y I P I 0 0 6 9 8 5 7 0 . 2 P R E D I C T E D : s im i l a r to c i l iary root le t co i l ed -co i l , 4 Y root le t in 95 Access ion # Name Peptides Detected in R O S Disk Mem P M Sol I P I 0 0 6 9 8 5 8 9 . 1 S i m i l a r to p h o s p h o g l y c e r a t e m u t a s e 13 Y Y Y Y I P I 0 0 6 9 8 5 9 4 . 1 Rab G D P d issoc ia t i on inh ib i tor a lpha 10 Y Y Y I P I 0 0 6 9 8 6 7 3 . 1 5 ' - nuc l eo t i dase p recu rso r 17 Y Y Y Y I P I 0 0 6 9 8 8 0 5 . 1 33 k D a pro te in ( P R E D I C T E D : s im i l a r to s y n t a x i n 3 , 5 Y Y Y Y Y par t ia l ) I P I 0 0 6 9 8 8 4 3 . 1 A l p h a - S 2 - c a s e i n p recu rso r Hypo the t i ca l p ro te in M G C 1 2 8 0 7 4 3 Y Y I P I 0 0 6 9 9 0 0 2 . 1 (Ac id ic Leuc ine - r i ch Nuc lea r Phosph 'oprote in 3 2 Fami l y M e m b e r B) Hypo the t i ca l p ro te in ( P R E D I C T E D : s im i la r to I m m u n o g l o b u l i n l a m b d a - l i k e po l ypep t i de 1 p recu rso r 3 Y I P I 0 0 6 9 9 0 1 1 . 3 ( I m m u n o g l o b u l i n - r e l a t e d 14.1 prote in) ( I m m u n o g l o b u l i n o m e g a po lypep t ide ) ( L a m b d a 5) ( C D 1 7 9 b an t igen) ) 2 Y Y I P I 0 0 6 9 9 1 0 9 . 2 Ma la te d e h y d r o g e n a s e , c y t o p l a s m i c 9 Y Y Y Y I P I 0 0 6 9 9 2 9 6 . 1 75 k D a pro te in (s im i la r to R a s G A P - a c t i v a t i n g - l i k e 3 Y Y pro te in 1) I P I 0 0 6 9 9 3 9 9 . 2 P R E D I C T E D : s im i la r to H is tone d e a c e t y l a s e 11 2 Y I P I 0 0 6 9 9 6 2 2 . 1 9 0 - k D a hea t shock prote in a l pha 35 Y Y Y Y Y I P I 0 0 6 9 9 6 9 8 . 1 Be ta - l ac tog lobu l i n p recu rso r 2 Y I P I 0 0 6 9 9 7 0 0 . 1 Cof i l in -1 4 Y I P I 0 0 6 9 9 7 0 8 . 1 6 5 k D a pro te in (Pro te in k i nase C a lpha type) 7 Y Y Y I P I 0 0 6 9 9 7 1 7 . 1 D - 3 - p h o s p h o g l y c e r a t e d e h y d r o g e n a s e 5 Y Y Y I P I 0 0 6 9 9 7 2 3 . 1 Hypo the t i ca l p ro te in (RAN b ind ing prote in 6) 2 Y Y I P I 0 0 6 9 9 8 3 9 . 2 P R E D I C T E D : s im i la r to i s o c h o r i s m a t a s e d o m a i n con ta in ing 1 i so fo rm 4 2 Y I P I 0 0 6 9 9 8 6 6 . 2 P R E D I C T E D : s im i la r to G a l e c t i n - 5 2 Y Y I P I 0 0 6 9 9 8 8 7 . 1 O x y g e n - r e g u l a t e d pro te in 1 (Ret in i t is P i g m e n t o s a 1 15 Y Y Y Y p ro te in ) I P I 0 0 6 9 9 9 3 7 . 1 Re t ina G p ro te in - coup led recep to r k i nase 7 5 Y Y I P I 0 0 6 9 9 9 9 5 . 1 P R E D I C T E D : s im i l a r to C G 8 7 6 8 - P A i so fo rm 1 4 Y Y I P I 0 0 7 0 0 1 1 2 . 1 Fat ty ac i d -b i nd ing p ro te in , bra in 2 Y I P I 0 0 7 0 0 2 3 5 . 1 P R E D I C T E D : s im i l a r to ret in i t is p i g m e n t o s a 1-l ike 1 21 Y I P I 0 0 7 0 0 4 3 1 . 1 Hypo the t i ca l p ro te in M G C 1 2 8 3 1 8 (Ca l re t in in ) 10 Y Y Y Y I P I 0 0 7 0 0 4 5 3 . 1 P R E D I C T E D : s im i l a r to c - K - r a s 2 pro te in i so fo rm b 7 Y Y I P I 0 0 7 0 0 4 7 1 . 1 5 9 k D a pro te in (s im i la r to cy toke ra t i n t y p e II) 3 Y Y I P I 0 0 7 0 0 5 2 1 . 1 51 k D a pro te in ( he te rogeneous nuc lea r 8 Y Y Y r i bonuc leop ro te in K) I P I 0 0 7 0 0 6 2 2 . 3 E n d o p i n - 1 p recu rso r 2 Y I P I 0 0 7 0 0 7 9 9 . 2 P R E D I C T E D : s im i la r to M y o s i n - 6 (Myos in V I ) , par t ia l 7 Y Y Y I P I 0 0 7 0 1 0 2 3 . 2 P R E D I C T E D : s im i la r to ac id ic ( leuc ine- r i ch ) nuc lea r 2 Y p h o s p h o p r o t e i n 32 fam i l y , m e m b e r E I P I 0 0 7 0 1 0 9 8 . 1 P R E D I C T E D : s im i la r to H e x o k i n a s e t ype II 3 Y I P I 0 0 7 0 1 1 5 0 . 1 Recove r i n 17 Y Y Y Y Y I P I 0 0 7 0 1 2 0 0 . 1 S p l i c e I so fo rm C N G 4 D of 2 4 0 k D a pro te in of rod 38 Y Y Y Y Y p h o t o r e c e p t o r C N G - c h a n n e l I P I 0 0 7 0 1 2 1 4 . 1 4 3 k D a pro te in (Ar res t in 3 , re t ina l (X -a r res t i n ) ) 6 Y Y Y I P I 0 0 7 0 1 6 4 2 . 2 P R E D I C T E D : s im i la r to 6 - p h o s p h o g l u c o n a t e 7 Y Y d e h y d r o g e n a s e , d e c a r b o x y l a t i n g I P I 0 0 7 0 2 0 0 2 . 1 Cyc l i c nuc leo t i de -ga ted ca t ion channe l a l pha 3 5 Y I P I 0 0 7 0 2 0 9 8 . 1 Pep t idy lp ro ly l i s o m e r a s e B P R E D I C T E D : s im i l a r to pro te in p h o s p h a t a s e 3 2 Y I P I 0 0 7 0 2 1 7 5 . 1 ( fo rmer l y 2 B ) , ca ta ly t i c subun i t , g a m m a i so fo rm (ca lc ineur in A g a m m a ) , par t ia l 7 Y Y I P I 0 0 7 0 2 3 1 4 . 1 G e n e r a l v e s i c u l a r t r anspo r t fac to r p i 15 3 Y Y I P I 0 0 7 0 2 4 5 6 . 1 6 0 S ac id ic r i b o s o m a l pro te in P2 3 Y I P I 0 0 7 0 2 4 6 8 . 1 Ze ta - c r ys ta l l i n 2 Y Y 96 Access ion # Name Peptides Detected in R O S Disk Mem P M Sol 3 Y 2 Y 2 Y 11 Y Y Y Y 2 Y 2 Y 2 Y Y Y 5 Y Y Y Y 9 Y Y Y Y Y 35 Y 12 Y Y Y Y Y 4 Y Y 19 Y Y Y Y Y 2 Y 10 Y Y Y Y 2 Y 2 Y Y 4 Y Y 8 Y Y Y Y 8 Y Y Y Y 2 Y 25 Y Y Y Y Y 8 Y Y 2 Y 3 Y Y Y 6 Y Y Y 3 Y Y 3 Y Y Y 3 Y Y 8 Y 2 Y 19 Y Y Y Y Y 3 Y Y 10 Y Y Y Y 2 Y Y Y Y 4 Y Y Y Y Y 26 Y Y Y Y 14 Y Y Y Y Y 4 Y 2 Y 5 Y Y I P I 0 0 7 0 2 5 3 2 . 2 I P I 0 0 7 0 2 5 6 5 . 1 I P I 0 0 7 0 2 7 1 8 . 1 I P I 0 0 7 0 2 7 6 5 . 1 I P I 0 0 7 0 2 7 6 8 . 1 I P I 0 0 7 0 2 7 8 1 . 1 I P I 0 0 7 0 2 8 5 8 . 1 I P I 0 0 7 0 2 9 2 8 . 1 I P I 0 0 7 0 2 9 5 0 . 1 I P I 0 0 7 0 2 9 7 0 . 1 I P I 0 0 7 0 3 1 1 0 . 1 I P I 0 0 7 0 3 1 2 9 . 2 I P I 0 0 7 0 3 1 6 0 . 1 I P I 0 0 7 0 3 2 1 7 . 1 I P I 0 0 7 0 3 4 3 6 . 1 I P I 0 0 7 0 3 4 6 8 . 1 I P I 0 0 7 0 3 4 7 0 . 1 I P I 0 0 7 0 3 5 4 7 . 1 I P I 0 0 7 0 3 5 9 2 . 1 I P I 0 0 7 0 3 6 6 5 . 1 I P I 0 0 7 0 3 7 3 1 . 1 I P I 0 0 7 0 3 8 7 3 . 1 I P I 0 0 7 0 3 9 9 2 . 1 I P I 0 0 7 0 4 0 0 6 . 1 I P I 0 0 7 0 4 0 8 2 . 1 I P I 0 0 7 0 4 1 5 5 . 1 I P I 0 0 7 0 4 2 0 0 . 1 I P I 0 0 7 0 4 2 3 2 . 1 I P I 0 0 7 0 4 2 5 7 . 2 I P I 0 0 7 0 4 3 1 4 . 1 I P I 0 0 7 0 4 3 2 5 . 1 I P I 0 0 7 0 4 3 5 3 . 2 I P I 0 0 7 0 4 5 2 3 . 1 I P I 0 0 7 0 4 7 5 2 . 1 I P I 0 0 7 0 4 7 5 4 . 1 I P I 0 0 7 0 4 8 3 6 . 1 I P I 0 0 7 0 5 1 5 9 . 2 I P I 0 0 7 0 5 2 6 5 . 2 I P I 0 0 7 0 5 2 6 9 . 2 I P I 0 0 7 0 5 3 3 4 . 1 I P I 0 0 7 0 5 3 7 8 . 1 P R E D I C T E D : s im i la r to S H 3 - d o m a i n G R B 2 - l i k e 2 (Endoph i l i n 1) Hypo the t i ca l p ro te in ( P R E D I C T E D : s im i la r to S E C 2 2 ves i c le t ra f f i ck ing p ro te in - l i ke 1) D i h y d r o x y a c e t o n e k inase DJ -1 p ro te in A l ka l i ne p h o s p h a t a s e , t i s sue -nonspec i f i c i s o z y m e p recu rso r Isoc i t ra te d e h y d r o g e n a s e 1 ( N A D P + ) , so lub le 10 k D a heat s h o c k p ro te in , m i tochondr ia l S y n a p t o t a g m i n - 1 S i m i l a r to g l u t a t h i o n e - S - t r a n s f e r a s e , m u 5 Sp l i ce I so fo rm B of S -a r res t i n 1 4 - 3 - 3 pro te in z e t a / d e l t a V o l t a g e - d e p e n d e n t an ion -se lec t i ve channe l p ro te in 1 G u a n i n e nuc leo t i de -b ind ing prote in G ( I ) / G ( S ) / G ( T ) beta subun i t 1 21 k D a pro te in ( 6 0 S r i bosoma l pro te in L9) R a s - r e l a t e d pro te in R a b - 3 A 8 k D a pro te in (ATP s y n t h a s e e c h a i n , m i tochondr ia l ) P R E D I C T E D : s im i l a r to ub iqu i t i n -con juga t ing e n z y m e E 2 N i so fo rm 1 P R E D I C T E D : s im i l a r to Ca lb ind in P R E D I C T E D : s im i l a r to h ippoca lc in - l i ke 1 i so fo rm 1 R a s - r e l a t e d pro te in R a p - I A p recu rso r 4 0 k D a pep t idy l -p ro l y l c i s - t rans i s o m e r a s e R h o d o p s i n k i nase p recu rso r P R E D I C T E D : s im i l a r to g u a n i n e nuc leo t i de -b ind ing p ro te in , b e t a - 3 subun i t 3 2 k D a pro te in (s im i la r to L imb ic s y s t e m - a s s o c i a t e d m e m b r a n e pro te in p recurso r ) S o d i u m / p o t a s s i u m - t r a n s p o r t i n g A T P a s e be ta -2 cha in P R E D I C T E D : s im i la r to A D P - r i b o s y l a t i o n fac to r - l i ke 2 -l ike 1 i so fo rm 1 i so fo rm 3 P R E D I C T E D : s im i l a r to g lu ta th ione S - t r a n s f e r a s e M l i so fo rm 2 i so fo rm 3 Leuc ine z i p p e r t ransc r ip t i on fac to r - l i ke 1 M G C 1 2 8 6 1 2 pro te in ( P R E D I C T E D : s im i l a r to cel l d i v i s ion cyc le 4 2 , par t ia l ) Sp l i ce I so fo rm 1 of S e r i n e / t h r e o n i n e - p r o t e i n p h o s p h a t a s e 2 B ca ta ly t i c subun i t a l pha i so fo rm W A R S pro te in ( t ryptophanyl - tRIMA s y n t h e t a s e ) T u b u l i n , a l pha 1 G u a n i n e nuc leo t i de -b ind ing prote in G ( I ) / G ( S ) / G ( 0 ) g a m m a - T 2 subun i t p recu rso r Hypo the t i ca l p ro te in M G C 1 2 7 5 9 7 (s im i la r to R a s -re la ted prote in R a b - 7 ) B l u e - s e n s i t i v e ops in Heat shock 2 7 k D a prote in 1 P R E D I C T E D : s im i l a r to S o d i u m / p o t a s s i u m -t ranspo r t i ng A T P a s e a l p h a - 1 cha in p recu rso r ( S o d i u m p u m p 1) ( N a + / K + A T P a s e 1) , par t ia l P R E D I C T E D : s im i l a r to B a r d e t - B i e d l s y n d r o m e 1 S i m i l a r to Y 5 5 F 3 A M . 9 S e p t i n - 7 A D P / A T P t r ans l ocase 3 97 Access ion # Name Peptides Detected in R O S Disk Mem P M Sol P R E D I C T E d : s im i l a r to H e a t - s h o c k pro te in 105 k D a I P I 0 0 7 0 5 4 7 7 . 1 (hea t s h o c k H O k D a prote in) (An t igen N Y - C O - 2 5 ) i so fo rm 1 2 Y I P I 0 0 7 0 5 6 0 3 . 2 P R E D I C T E D : s im i la r to Ret i cu lon 4 2 Y I P I 0 0 7 0 5 6 5 7 . 1 Sp l i ce I so fo rm A of S -a r res t i n L O C 5 2 0 1 7 0 p ro te in ( P R E D I C T E D : s im i l a r to Ub iqu i t in 38 Y Y Y Y Y I P I 0 0 7 0 5 7 4 9 . 4 c a r b o x y l - t e r m i n a l hyd ro lase i s o z y m e L3 ( U C H - L 3 ) (Ub iqu i t in t h i o l es te rase L3)) 2 Y Y I P I 0 0 7 0 5 8 1 5 . 1 Bra in ac id so lub le pro te in 1 3 Y Y I P I 0 0 7 0 5 8 9 9 . 2 P R E D I C T E D : s im i la r to t ubu l i n , a l pha 1 22 Y Y Y Y I P I 0 0 7 0 5 9 3 3 . 1 P R E D I C T E D : s im i l a r to c h r o m a t i n mod i f y i ng pro te in 6 2 Y Y Y I P I 0 0 7 0 5 9 4 1 . 1 R A N , m e m b e r R A S o n c o g e n e fami l y 3 Y Y I P I 0 0 7 0 6 0 0 2 . 1 A n n e x i n A 2 2 Y I P I 0 0 7 0 6 0 9 4 . 1 A l p h a - S l - c a s e i n p recu rso r 3 Y Y Y Y I P I 0 0 7 0 6 3 1 7 . 1 P R E D I C T E D : s im i l a r to i m m u n o g l o b u l i n s u p e r f a m i l y , m e m b e r 4 D , par t ia l 5 Y Y Y I P I 0 0 7 0 6 4 5 1 . 1 Hypo the t i ca l p ro te in M G C 1 2 8 0 5 9 ( A D P - R i b o s y l a t i o n 2 Y Fac to r 4) I P I 0 0 7 0 6 5 9 4 . 1 Tubu l i n b e t a - 2 C cha in 21 Y Y Y Y Y I P I 0 0 7 0 6 8 1 5 . 1 Fasc in h o m o l o g 1, ac t i n -bund l i ng pro te in 6 Y Y I P I 0 0 7 0 6 9 4 2 . 1 T r i o s e p h o s p h a t e i s o m e r a s e 1 13 Y Y Y Y Y I P I 0 0 7 0 7 0 5 6 . 1 Sp l i ce I so fo rm 1 of S o d i u m / p o t a s s i u m / c a l c i u m 14 Y Y Y Y Y e x c h a n g e r 1 I P I 0 0 7 0 7 0 9 5 . 2 A l p h a - E n o l a s e 31 Y Y Y Y Y I P I 0 0 7 0 7 1 1 4 . 1 P R E D I C T E D : s im i l a r to C G 1 0 2 3 7 - P B , i so fo rm B 2 Y I P I 0 0 7 0 7 4 9 0 . 1 P R E D I C T E D : s im i l a r to S o d i u m / p o t a s s i u m / c a l c i u m 3 Y Y e x c h a n g e r 2 p recu rso r I P I 0 0 7 0 7 5 8 1 . 2 P R E D I C T E D : s im i l a r to M y o s i n - 6 8 Y Y Y I P I 0 0 7 0 7 5 9 3 . 1 4 2 k D a pro te in 2 Y Y I P I 0 0 7 0 7 7 5 1 . 2 E longa t ion fac to r 2 3 Y Y I P I 0 0 7 0 7 9 2 1 . 1 Phosduc in 8 Y Y Y Y I P I 0 0 7 0 7 9 6 6 . 2 P R E D I C T E D : s im i l a r to e m b i g i n h o m o l o g , par t ia l 5 Y Y Y I P I 0 0 7 0 8 0 2 7 . 1 N A D H - u b i q u i n o n e o x i d o r e d u c t a s e 75 k D a subun i t , 4 Y Y Y Y m i tochond r i a l p recu rso r I P I 0 0 7 0 8 0 3 0 . 2 Hypo the t i ca l pro te in ( A c y l p h o s p h a t a s e 1) 2 Y I P I 0 0 7 0 8 0 6 9 . 1 G u a n i n e nuc leo t i de -b ind ing prote in G ( t ) , a l p h a - 2 18 Y Y Y Y subun i t I P I 0 0 7 0 8 1 2 5 . 1 14 k D a pro te in ( P R E D I C T E D : s im i la r to R a s - r e l a t e d 9 Y pro te in R a b - 1 0 ) I P I 0 0 7 0 8 1 8 9 . 2 P R E D I C T E D : s im i l a r to H is tone H I . 2 4 Y I P I 0 0 7 0 8 3 4 2 . 1 P R E D I C T E D : s im i l a r to hea t shock 7 0 k D a pro te in 6 6 Y Y Y I P I 0 0 7 0 8 3 9 8 . 1 S e r u m a l b u m i n p recu rso r 16 Y Y Y Y Y I P I 0 0 7 0 8 5 2 6 . 1 Hea t s h o c k c o g n a t e 71 k D a pro te in 29 Y Y I P I 0 0 7 0 8 5 8 2 . 1 V o l t a g e - d e p e n d e n t a n i o n - s e l e c t i v e channe l p ro te in 3 3 Y Y I P I 0 0 7 0 8 6 9 1 . 1 Hypo the t i ca l pro te in M G C 1 2 7 3 9 9 2 Y I P I 0 0 7 0 8 7 6 1 . 1 C a r b o n y l r educ tase 1 2 Y I P I 0 0 7 0 8 7 6 7 . 2 P R E D I C T E D : s im i l a r to act in re la ted prote in 2 / 3 3 Y Y c o m p l e x , subun i t 4 I P I 0 0 7 0 8 9 8 6 . 2 P R E D I C T E D : s im i l a r to ion t r a n s p o r t e r p ro te in 2 Y I P I 0 0 7 0 9 0 1 4 . 1 2 4 k D a pro te in ( P R E D I C T E D : s im i l a r to R A B 3 D , 8 Y Y m e m b e r R A S o n c o g e n e fami l y ) I P I 0 0 7 0 9 1 1 7 . 2 P R E D I C T E D : s im i l a r to R a s - r e l a t e d prote in M - R a s 3 Y I P I 0 0 7 0 9 1 6 2 . 1 G l y c i n a m i d e r ibonuc leo t ide f o r m y l t r a n s f e r a s e 2 Y I P I 0 0 7 0 9 2 1 9 . 1 M y o s i n - 1 0 3 Y I P I 0 0 7 0 9 2 6 5 . 1 10 k D a pro te in (s im i la r to R a s - r e l a t e d pro te in R a b - 7 ) 3 Y Y Y Y I P I 0 0 7 0 9 4 0 3 . 1 H i s tone -b i nd ing pro te in R B B P 7 ( r e t i nob las toma 2 Y b ind ing pro te in 7) 98 Access ion # Name Peptides Detected in R O S Disk Mem P M Sol 4 Y Y Y Y 19 Y Y Y Y 3 Y Y 2 Y Y 4 Y Y 2 Y Y 15 Y Y Y Y 4 Y Y 8 Y Y Y 3 Y 2 Y 4 Y Y 5 Y Y 5 Y Y Y 2 Y 17 Y Y Y Y 4 Y Y 2 Y 2 Y 2 Y 9 Y Y Y Y Y 8 Y Y Y Y 8 Y Y Y Y Y 11 Y Y Y Y 2 Y 9 Y Y Y Y Y 22 Y Y Y Y 2 Y 57 Y Y Y Y Y 16 Y Y Y Y 32 Y Y Y Y Y 7 Y Y Y Y 4 Y Y Y 4 Y-, 32 Y Y Y Y Y 4 Y 3 Y Y 4 Y Y 2 Y 4 Y Y I P I 0 0 7 0 9 4 0 7 . 2 I P I 0 0 7 0 9 4 3 5 . 1 I P I 0 0 7 0 9 4 6 5 . 1 I P I 0 0 7 0 9 5 3 3 . 1 I P I 0 0 7 0 9 5 7 3 . 2 I P I 0 0 7 0 9 6 6 5 . 1 I P I 0 0 7 0 9 6 8 9 . 2 I P I 0 0 7 0 9 8 0 5 . 1 I P I 0 0 7 0 9 8 2 2 . 2 I P I 0 0 7 0 9 8 4 0 . 1 I P I 0 0 7 1 0 1 0 1 . 1 I P I 0 0 7 1 0 1 1 4 . 1 I P I 0 0 7 1 0 1 2 3 . 1 I P I 0 0 7 1 0 1 2 9 . 1 I P I 0 0 7 1 0 1 7 5 . 1 I P I 0 0 7 1 0 1 9 0 . 1 I P I 0 0 7 1 0 3 2 6 . 1 I P I 0 0 7 1 0 3 5 4 . 1 I P I 0 0 7 1 0 3 6 6 . 1 I P I 0 0 7 1 0 4 5 0 . 2 I P I 0 0 7 1 0 5 1 5 . 1 I P I 0 0 7 1 0 7 1 4 . 2 I P I 0 0 7 1 0 7 2 6 . 1 I P I 0 0 7 1 0 7 2 7 . 1 I P I 0 0 7 1 0 7 8 0 . 1 I P I 0 0 7 1 0 7 8 3 . 1 I P I 0 0 7 1 0 8 9 5 . 1 I P I 0 0 7 1 0 9 0 4 . 1 I P I 0 0 7 1 1 0 2 6 . 1 I P I 0 0 7 1 1 0 3 1 . 1 I P I 0 0 7 1 1 0 9 4 . 1 I P I 0 0 7 1 1 1 7 2 . 2 I P I 0 0 7 1 1 2 1 9 . 1 I P I 0 0 7 1 1 2 4 1 . 2 I P I 0 0 7 1 1 3 1 9 . 1 I P I 0 0 7 1 1 3 6 8 . 1 I P I 0 0 7 1 1 3 8 6 . 1 I P I 0 0 7 1 1 4 0 4 . 3 I P I 0 0 7 1 1 4 4 1 . 1 I P I 0 0 7 1 1 4 5 5 . 1 P R E D I C T E D : s im i l a r to e n d o p l a s m i c re t i cu lum pro te in 2 9 p recu rso r 9 0 - k D a hea t shock prote in beta Pro te in d i s u l f i d e - i s o m e r a s e p recu rso r 18 k D a pro te in ( P R E D I C T E D : s im i l a r to A D P -r ibosy la t ion fac to r - l i ke 8 A ) Hypo the t i ca l p ro te in M G C 1 2 7 5 9 6 ( F K 5 0 6 - B I N D I N G P R O T E I N 4 ) P R E D I C T E D : s im i l a r to D - d o p a c h r o m e t a u t o m e r a s e P R E D I C T E D : s im i l a r to Potent ia l p h o s p h o l i p i d -t r anspo r t i ng A T P a s e IB S i m i l a r to Mu-c rys ta l l i n h o m o l o g P R E D I C T E D : s im i l a r to g l u t a m a t e - a m m o n i a l igase 21 k D a pro te in ( P R E D I C T E D : s im i l a r to g l y o x y l a s e 1) V a c u o l a r A T P s y n t h a s e subun i t E High mobi l i t y g r o u p pro te in B l P R E D I C T E D : s im i l a r to R A B 2 B pro te in i so fo rm 1 P R E D I C T E D : s im i l a r to B a r d e t - B i e d l s y n d r o m e 5 i so fo rm 1 P R E D I C T E D : s im i l a r to C o p i n e - 1 (Cop ine I) i so fo rm 3 4 4 k D a p r o t e i n ( P R E D I C T E D : s im i la r to g u a n i n e nuc leo t i de -b ind ing p ro te in , be ta -5 subun i t i so fo rm 1) Neu ro t r im in A T P c i t ra te l yase P R E D I C T E D : s im i l a r to G l y c e r a l d e h y d e - 3 - p h o s p h a t e d e h y d r o g e n a s e , l i ver P R E D I C T E D : s im i l a r to A l p h a - m a n n o s i d a s e II Neura l cel l a d h e s i o n mo lecu le 1, 140 k D a i so fo rm p r e c u r s o r P R E D I C T E D : s im i l a r to F r u c t o s e - b i s p h o s p h a t e a l d o l a s e C ( B r a i n - t y p e a ldo lase ) i so fo rm 1 C h a p e r o n i n con ta in ing T C P 1 , subun i t 8 Hypo the t i ca l p ro te in M G C 1 2 8 1 4 2 (Trans i t iona l e n d o p l a s m i c re t i cu lum A T P a s e ) 19 k D a pro te in ( P R E D I C T E D : s im i l a r to R a s - r e l a t e d p ro te in R a b - 2 1 ) H e m o g l o b i n a l pha subun i t P h o s p h o g l y c e r a t e k i n a s e 1 P R E D I C T E D : s im i l a r to S - p h a s e k i n a s e - a s s o c i a t e d p ro te in I A i so fo rm b Rod c G M P - s p e c i f i c 3 ' , 5 ' - cyc l i c p h o s p h o d i e s t e r a s e b e t a -subun i t p recu rso r Hea t shock 70 k D a pro te in I A P R E D I C T E D : s im i l a r to Py ruva te k i n a s e , i s o z y m e s M 1 / M 2 ( P y r u v a t e k i nase musc le i s o z y m e ) (Cy toso l i c t hy ro id h o r m o n e - b i n d i n g pro te in) ( C T H B P ) ( T H B P 1 ) , par t ia l P R E D I C T E D : s im i l a r to 1 4 - 3 - 3 pro te in the ta P R E D I C T E D : s im i la r to s t r o m a l cel l de r i ved fac to r recep to r 1 i so fo rm 2 Hypo the t i ca l p ro te in (T ransa ldo lase 1) G u a n i n e nuc leo t i de -b ind ing prote in G ( t ) , a l p h a - 1 subun i t Hypo the t i ca l p ro te in ( P R E D I C T E D : s im i la r to r ad i x i n , par t ia l ) Nuc leos ide d i p h o s p h a t e k i nase N B R - B T e t r a s p a n i n - 1 8 s im i l a r to Neut ra l a m i n o ac id t r anspo r te r A Hypo the t i ca l p ro te in M G C 1 2 8 1 4 7 (s im i la r to t h r e o n y l -t R N A s y n t h e t a s e ) 99 Access ion # Name Peptides Detected in R O S Disk Mem P M Sol 3 Y Y 3 Y 104 Y Y Y Y 7 Y Y Y 3 Y Y 2 Y 3 Y Y Y 2 Y Y 4 Y Y Y 12 Y Y Y Y 3 Y Y 17 Y Y Y Y Y 2 Y 4 Y Y Y 23 Y Y Y Y 14 Y Y Y Y 2 Y Y 7 Y Y Y Y 12 Y Y Y 14 Y Y Y Y Y 5 Y Y Y 9 Y Y Y Y Y 6 Y Y Y Y Y 4 Y Y 12 Y Y Y Y Y 2 Y 2 Y 2 Y 2 Y 6 Y Y 4 Y 3 Y 3 Y 3 Y Y 10 Y Y 3 Y Y 19 Y Y Y Y Y 3 Y Y Y 6 Y 2 Y 7 Y Y Y I P I 0 0 7 1 1 4 7 1 . 1 I P I 0 0 7 1 1 4 7 9 . 1 I P I 0 0 7 1 1 5 1 8 . 1 I P I 0 0 7 1 1 5 3 7 . 1 I P I 0 0 7 1 1 7 7 7 . 1 I P I 0 0 7 1 1 8 2 6 . 1 I P I 0 0 7 1 1 9 0 0 . 1 I P I 0 0 7 1 2 1 3 4 . 1 I P I 0 0 7 1 2 1 9 2 . 2 I P I 0 0 7 1 2 2 5 0 . 1 I P I 0 0 7 1 2 2 5 2 . 1 I P I 0 0 7 1 2 3 9 6 . 2 I P I 0 0 7 1 2 5 9 0 . 1 I P I 0 0 7 1 2 6 4 8 . 2 I P I 0 0 7 1 2 6 7 9 . 1 I P I 0 0 7 1 2 6 9 5 . 1 I P I 0 0 7 1 2 7 3 9 . 2 I P I 0 0 7 1 2 7 7 5 . 1 I P I 0 0 7 1 2 8 1 9 . 2 I P I 0 0 7 1 2 8 3 8 . 1 I P I 0 0 7 1 2 8 5 8 . 1 I P I 0 0 7 1 2 8 7 3 . 1 I P I 0 0 7 1 3 1 1 2 . 1 I P I 0 0 7 1 3 1 1 5 . 1 I P I 0 0 7 1 3 1 3 7 . 1 I P I 0 0 7 1 3 1 8 5 . 1 I P I 0 0 7 1 3 4 8 4 . 2 I P I 0 0 7 1 3 4 9 6 . 1 I P I 0 0 7 1 3 5 0 5 . 1 I P I 0 0 7 1 3 5 4 7 . 1 I P I 0 0 7 1 3 6 3 2 . 1 I P I 0 0 7 1 3 6 7 2 . 2 I P I 0 0 7 1 3 6 7 6 . 2 I P I 0 0 7 1 3 7 5 6 . 1 I P I 0 0 7 1 3 7 6 0 . 2 I P I 0 0 7 1 3 7 8 0 . 3 I P I 0 0 7 1 3 8 1 4 . 1 I P I 0 0 7 1 3 8 1 5 . 1 I P I 0 0 7 1 3 8 3 5 . 2 I P I 0 0 7 1 3 9 2 3 . 1 I P I 0 0 7 1 3 9 5 3 . 2 S p l i c e I so fo rm 2 of V a c u o l a r p ro ton t rans loca t i ng A T P a s e 116 k D a subun i t a i so fo rm 1 T a r g e t of m y b l A B C t r a n s p o r t e r ( A B C R or R im Prote in) P R E D I C T E D : s im i l a r to G u a n i n e nuc leo t i de -b ind ing pro te in G ( i ) , a l p h a - 2 subun i t (Adeny la te c y c l a s e -inh ib i t ing G a lpha prote in) i so fo rm 1 Ret ina l p i g m e n t ep i t he l i um-spec i f i c 65 k D a pro te in 57 k D a pro te in Ca l c i neu r i n subun i t B i so fo rm 1 Hypo the t i ca l p ro te in ( chaperon in con ta in ing T C P 1 , subun i t 4 (de l ta) ) P R E D I C T E D : s im i l a r to B a r d e t - B i e d l s y n d r o m e 4 pro te in M D H 2 pro te in (Ma la te d e h y d r o g e n a s e ) A T P s y n t h a s e D c h a i n , m i tochondr ia l Tubu l i n b e t a - 3 cha in D e h y d r o g e n a s e / r e d u c t a s e S D R fami l y m e m b e r 11 p recu rso r P R E D I C T E D : s im i l a r to M a n n o s y l - o l i g o s a c c h a r i d e g l u c o s i d a s e c G M P - g a t e d ca t ion channe l a lpha 1 Ce l l u l a r r e t i na l dehyde -b i nd i ng pro te in Pro te in S 1 0 0 - B E longa t ion fac to r 1 -a lpha 1 P R E D I C T E D : s im i la r to g u a n i n e nuc leo t i de -b ind ing p ro te in , beta 2 i so fo rm 1 A c t i n , c y t o p l a s m i c 2 A D P / A T P t r ans l ocase 2 R h o d o p s i n P e r o x i r e d o x i n - 2 P R E D I C T E D : s im i l a r to t h io redox in - l i ke 6 A s p a r t a t e a m i n o t r a n s f e r a s e , m i tochond r i a l p recu rso r 3 2 k D a pro te in ( P R E D I C T E D : s im i l a r to Pro te in k i nase C , eps i l on t ype) P R E D I C T E D : s im i la r to g l y o x a l a s e 1 P R E D I C T E D : s im i la r to A T P - b i n d i n g c a s s e t t e , s u b -fam i l y A , m e m b e r 7 i so fo rm a i so fo rm 1 Prepro c o m p l e m e n t c o m p o n e n t C 3 p recu rso r S u p e r o x i d e d i s m u t a s e Hypo the t i ca l p ro te in (s im i la r to Ub iqu i t i n - l i ke pro te in 3 ( H C G - 1 pro te in) ) M i t o g e n - a c t i v a t e d pro te in k i nase 1 P R E D I C T E D : s im i l a r to Prote in k i n a s e C a n d case in k i n a s e subs t ra te in n e u r o n s pro te in 1 P l a s m a m e m b r a n e c a l c i u m - t r a n s p o r t i n g A T P a s e Rab G D P d issoc ia t i on inh ib i to r beta Hypo the t i ca l p ro te in ( P R E D I C T E D : s im i l a r to Apo l i pop ro te i n D p recu rso r (Apo -D ) ) G l y c e r a l d e h y d e - 3 - p h o s p h a t e d e h y d r o g e n a s e Hypo the t i ca l p ro te in ( P R E D I C T E D : s im i l a r to r hoB g e n e ) L O C 6 1 7 6 0 0 pro te in ( R a s - R e l a t e d Prote in R a b - 3 C ) P R E D I C T E D : s im i l a r to p r e - B - c e l l l e u k e m i a t ransc r ip t i on fac to r in te rac t ing pro te in 1 P R E D I C T E D : s im i l a r to R A B 3 5 , m e m b e r R A S o n c o g e n e f am i l y 100 Access ion # Name Peptides Detected in R O S Disk Mem P M Sol I P I 0 0 7 1 3 9 8 9 . 1 P R E D I C T E D : s im i l a r to T u m o r d i f fe rent ia l ly e x p r e s s e d pro te in 1 i so fo rm 1 3 Y Y I P I 0 0 7 1 4 0 0 4 . 1 Hypo the t i ca l p ro te in (s im i la r to po lypos is locus pro te in 1- l ike 1) 2 Y Y Y Y I P I 0 0 7 1 4 0 4 8 . 1 Go lg in sub fam i l y A m e m b e r 7 5 Y Y I P I 0 0 7 1 4 2 9 4 . 1 S - a d e n o s y l h o m o c y s t e i n e hyd ro lase 5 Y Y Y I P I 0 0 7 1 4 4 4 6 . 1 G l u c o s e p h o s p h a t e i s o m e r a s e 23 Y Y Y Y Y I P I 0 0 7 1 4 4 5 4 . 2 P R E D I C T E D : s im i la r to A l p h a adduc in 3 Y I P I 0 0 7 1 4 4 7 6 . 2 Hypo the t i ca l p ro te in M G C 1 3 4 1 4 7 ( I m m u n o g l o b u l i n 5 Y Y Y s u p e r f a m i l y , m e m b e r 4) I P I 0 0 7 1 4 6 2 4 . 1 C C T 3 pro te in 7 Y Y Y I P I 0 0 7 1 4 6 5 6 . 1 S o l u t e car r ie r f am i l y 2 , fac i l i ta ted g l ucose t r a n s p o r t e r m e m b e r 1 Hypo the t i ca l p ro te in ( P R E D I C T E D : s im i la r to 6 Y Y Y Y I P I 0 0 7 1 4 6 8 3 . 1 D ihyd rop te r i d i ne reduc tase ( H D H P R ) (Qu ino id d ihyd rop te r i d i ne reduc tase ) ) P R E D I C T E D : s im i l a r to G a m m a e n o l a s e ( 2 - p h o s p h o - D -3 Y Y I P I 0 0 7 1 4 7 6 4 . 1 g l yce ra te h y d r o - l y a s e ) (Neura l e n o l a s e ) ( N e u r o n -spec i f i c e n o l a s e ) ( N S E ) (Eno lase 2) i so fo rm 2 16 Y Y Y Y I P I 0 0 7 1 4 7 6 5 . 1 P R E D I C T E D : s im i l a r to L a n C - l i k e pro te in 1 2 Y I P I 0 0 7 1 4 8 6 8 . 2 P R E D I C T E D : s im i l a r to Prote in F A M 3 C p recu rso r (Pro te in G S 3 7 8 6 ) i so fo rm 2 3 Y Y I P I 0 0 7 1 4 9 1 0 . 2 Hypo the t i ca l p ro te in ( P R E D I C T E D : s im i l a r to apop tos i s 2 Y inh ib i to r 5 , par t ia l ) I P I 0 0 7 1 4 9 1 4 . 1 2 7 2 k D a pro te in (Spec t r i n beta) 13 Y I P I 0 0 7 1 5 1 0 1 . 1 Lac ta te d e h y d r o g e n a s e B 19 Y Y Y Y Y I P I 0 0 7 1 5 2 1 0 . 1 7 0 k D a pro te in 2 Y I P I 0 0 7 1 5 2 1 1 . 2 P R E D I C T E D : s im i l a r to S y n a p t i c ves i c le m e m b r a n e pro te in V A T - 1 h o m o l o g 2 Y I P I 0 0 7 1 5 2 1 8 . 1 Hypo the t i ca l p ro te in ( L S M 3 h o m o l o g , U6 sma l l nuc lea r 2 Y R N A a s s o c i a t e d ) I P I 0 0 7 1 5 4 1 5 . 1 D i m e t h y l a r g i n i n e d i m e t h y l a m i n o h y d r o l a s e 2 2 Y I P I 0 0 7 1 5 6 9 0 . 1 Hypo the t i ca l p ro te in M G C 1 2 8 3 6 3 (s im i la r to ca ten in 2 Y ( c a d h e r i n - a s s o c i a t e d p ro te in ) , a l pha 1, 1 0 2 k D a ) I P I 0 0 7 1 5 7 7 0 . 1 A D P - r i b o s y l a t i o n fac to r 1 4 Y I P I 0 0 7 1 5 7 9 9 . 1 M G C 1 2 8 2 7 9 pro te in ( P R E D I C T E D : s im i la r to 4 Y Y G l y c e r a l d e h y d e - 3 - p h o s p h a t e d e h y d r o g e n a s e ) I P I 0 0 7 1 5 8 5 3 . 1 S o l u t e ca r r i e r f am i l y 16 m e m b e r 1 7 Y Y Y I P I 0 0 7 1 6 0 7 7 . 2 Hypo the t i ca l p ro te in M G C 1 3 3 6 5 1 (S im i l a r to A D P - 7 Y Y Y Y r i bosy la t ion fac to r - l i ke 3) I P I 0 0 7 1 6 1 0 5 . 1 Hypo the t i ca l p ro te in (Ba rde t -B ied l s y n d r o m e 2) 14 Y Y Y Y I P I 0 0 7 1 6 1 1 6 . 1 R i b o s o m a l pro te in S 2 8 3 Y I P I 0 0 7 1 6 1 2 1 . 1 P i g m e n t e p i t h e l i u m - d e r i v e d fac to r p recu rso r 5 Y Y I P I 0 0 7 1 6 1 3 0 . 1 P R E D I C T E D : s im i l a r to ub iqu i t i n -ac t i va t ing e n z y m e E l i so fo rm 2 8 Y Y Y I P I 0 0 7 1 6 1 7 5 . 2 P R E D I C T E D : s im i la r to R a s - r e l a t e d prote in R a b - 4 B 3 Y I P I 0 0 7 1 6 1 8 3 . 1 4 3 k D a pro te in (s im i la r to ha loac id d e h a l o g e n a s e - l i k e 3 Y Y h y d r o l a s e d o m a i n con ta in ing 2) I P I 0 0 7 1 6 1 8 6 . 1 81 k D a pro te in ( P R E D I C T E D : s im i la r to Ge lso l i n ) 3 Y Y I P I 0 0 7 1 6 2 4 6 . 1 C a r b o n i c a n h y d r a s e 2 8 Y Y Y Y Y I P I 0 0 7 1 6 3 0 4 . 2 P R E D I C T E D : s im i l a r to ta rge t of m y b l - l i k e 2 164 k D a pro te in ( P R E D I C T E D : s im i l a r to M y o s i n - 5 A 2 Y I P I 0 0 7 1 6 3 1 4 . 1 ( M y o s i n V a ) (D i lu te m y o s i n h e a v y c h a i n , n o n - m u s c l e ) , par t ia l ) 2 Y I P I 0 0 7 1 6 3 7 6 . 3 R a s - r e l a t e d pro te in R a b - 4 A 5 Y Y I P I 0 0 7 1 6 3 9 9 . 1 G u a n y l a t e k i n a s e 6 Y Y Y I P I 0 0 7 1 6 4 5 5 . 1 H e m o g l o b i n beta subun i t 9 Y Y Y Y Y I P I 0 0 7 1 6 7 1 6 . 1 P R E D I C T E D : s im i l a r to A l a n y l - t R N A s y n t h e t a s e 5 Y Y I P I 0 0 7 1 6 8 2 7 . 1 Bra in c rea t i ne k i nase 16 Y Y Y Y 101 Access ion # Name Peptides Detected in ROS Disk Mem PM Sol Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y I P I 0 0 7 1 6 8 4 3 . 1 I P I 0 0 7 1 6 9 1 6 . 1 I P I 0 0 7 1 6 9 7 4 . 1 I P I 0 0 7 1 6 9 8 9 . 1 I P I 0 0 7 1 7 0 4 6 . 2 I P I 0 0 7 1 7 2 0 7 . 2 I P I 0 0 7 1 7 2 3 4 . 2 I P I 0 0 7 1 7 2 7 2 . 1 I P I 0 0 7 1 7 4 6 5 . 1 I P I 0 0 7 1 7 5 7 3 . 1 I P I 0 0 7 1 7 6 2 1 . 1 I P I 0 0 7 1 7 6 3 8 . 1 I P I 0 0 7 1 7 6 5 5 . 1 I P I 0 0 7 1 7 6 8 5 . 1 I P I 0 0 7 1 7 7 0 4 . 1 I P I 0 0 7 1 7 8 2 8 . 1 I P I 0 0 7 1 7 8 8 4 . 1 I P I 0 0 7 1 8 0 3 5 . 1 I P I 0 0 7 1 8 1 0 9 . 1 I P I 0 0 7 1 8 1 4 9 . 1 I P I 0 0 7 1 8 1 5 2 . 1 I P I 0 0 7 1 8 1 5 5 . 2 I P I 0 0 7 1 8 1 8 5 . 1 I P I 0 0 7 1 8 1 9 7 . 1 I P I 0 0 7 1 8 2 2 0 . 2 I P I 0 0 7 1 8 2 6 1 . 1 I P I 0 0 7 1 8 2 7 1 . 2 I P I 0 0 7 1 8 2 9 1 . 2 I P I 0 0 7 1 8 3 4 5 . 1 I P I 0 0 7 1 8 3 8 3 . 1 I P I 0 0 7 1 8 4 8 0 . 2 I P I 0 0 7 1 8 5 9 9 . 1 I P I 0 0 7 1 8 6 4 0 . 2 I P I 0 0 7 2 0 7 6 1 . 1 I P I 0 0 7 2 1 0 9 1 . 1 I P I 0 0 7 2 1 1 1 8 . 1 I P I 0 0 7 2 1 2 2 7 . 1 G u a n i n e nuc leo t i de -b ind ing prote in G ( o ) , a l pha subun i t 1 14 k D a pro te in L- lac ta te d e h y d r o g e n a s e A cha in Ret ina l g u a n y l y l c y c l a s e 1 p recu rso r P R E D I C T E D : s im i l a r to regu la to r of G pro te in s igna l ing 9 -b ind ing pro te in ( R 9 A P ) P R E D I C T E D : s im i l a r to cy t i dy la te k i n a s e P R E D I C T E D : s im i l a r to 78 k D a g l u c o s e - r e g u l a t e d pro te in p r e c u r s o r M G C 1 2 8 3 5 6 pro te in ( P R E D I C T E D : s im i l a r to C y t o c h r o m e c, soma t i c ) Hypo the t i ca l p ro te in M G C 1 2 7 5 1 7 ( S A R I gene h o m o l o g A ) Pur ine nuc leos ide p h o s p h o r y l a s e C o n e c G M P - s p e c i f i c 3 ' , 5 ' - cyc l i c p h o s p h o d i e s t e r a s e a l p h a ' - s u b u n i t Ferr i t in l ight cha in 2 ' , 3 ' - c yc l i c - nuc l eo t i de 3 ' - p h o s p h o d i e s t e r a s e Hypo the t i ca l p ro te in M G C 1 2 8 1 1 0 (s im i la r to S t r e s s -i n d u c e d - p h o s p h o p r o t e i n 1 (STI1 ) ( H s c 7 0 / H s p 9 0 -o rgan i z i ng pro te in) (Hop) ( T r a n s f o r m a t i o n - s e n s i t i v e pro te in IEF S S P 3 5 2 1 ) ) H i s t one -b i nd ing pro te in R B B P 4 Rod ou te r s e g m e n t m e m b r a n e pro te in 1 ( R o m - 1 ) A T P s y n t h a s e beta c h a i n , m i tochondr ia l p recu rso r P R E D I C T E D : s im i la r to cel l l ine N K 1 4 de r i ved t r a n s f o r m i n g o n c o g e n e (Rab 8 h o m o l o g ) T r a n s k e t o l a s e 2 7 k D a pro te in ( N A D H - u b i q u i n o n e o x i d o r e d u c t a s e 24 k D a subun i t , m i tochondr ia l p recu rso r ) R A B 1 1 A , m e m b e r R A S o n c o g e n e fam i l y P R E D I C T E D : s im i l a r to P romin in 1 p recu rso r 12 k D a pro te in F-box a n d leuc ine - r i ch repea t pro te in 20 P R E D I C T E D : s im i l a r to 6 - p h o s p h o f r u c t o k i n a s e , l i ver t y p e Ret ina l rod r h o d o p s i n - s e n s i t i v e c G M P 3 ' , 5 ' - cyc l i c p h o s p h o d i e s t e r a s e de l t a - subun i t m y o s i n , l ight pep t ide 6 , a l ka l i , s m o o t h m u s c l e a n d n o n - m u s c l e P R E D I C T E D : s im i l a r to R A B 14 , m e m b e r R A S o n c o g e n e fam i l y U n c - 1 1 9 h o m o l o g (Ret ina l p ro te in 4) Hypo the t i ca l p ro te in (s im i la r to T - c o m p l e x pro te in 1, a l pha subun i t ) P R E D I C T E D : s im i l a r to G u a n i n e nuc leo t i de -b ind ing pro te in G ( z ) , a l pha subun i t Hypo the t i ca l p ro te in M G C 1 2 8 6 1 0 (s im i la r to H y d r o x y a c y l g l u t a t h i o n e hyd ro lase ( G l y o x a l a s e II) ( G L X II)) P R E D I C T E D : s im i l a r to ta l in 2 P R E D I C T E D : s im i l a r to G T P a s e N R a s (T rans fo rm ing pro te in N -Ras ) i so fo rm 3 P R E D I C T E D : s im i l a r to A T P a s e , a m i n o p h o s p h o l i p i d t r a n s p o r t e r - l i k e , c l ass I, t ype 8 A , m e m b e r 2 P R E D I C T E D : s im i l a r to a l d e h y d e d e h y d r o g e n a s e 9 A 1 i so fo rm 1 P R E D I C T E D : s im i l a r to N - e t h y l m a l e i m i d e - s e n s i t i v e fac to r i so fo rm 4 12 8 16 35 9 2 5 2 2 4 27 2 4 3 11 17 7 7 2 14 12 2 5 3 3 4 10 6 6 4 7 7 3 3 17 102 Access ion # Name Peptides Detected in R O S Disk Mem P M Sol I P I 0 0 7 2 1 2 8 4 . 1 I P I 0 0 7 2 1 4 6 3 . 1 I P I 0 0 7 2 1 6 9 9 . 1 I P I 0 0 7 2 1 7 3 1 . 1 I P I 0 0 7 2 2 0 4 4 . 1 I P I 0 0 7 2 2 8 1 1 . 1 I P I 0 0 7 2 3 4 0 4 . 1 I P I 0 0 7 2 3 8 0 3 . 1 I P I 0 0 7 2 3 9 1 5 . 1 I P I 0 0 7 2 4 0 2 4 . 1 I P I 0 0 7 2 4 1 8 9 . 1 I P I 0 0 7 2 4 2 7 4 . 1 I P I 0 0 7 2 4 3 6 0 . 1 I P I 0 0 7 2 4 7 2 5 . 1 I P I 0 0 7 2 5 1 9 3 . 1 I P I 0 0 7 2 5 5 6 4 . 1 I P I 0 0 7 2 5 5 7 3 . 1 I P I 0 0 7 2 5 8 1 0 . 1 I P I 0 0 7 2 5 9 3 0 . 1 I P I 0 0 7 2 6 1 3 1 . 1 I P I 0 0 7 2 6 3 2 2 . 1 I P I 0 0 7 2 6 3 4 3 . 1 I P I 0 0 7 2 7 0 5 0 . 2 I P I 0 0 7 2 7 3 1 4 . 1 I P I 0 0 7 2 7 6 4 5 . 1 I P I 0 0 7 2 7 7 1 2 . 1 P R E D I C T E D : s im i l a r to ca lmodu l i n 1 P R E D I C T E D : s im i l a r to P h o s p h a t i d y l e t h a n o l a m i n e -b ind ing pro te in P R E D I C T E D : s im i l a r to t ubu l i n , be ta 5 i so fo rm 4 Hypo the t i ca l p ro te in F U 3 6 8 1 2 P R E D I C T E D : s im i l a r to B-cel l r e c e p t o r - a s s o c i a t e d pro te in 37 i so fo rm 1 (Proh ib i t in -2 ) P R E D I C T E D : s im i l a r to R a s - r e l a t e d pro te in R a l - B , par t ia l P R E D I C T E D : s im i l a r to phospha t i dy l i nos i t o l - b i nd ing c la thr in a s s e m b l y p ro te in , par t ia l P R E D I C T E D : s im i l a r to ca lnex in P R E D I C T E D : s im i la r to P romin in 1 p recu rso r (P rom in in - l i ke pro te in 1) (An t igen A C 1 3 3 ) ( C D 1 3 3 a n t i g e n ) , par t ia l P R E D I C T E D : s im i la r to 6 0 S r i bosoma l pro te in L23a P R E D I C T E D : s im i la r to Pro te in d i s u l f i d e - i s o m e r a s e A 4 p r e c u r s o r (Pro te in E R p - 7 2 ) ( E R p 7 2 ) i so fo rm 2 P R E D I C T E D : s im i l a r to R A B 5 B , m e m b e r R A S o n c o g e n e fam i l y i so fo rm 1 P R E D I C T E D : s im i la r to M i c r o t u b u l e - a s s o c i a t e d pro te in I A ( M A P I A ) (Pro l i fe ra t ion- re la ted prote in p80 ) i so fo rm 4 P R E D I C T E D : s im i la r to Tubu l in a l p h a - 2 cha in ( A l p h a -tubu l in 2) i so fo rm 15 P R E D I C T E D : s im i la r to Per ipher in (Ret ina l d e g e n e r a t i o n s low prote in) i so fo rm 2 3 2 k D a pro te in (Py r idoxa l (Py r i dox ine , v i t am in B6 ) p h o s p h a t a s e ) P R E D I C T E D : s im i la r to t r a n s m e m b r a n e pro te in 6 3 B i so fo rm 3 P R E D I C T E D : s im i la r to t ype II kera t in K b 9 P R E D I C T E D : s im i la r to 78 k D a g l u c o s e - r e g u l a t e d p ro te in p recu rso r P R E D I C T E D : s im i la r to R a s - r e l a t e d pro te in R a b - 9 A ( R a b - 9 ) i so fo rm 1 P R E D I C T E D : s im i la r to v e s i c l e - a s s o c i a t e d m e m b r a n e pro te in 2 / 3 ( V A M P - 2 / 3 ) P R E D I C T E D : s im i la r to h e t e r o g e n e o u s nuc lea r r i bonuc leop ro te in D i so fo rm c i so fo rm 8 Hypo the t i ca l p ro te in ( R A S - R E L A T E D P R O T E I N R A B - 1 B ) P R E D I C T E D : s im i l a r to A T P s y n t h a s e a lpha c h a i n , m i tochond r i a l p recu rso r i so fo rm 1 P R E D I C T E D : s im i l a r to ka r yophe r i n beta 1 i so fo rm 5 P R E D I C T E D : s im i l a r to A l p h a - l - a n t i c h y m o t r y p s i n p r e c u r s o r P R E D I C T E D : s im i la r to P h o s p h o g l u c o m u t a s e 1 ( G l u c o s e p h o s p h o m u t a s e 1) ( P G M 1) , par t ia l P R E D I C T E D : s im i l a r to M i c r o t u b u l e - a s s o c i a t e d pro te in I B ( M A P I B ) i so fo rm 4 P R E D I C T E D : s im i l a r to Ras G T P a s e - a c t i v a t i n g pro te in 4 ( R a s G A P - a c t i v a t i n g - l i k e prote in 2) ( C a l c i u m -p r o m o t e d R a s inac t i va to r ) i so fo rm 6 P R E D I C T E D : s im i l a r to A D P - r i b o s y l a t i o n fac to r - l i ke pro te in 6 i so fo rm 1 ( B B S 3 ) P R E D I C T E D : s im i l a r to t r a n s m e m b r a n e pro te in 3 0 A s im i l a r to C a t h e p s i n D p recu rso r i so fo rm 4 N G , N G - d i m e t h y l a r g i n i n e d i m e t h y l a m i n o h y d r o l a s e 1 P R E D I C T E D : s im i l a r to inos ine m o n o p h o s p h a t e d e h y d r o g e n a s e 1 i so fo rm a P R E D I C T E D : s im i l a r to c i l ia ry root le t c o i l e d - c o i l , root le t in 4 9 20 5 2 3 2 2 10 2 3 6 3 22 15 2 6 2 7 2 3 3 9 6 2 2 Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y > > > > > > > > >• > >- > > > Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y I P I 0 0 7 2 7 7 9 5 . 1 I P I 0 0 7 2 7 9 5 5 . 1 I P I 0 0 7 2 8 1 3 3 . 1 I P I 0 0 7 2 8 1 8 6 . 1 I P I 0 0 7 2 8 5 7 6 . 1 I P I 0 0 7 2 8 7 1 0 . 1 I P I 0 0 7 2 8 7 9 1 . 1 I P I 0 0 7 2 9 0 5 7 . 1 I P I 0 0 7 2 9 0 6 7 . 1 4 19 11 3 2 2 4 15 2 Y Y Y Y Y Y Y < < < < < Y Y Y Y Y < -<. < •< 103 Access ion # Name Peptides Detected in R O S Disk Mem P M Sol I P I 0 0 7 2 9 1 7 3 . 1 I P I 0 0 7 2 9 3 1 7 . 1 I P I 0 0 7 2 9 5 8 0 . 1 P R E D I C T E D : s im i l a r to c i l iary root le t co i l ed -co i l , root le t in P R E D I C T E D : s im i l a r to d y n e i n , c y t o p l a s m i c , h e a v y po l ypep t i de 1 i so fo rm 1 P R E D I C T E D : s im i l a r to C G 8 7 6 8 - P A i so fo rm 2 2 25 3 Y Y Y Y Y I P I 0 0 7 3 0 2 1 9 . 1 I P I 0 0 7 3 0 2 6 9 . 1 I P I 0 0 7 3 0 4 0 2 . 1 I P I 0 0 7 3 0 6 9 1 . 2 I P I 0 0 7 3 0 9 5 0 . 1 I P I 0 0 7 3 0 9 7 9 . 1 I P I 0 0 7 3 1 1 4 8 . 1 I P I 0 0 7 3 1 2 0 2 . 1 I P I 0 0 7 3 1 3 2 2 . 1 I P I 0 0 7 3 1 3 9 1 . 1 I P I 0 0 7 3 1 6 0 4 . 1 I P I 0 0 7 3 1 6 4 5 . 1 I P I 0 0 7 3 1 9 5 6 . 1 I P I 0 0 7 3 2 0 7 7 . 1 I P I 0 0 7 3 2 0 8 1 . 1 I P I 0 0 7 3 2 1 6 2 . 1 I P I 0 0 7 3 2 2 0 5 . 1 I P I 0 0 7 3 2 3 5 7 . 1 I P I 0 0 7 3 2 4 1 0 . 1 I P I 0 0 7 3 2 4 1 7 . 1 I P I 0 0 7 3 2 5 6 6 . 1 I P I 0 0 7 3 2 7 0 3 . 1 I P I 0 0 7 3 2 7 4 7 . 1 I P I 0 0 7 3 2 8 0 7 . 1 I P I 0 0 7 3 2 9 6 8 . 1 I P I 0 0 7 3 3 0 3 8 . 1 P R E D I C T E D : s im i l a r to so lu te ca r r i e r f am i l y 25 (m i tochond r ia l ca r r ie r , A r a l a r ) , m e m b e r 12 i so fo rm 4 P R E D I C T E D : s im i l a r to 4 0 S r i bosoma l pro te in S 4 , X i so fo rm P R E D I C T E D : s im i l a r to 6 - p h o s p h o f r u c t o k i n a s e , m u s c l e t y p e ( P h o s p h o f r u c t o k i n a s e l ) ( P h o s p h o h e x o k i n a s e ) ( P h o s p h o f r u c t o - l - k i n a s e i s o z y m e A ) ( P F K - A ) ( P h o s p h o f r u c t o k i n a s e - M ) i so fo rm 5 Hypo the t i ca l p ro te in P R E D I C T E D : s im i l a r to g e r m i n a l h i s tone H4 gene P R E D I C T E D : s im i l a r to F u m a r a t e h y d r a t a s e , m i tochond r i a l p recu rso r ( F u m a r a s e ) i so fo rm 3 P R E D I C T E D : s im i l a r to X - l i n k e d j uven i l e re t inosch is is p ro te in , par t ia l P R E D I C T E D : s im i la r to T I P 1 2 0 pro te in i so fo rm 1 P R E D I C T E D : s im i l a r to Dynac t i n -1 ( 1 5 0 k D a d y n e i n -a s s o c i a t e d po lypep t ide ) ( D P - 1 5 0 ) ( D A P - 1 5 0 ) ( p l 5 0 -g lued ) i so fo rm 1 P R E D I C T E D : s im i l a r to D i h y d r o p y r i m i d i n a s e - r e l a t e d pro te in 1 ( D R P - 1 ) (Co l laps in r e s p o n s e m e d i a t o r p ro te in 1) ( C R M P - 1 ) , par t ia l P R E D I C T E D : s im i la r to a l p h a - 2 - m a c r o g l o b u l i n p recu rso r i so fo rm 5 18 k D a pro te in (Pep t idy l -p ro ly l c i s - t r ans i s o m e r a s e A) P R E D I C T E D : s im i la r to Heat s h o c k 70 k D a pro te in 4 L ( O s m o t i c s t ress pro te in 9 4 ) (Heat s h o c k 7 0 - r e l a t e d p ro te in A P G - 1 ) i so fo rm 4 P R E D I C T E D : s im i l a r to Nuc leos ide d i p h o s p h a t e k i n a s e B ( N D K B) ( N D P k inase B ) ( C - m y c pu r i ne -b ind ing t ransc r ip t i on fac to r PUF) i so fo rm 2 P R E D I C T E D : s im i l a r to nuc leo l i n - re la ted pro te in i so fo rm 8 P R E D I C T E D : s im i la r to c i t ra te s y n t h a s e p r e c u r s o r , i so fo rm a i so fo rm 5 P R E D I C T E D : s im i l a r to K e r a t i n , t y p e I cy toske le ta l 14 (Cy toke ra t i n 14) ( K 1 4 ) (CK 14) i so fo rm 5 P R E D I C T E D : s im i l a r to a d a p t o r - r e l a t e d pro te in c o m p l e x 1 beta 1 subun i t i so fo rm b i so fo rm 5 P R E D I C T E D : s im i l a r to 5 - a m i n o i m i d a z o l e - 4 -c a r b o x a m i d e r ibonuc leo t ide f o r m y l t r a n s f e r a s e / I M P c y c l o h y d r o l a s e , par t ia l P R E D I C T E D : s im i l a r to kera t in 4 i so fo rm 9 P R E D I C T E D : s im i l a r to P r o t e a s o m e subun i t a l pha t ype 6 ( P r o t e a s o m e iota cha in ) (Mu l t i ca ta ly t i c e n d o p e p t i d a s e c o m p l e x iota cha in ) i so fo rm 3 P R E D I C T E D : s im i l a r to S K B 1 h o m o l o g i so fo rm 11 P R E D I C T E D : s im i l a r to C H - T O G pro te in P R E D I C T E D : s im i l a r to hea t s h o c k 7 0 k D a pro te in 4 i so fo rm a i so fo rm 1 P R E D I C T E D : s im i l a r to ca rbon i c a n h y d r a s e X I V p recu rso r i so fo rm 2 P R E D I C T E D : s im i l a r to prote in k i nase C , de l ta i so fo rm 3 2 2 7 2 2 2 3 7 4 5 9 10 11 2 4 3 2 3 2 4 2 2 2 7 2 2 Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y I P I 0 0 7 3 3 0 5 5 . 1 I P I 0 0 7 3 3 2 0 8 . 1 P R E D I C T E D : s im i l a r to C h a r g e d mu l t i ves i cu l a r body pro te in 4b ( C h r o m a t i n mod i f y ing pro te in 4b ) ( C H M P 4 b ) i so fo rm 1 P R E D I C T E D : s im i l a r to m y o s i n , h e a v y po l ypep t i de 14 i so fo rm 3 4 4 Y Y Y Y 104 Access ion # Name Peptides Detected in R O S Disk Mem P M Sol P R E D I C T E D : s im i l a r to T - c o m p l e x pro te in 1, e ta I P I 0 0 7 3 3 2 1 5 . 1 subun i t ( T C P - l - e t a ) ( C C T - e t a ) (H IV -1 Nef in te rac t ing prote in) i so fo rm 15 3 Y Y I P I 0 0 7 3 3 2 5 6 . 1 P R E D I C T E D : s im i l a r to P u r o m y c i n - s e n s i t i v e a m i n o p e p t i d a s e ( P S A ) i so fo rm 2 12 Y Y Y I P I 0 0 7 3 3 2 8 0 . 1 P R E D I C T E D : s im i l a r to pro te in p h o s p h a t a s e 3 , 8 Y Y Y ca ta ly t i c subun i t , be ta i so fo rm i so fo rm 8 I P I 0 0 7 3 3 5 0 4 . 1 P R E D I C T E D : s im i l a r to N a + / K + - A T P a s e a l pha 3 34 Y Y subun i t i so fo rm 6 I P I 0 0 7 3 3 5 4 9 . 1 P R E D I C T E D : s im i l a r to ac t i n in , a l pha 4 i so fo rm 8 3 Y Y I P I 0 0 7 3 3 6 0 9 . 1 P R E D I C T E D : s im i l a r to kera t in c o m p l e x 2 , bas i c , g e n e 6a i so fo rm 3 5 Y Y I P I 0 0 7 3 3 6 2 7 . 1 P R E D I C T E D : s im i l a r to ca ten in ( c a d h e r i n - a s s o c i a t e d 2 Y p ro te in ) , be ta 1, 8 8 k D a i so fo rm 9 I P I 0 0 7 3 3 7 3 4 . 1 P R E D I C T E D : s im i la r to ubiqui t in C i so fo rm 1 7 Y Y Y Y I P I 0 0 7 3 3 7 4 7 . 1 P R E D I C T E D : s im i l a r to p h o s p h o f r u c t o k i n a s e , p la te le t i so fo rm 3 8 Y Y Y I P I 0 0 7 3 3 7 6 9 . 1 P R E D I C T E D : s im i l a r to a n n e x i n VII i so fo rm 2 i so fo rm 3 2 Y I P I 0 0 7 3 3 8 2 2 . 1 P R E D I C T E D : s im i l a r to h e m e b ind ing prote in 1 3 Y Y Y I P I 0 0 7 3 3 8 4 1 . 1 S i m i l a r to h e t e r o g e n e o u s nuc lea r r i bonuc leopro te in A 2 / B 1 i so fo rm 2 2 Y I P I 0 0 7 3 3 9 0 5 . 1 P R E D I C T E D : s im i l a r to D y n a m i n - 1 ( D 1 0 0 ) 16 Y Y Y Y ( D y n a m i n , bra in) ( B - d y n a m i n ) i so fo rm 16 I P I 0 0 7 3 3 9 7 0 . 1 P R E D I C T E D : s im i l a r to c a l u m e n i n p recu rso r i so fo rm 3 P R E D I C T E D : s im i l a r to C o a t o m e r a l pha subun i t 2 Y I P I 0 0 7 3 4 0 8 4 . 1 ( A l p h a - c o a t pro te in) ( A l p h a - C O P ) ( H E P C O P ) ( H E P -C O P ) i so fo rm 19 6 Y I P I 0 0 7 3 4 1 0 4 . 1 P R E D I C T E D : s im i l a r to R A B 1 , m e m b e r R A S o n c o g e n e fam i l y i so fo rm 2 12 Y Y Y I P I 0 0 7 3 4 2 7 3 . 1 P R E D I C T E D : s im i l a r to d y n a m i n 2 i so fo rm 24 5 Y I P I 0 0 7 3 4 2 9 3 . 1 P R E D I C T E D : s im i la r to heat shock 7 0 k D pro te in 4 Y Y Y b ind ing pro te in P R E D I C T E D : s im i l a r to Spec t r i n a l pha c h a i n , bra in I P I 0 0 7 3 4 3 1 7 . 1 ( S p e c t r i n , non -e r y th ro i d a l pha cha in ) (A lpha- I I spec t r in ) (Fodr in a l pha cha in ) i so fo rm 9 19 Y I P I 0 0 7 3 4 4 2 7 . 1 P R E D I C T E D : s im i l a r to a d e n y l o s u c c i n a t e l yase i so fo rm 2 P R E D I C T E D : s im i la r to B a n d 4 . 1 - l i k e pro te in 3 ( 4 . I B ) 3 Y I P I 0 0 7 3 4 4 3 6 . 1 (D i f fe rent ia l ly e x p r e s s e d in a d e n o c a r c i n o m a of the lung pro te in 1) ( D A L - 1 ) ( D A L 1 P ) ( m D A L - 1 ) i so fo rm 7 2 Y I P I 0 0 7 3 4 5 2 8 . 1 P R E D I C T E D : s im i l a r to C G 1 5 3 2 - P A i so fo rm 3 2 Y Y I P I 0 0 7 4 2 6 2 3 . 1 Hypo the t i ca l p ro te in (Cy toso l a m i n o p e p t i d a s e ) 2 Y Y I P I 0 0 7 4 4 3 8 0 . 1 73 k D a pro te in (so lu te ca r r i e r f am i l y 4 4 , m e m b e r 1) 2 Y Y Y I P I 0 0 7 5 9 4 3 6 . 1 Hypo the t i ca l p ro te in ( Ino rgan ic p y r o p h o s p h a t a s e ) 12 Y Y Y 105 A P P E N D I X II - Functions of ROS proteins sorted into various categories Accession # Name Category Function/Comments IPI00716989 1 Retinal guanylyl cyclase 1 precursor Phototransduction Phototransduction/ Visual Cycle IPI00696393 1 Retinal guanylyl cyclase 2 precursor Phototransduction Phototransduction/ Visual Cycle IPI00711319 1 Guanine nucleotide-binding protein G(t), alpha-1 subunit Phototransduction Phototransduction/ Visual Cycle IPI00703160 1 Guanine nucleotide-binding protein G(I)/G(S)/G(T) beta subunit 1 Phototransduction Phototransduction/ Visual Cycle IPI00695759 1 Guanine nucleotide-binding protein G(T) gamma-T l subunit precursor 44 kDa protein (PREDICTED: similar to guanine Phototransduction Phototransduction/ Visual Cycle IPI00710190 1 nucleotide-binding protein beta-5 subunit isoform 1) Phototransduction Phototransduction/ Visual Cycle IPI00694482 1 Regulator of G-protein signaling 9 (RGS9) Phototransduction Phototransduction/ Visual Cycle IPI00717046 2 PREDICTED: similar to regulator of G protein signaling 9-binding protein (R9AP) Phototransduction Phototransduction/ Visual Cycle IPI00711518 1 ABC transporter (ABCR or Rim Protein) Phototransduction Phototransduction/ Visual Cycle IPI00690805 1 Rod cGMP-specific 3',5'-cyclic phosphodiesterase alpha-subunit Phototransduction Phototransduction/ Visual Cycle IPI00711026 1 Rod cGMP-specific 3',5'-cyclic phosphodiesterase beta-subunit Phototransduction Phototransduction/ Visual Cycle IPI00707056 1 Splice Isoform 1 of Sodium/potassium/calcium exchanger 1 Phototransduction Phototransduction/ Visual Cycle IPI00701150 1 Recoverin Phototransduction Phototransduction/ Visual Cycle IPI00688887 1 Hypothetical protein (similar to alpha isoform of regulatory subunit A, protein phosphatase 2) Phototransduction Phototransduction/ Visual Cycle IPI00712873 1 Rhodopsin Phototransduction Phototransduction/ Visual Cycle IPI00707921 1 Phosducin Phototransduction Phototransduction/ Visua Cycle IPI00705657 1 Splice Isoform A of S-arrestin Phototransduction Phototransduction/ Visua Cycle IPI00702970 1 Splice Isoform B of S-arrestin Phototransduction Phototransduction/ Visua Cycle IPI00703873 1 Rhodopsin kinase precursor Phototransduction Phototransduction/ Visua Cycle IPI00712679 1 cGMP-gated cation channel alpha 1 Phototransduction Phototransduction/ Visua Cycle IPI00701200 1 Splice Isoform CNG4D of 240 kDa protein of rod photoreceptor CNG-channel Phototransduction Phototransduction/ Visua Cycle IPI00693264 1 Photoreceptor outer segment all-trans retinol dehydrogenase Interphotoreceptor retinoid-binding protein precursor (IRBP) Phototransduction Phototransduction/ Visua Cycle IPI00687859 1 Phototransduction Phototransduction/ Visua Cycle IPI00712695 1 Cellular retinaldehyde-binding protein Phototransduction Phototransduction/ Visua1 Cycle IPI00711777 1 Retinal pigment epithelium-specific 65 kDa protein Phototransduction Phototransduction/ Visual Cycle IPI00721284 1 PREDICTED: similar to calmodulin 1 Phototransduction Phototransduction/ Visual Cycle IPI00707490 1 PREDICTED: similar to Sodium/potassium/calcium exchanger 2 Phototransduction Phototransduction/ Visua! Cycle IPI00699937 1 Retina G protein-coupled receptor kinase 7 Phototransduction Phototransduction/ Visual Cycle IPI00708069 1 Guanine nucleotide-binding protein G(t), alpha-2 subunit Phototransduction Phototransduction/ Visua Cycle IPI00704523 1 Guanine nucleotide-binding protein G(I ) /G(S)/G(0) gamma-T2 subunit Phototransduction Phototransduction/ Visual Cycle IPI00701214 1 43 kDa protein (Arrestin 3, retinal (X-arrestin)) Phototransduction Phototransduction/ Visual Cycle IPI00702002 1 Cyclic nucleotide-gated cation channel alpha 3 Phototransduction Phototransduction/ Visua Cycle IPI00696922 1 Red opsin Phototransduction Phototransduction/ Visua Cycle IPI00704754 1 Blue-sensitive opsin Phototransduction Phototransduction/ Visua Cycle IPI00717621 1 Cone cGMP-specific 3',5'-cyclic phosphodiesterase alpha'-subunit Phototransduction Phototransduction/ Visual Cycle IPI00718149 1 NADH-ubiquinone oxidoreductase 24 kDa subunit, mitochondrial Metabolism Electron transport 106 A c c e s s i o n # N a m e C a t e g o r y Function/Comments I P I 0 0 7 0 8 0 2 7 . I P I 0 0 7 0 2 7 1 8 . I P I 0 0 7 3 4 4 2 7 . I P I 0 0 7 1 3 8 1 4 . I P I 0 0 7 1 5 7 9 9 . I P I 0 0 7 1 0 3 6 6 . I P I 0 0 6 9 9 7 1 7 . I P I 0 0 7 1 0 8 9 5 . I P I 0 0 6 9 8 5 8 9 . I P I 0 0 7 3 0 9 7 9 . I P I 0 0 6 8 6 9 8 4 . 3 I P I 0 0 6 9 4 2 0 2 . I P I 0 0 7 3 2 1 6 2 . I P I 0 0 7 1 0 3 5 4 . I P I 0 0 6 9 0 8 7 0 . I P I 0 0 7 1 1 0 9 4 . 1 I P I 0 0 4 7 9 1 8 6 . I P I 0 0 7 1 6 9 7 4 . I P I 0 0 7 1 5 1 0 1 . I P I 0 0 7 0 6 9 4 2 . I P I 0 0 7 3 3 7 4 7 . I P I 0 0 7 3 0 4 0 2 . 1 I P I 0 0 7 1 8 2 2 0 , I P I 0 0 7 1 4 4 4 6 . I P I 0 0 7 1 5 4 1 5 . 1 I P I 0 0 7 2 8 7 9 1 . 1 I P I 0 0 7 1 2 2 5 0 . I P I 0 0 6 9 9 1 0 9 . I P I 0 0 7 2 1 1 1 8 . I P I 0 0 6 9 4 4 7 2 . I P I 0 0 7 2 7 7 9 5 . I P I 0 0 7 1 6 8 2 7 . I P I 0 0 6 9 2 0 3 4 .1 I P I 0 0 6 8 9 5 1 5 . 2 I N A D H - u b i q u i n o n e o x i d o r e d u c t a s e 7 5 k D a s u b u n i t , m i t o c h o n d r i a l D i h y d r o x y a c e t o n e k i n a s e P R E D I C T E D : s i m i l a r t o a d e n y l o s u c c i n a t e l y a s e i s o f o r m 2 G l y c e r a l d e h y d e - 3 - p h o s p h a t e d e h y d r o g e n a s e M G C 1 2 8 2 7 9 p r o t e i n ( P R E D I C T E D : s i m i l a r t o . G l y c e r a l d e h y d e - 3 - p h o s p h a t e d e h y d r o g e n a s e ) s i m i l a r t o G l y c e r a l d e h y d e - 3 - p h o s p h a t e d e h y d r o g e n a s e , l i v e r D - 3 - p h o s p h o g l y c e r a t e d e h y d r o g e n a s e P h o s p h o g l y c e r a t e k i n a s e 1 S i m i l a r t o p h o s p h o g l y c e r a t e m u t a s e P R E D I C T E D : s i m i l a r t o F u m a r a t e h y d r a t a s e , m i t o c h o n d r i a l p r e c u r s o r ( F u m a r a s e ) i s o f o r m 3 P R E D I C T E D : s i m i l a r t o A l c o h o l d e h y d r o g e n a s e [ N A D P + ] ( A l d e h y d e r e d u c t a s e ) ( A l d o - k e t o r e d u c t a s e f a m i l y 1 m e m b e r A l ) L y s o p h o s p h o l i p a s e I P R E D I C T E D : s i m i l a r t o c i t r a t e s y n t h a s e p r e c u r s o r , i s o f o r m a i s o f o r m 5 A T P c i t r a t e l y a s e A c o n i t a t e h y d r a t a s e , m i t o c h o n d r i a l p r e c u r s o r P R E D I C T E D : s i m i l a r t o P y r u v a t e k i n a s e , i s o z y m e s M 1 / M 2 ( P y r u v a t e k i n a s e m u s c l e i s o z y m e ) ( C y t o s o l i c t h y r o i d h o r m o n e - b i n d i n g p r o t e i n ) ( C T H B P ) ( T H B P 1 ) , p a r t i a l p y r u v a t e k i n a s e 3 i s o f o r m 1 L - l a c t a t e d e h y d r o g e n a s e A c h a i n L a c t a t e d e h y d r o g e n a s e B | T r i o s e p h o s p h a t e i s o m e r a s e 1 P R E D I C T E D : s i m i l a r t o p h o s p h o f r u c t o k i n a s e , p l a t e l e t i s o f o r m 3 P R E D I C T E D : s i m i l a r t o 6 - p h o s p h o f r u c t o k i n a s e , m u s c l e t y p e ( P h o s p h o f r u c t o k i n a s e 1 ) ( P h o s p h o h e x o k i n a s e ) ( P h o s p h o f r u c t o - l - k i n a s e i s o z y m e A ) ( P F K - A ) ( P h o s p h o f r u c t o k i n a s e - M ) i s o f o r m 5 P R E D I C T E D : s i m i l a r t o 6 - p h o s p h o f r u c t o k i n a s e , l i v e r t y p e G l u c o s e p h o s p h a t e i s o m e r a s e D i m e t h y l a r g i n i n e d i m e t h y l a m i n o h y d r o l a s e 2 N G , N G - d i m e t h y l a r g i n i n e d i m e t h y l a m i n o h y d r o l a s e 1 M D H 2 p r o t e i n ( M a l a t e d e h y d r o g e n a s e ) M a l a t e d e h y d r o g e n a s e , c y t o p l a s m i c P R E D I C T E D : s i m i l a r t o a l d e h y d e d e h y d r o g e n a s e 9 A 1 i s o f o r m 1 P R E D I C T E D : s i m i l a r t o M o n o g l y c e r i d e l i p a s e P R E D I C T E D : s i m i l a r t o P h o s p h o g l u c o m u t a s e 1 ( G l u c o s e p h o s p h o m u t a s e 1 ) ( P G M 1 ) , p a r t i a l B r a i n c r e a t i n e k i n a s e C r e a t i n e k i n a s e , u b i q u i t o u s m i t o c h o n d r i a l p r e c u r s o r P R E D I C T E D : s i m i l a r t o F r u c t o s e - b i s p h o s p h a t e a l d o l a s e A ( M u s c l e - t y p e a l d o l a s e ) i s o f o r m 1 M e t a b o l i s m M e t a b o l i s m M e t a b o l i s m M e t a b o l i s m M e t a b o l i s m M e t a b o l i s m M e t a b o l i s m M e t a b o l i s m M e t a b o l i s m M e t a b o l i s m M e t a b o l i s m M e t a b o l i s m M e t a b o l i s m M e t a b o l i s m M e t a b o l i s m M e t a b o l i s m M e t a b o l i s m M e t a b o l i s m M e t a b o l i s m M e t a b o l i s m M e t a b o l i s m M e t a b o l i s m M e t a b o l i s m M e t a b o l i s m M e t a b o l i s m M e t a b o l i s m M e t a b o l i s m M e t a b o l i s m M e t a b o l i s m M e t a b o l i s m M e t a b o l i s m M e t a b o l i s m M e t a b o l i s m M e t a b o l i s m Electron transport (Glycerol utilization Purine biosynthesis Glycolysis Glycolysis Glycolysis L-Serine biosynthesis Glycolysis Glycolysis TCA cycle Alcohol metabolism Glycerophospholipid metabolism TCA cycle TCA cycle TCA cycle Glycolysis Glycolysis Glycolysis Glycolysis Glycolysis Glycolysis Glycolysis Glycolys is jdycolys is Dimethylarginine metabolism; Regulation of nitric oxide biosynthesis Dimethylarginine metabolism; Regulation of nitric oxide biosynthesis Glycolysis Glycolysis Carnitine biosynthesis Triglyceride lypolysis Glycolysis Electron transport Electron transport jGlycolysis 107 A c c e s s i o n # N a m e C a t e g o r y Function/Comments I P I 0 0 7 1 0 7 1 4 . 2 P R E D I C T E D : s im i l a r to F r u c t o s e - b i s p h o s p h a t e a l d o l a s e C ( B r a i n - t y p e a ldo lase ) i so fo rm 1 M e t a b o l i s m Glycolysis I P I 0 0 7 1 1 2 4 1 . 2 Hypo the t i ca l p ro te in (T ransa ldo lase 1) M e t a b o l i s m Pentose phosphate pathway I P I 0 0 7 2 9 0 5 7 . 1 P R E D I C T E D : s im i l a r to inos ine m o n o p h o s p h a t e d e h y d r o g e n a s e 1 M e t a b o l i s m G M P biosynthesis I P I 0 0 6 9 2 8 1 9 . 1 Inos i to l m o n o p h o s p h a t a s e M e t a b o l i s m Phosphatidyl inositol signaling I P I 0 0 6 9 4 3 8 3 . 2 s im i l a r to H y p o x a n t h i n e - g u a n i n e M e t a b o l i s m Purine salvage p h o s p h o r i b o s y l t r a n s f e r a s e I P I 0 0 6 9 1 4 4 0 . 2 P R E D I C T E D : s im i la r to d i m e r i c d ihydrod io l M e t a b o l i s m Electron transport d e h y d r o g e n a s e I P I 0 0 7 1 2 6 4 8 . 2 P R E D I C T E D : s im i l a r to M a n n o s y l - o l i g o s a c c h a r i d e g l u c o s i d a s e M e t a b o l i s m N-linked oligosaccharide processing I P I 0 0 6 8 7 2 1 1 . 1 H e x o k i n a s e 1 M e t a b o l i s m Glycolysis I P I 0 0 7 0 1 0 9 8 . 1 P R E D I C T E D : s im i l a r to H e x o k i n a s e t ype II M e t a b o l i s m Glycolysis I P I 0 0 6 9 4 6 1 2 . 1 A s p a r t a t e a m i n o t r a n s f e r a s e , c y t o p l a s m i c M e t a b o l i s m Aspartate catabolism I P I 0 0 7 1 3 1 3 7 . 1 A s p a r t a t e a m i n o t r a n s f e r a s e , m i tochondr ia l p recu rso r U b i q u i n o l - c y t o c h r o m e - c r educ tase c o m p l e x co re pro te in 2 T r a n s k e t o l a s e M e t a b o l i s m Aspartate catabolism I P I 0 0 6 9 7 8 2 7 . 1 M e t a b o l i s m Electron transport I P I 0 0 7 1 8 1 0 9 . 1 M e t a b o l i s m Pentose phosphate pathway / Glycolysis I P I 0 0 7 2 5 5 6 4 . 1 3 2 k D a pro te in (Py r idoxa l (Py r i dox ine , v i t a m i n M e t a b o l i s m Vitamin metabolism B6 ) p h o s p h a t a s e ) I P I 0 0 7 1 7 5 7 3 . 1 Pur ine nuc leos ide p h o s p h o r y l a s e M e t a b o l i s m Nucleic acid metabolism I P I 0 0 7 0 2 7 8 1 . 1 Isoc i t ra te d e h y d r o g e n a s e 1 ( N A D P + ) , so lub le M e t a b o l i s m Glutathione metabolism I P I 0 0 7 1 3 4 8 4 . 2 P R E D I C T E D : s im i l a r to g l y o x a l a s e 1 M e t a b o l i s m Glyoxal pathway I P I 0 0 7 0 9 8 4 0 . 1 21 k D a pro te in ( P R E D I C T E D : s im i la r to g l y o x y l a s e 1) Hypo the t i ca l p ro te in M G C 1 2 8 6 1 0 (s im i la r to M e t a b o l i s m Glyoxal pathway I P I 0 0 7 1 8 5 9 9 . 1 H y d r o x y a c y l g l u t a t h i o n e h y d r o l a s e ( G l y o x a l a s e II) ( G L X II)) M e t a b o l i s m Glyoxal pathway I P I 0 0 7 0 1 6 4 2 . 2 P R E D I C T E D : s im i l a r to 6 - p h o s p h o g l u c o n a t e M e t a b o l i s m Pentose phosphate pathway d e h y d r o g e n a s e , d e c a r b o x y l a t i n g I P I 0 0 7 0 9 8 2 2 . 2 s im i l a r to g l u t a m a t e - a m m o n i a l igase M e t a b o l i s m Nitrogen utilization I P I 0 0 7 0 7 0 9 5 . 2 A l p h a - E n o l a s e s im i l a r to G a m m a e n o l a s e ( 2 - p h o s p h o - D -M e t a b o l i s m Glycolysis I P I 0 0 7 1 4 7 6 4 . 1 g l yce ra te h y d r o - l y a s e ) (Neura l e n o l a s e ) ( N e u r o n - s p e c i f i c eno lase ) ( N S E ) (Eno lase 2) i so fo rm 2 M e t a b o l i s m Glycolysis I P I 0 0 7 5 9 4 3 6 . 1 Hypo the t i ca l p ro te in ( Ino rgan ic p y r o p h o s p h a t a s e ) M e t a b o l i s m Phosphate metabolism I P I 0 0 7 1 6 2 4 6 . 1 C a r b o n i c a n h y d r a s e 2 M e t a b o l i s m Carbon dioxide metabolism I P I 0 0 7 3 2 9 6 8 . 1 P R E D I C T E D : s im i la r to ca rbon i c a n h y d r a s e X I V M e t a b o l i s m Carbon dioxide metabolism p r e c u r s o r i so fo rm 2 I P I 0 0 6 9 9 8 3 9 . 2 P R E D I C T E D : s im i l a r to i s o c h o r i s m a t a s e d o m a i n con ta in ing 1 i so fo rm 4 M e t a b o l i s m Isochorismate metabolism I P I 0 0 7 1 7 2 7 2 . 1 M G C 1 2 8 3 5 6 pro te in ( P R E D I C T E D : s im i la r to M e t a b o l i s m Electron transfer C y t o c h r o m e c, soma t i c ) I P I 0 0 7 3 4 1 0 4 . 1 P R E D I C T E D : s im i l a r to R A B 1 , m e m b e r R A S o n c o g e n e f am i l y i so fo rm 2 V e s i c l e T ra f f i ck ing Vesic le membrane fusion I P I 0 0 7 2 7 0 5 0 . 2 Hypo the t i ca l p ro te in ( R A S - R E L A T E D P R O T E I N R A B - 1 B ) V e s i c l e T ra f f i ck ing Vesic le membrane fusion I P I 0 0 6 9 5 8 8 1 . 2 P R E D I C T E D : s im i l a r to R A B 2 , m e m b e r R A S o n c o g e n e fam i l y V e s i c l e T ra f f i ck ing Vesic le membrane fusion I P I 0 0 7 1 0 1 2 3 . 1 P R E D I C T E D : s im i l a r to R A B 2 B pro te in i so fo rm 1 V e s i c l e Tra f f i ck ing Vesicle membrane fusion I P I 0 0 7 0 3 4 3 6 . 1 R a s - r e l a t e d p ro te in R a b - 3 A V e s i c l e T ra f f i ck ing Vesic le membrane fusion I P I 0 0 7 1 3 8 3 5 . 2 L O C 6 1 7 6 0 0 pro te in ( R a s - R e l a t e d Prote in R a b -3 C ) V e s i c l e T ra f f i ck ing Vesic le membrane fusion I P I 0 0 7 0 9 0 1 4 . 1 2 4 k D a pro te in ( P R E D I C T E D : s im i la r to R A B 3 D , m e m b e r R A S o n c o g e n e fami l y ) V e s i c l e T ra f f i ck ing Vesic le membrane fusion 108 A c c e s s i o n # N a m e C a t e g o r y Function/Comments I P I 0 0 7 1 6 3 7 6 . 3 R a s - r e l a t e d pro te in R a b - 4 A Ves i c l e T ra f f i ck ing Vesic le membrane fusion I P I 0 0 7 1 6 1 7 5 . 2 P R E D I C T E D : s im i l a r to R a s - r e l a t e d pro te in R a b -4 B Ves i c l e T ra f f i ck ing Vesic le membrane fusion I P I 0 0 6 9 3 8 2 4 . 1 P R E D I C T E D : s im i l a r to R a s - r e l a t e d prote in R a b -5 A i so fo rm 1 V e s i c l e T ra f f i ck ing Vesic le membrane fusion I P I 0 0 7 2 4 2 7 4 . 1 P R E D I C T E D : s im i l a r to R A B 5 B , m e m b e r R A S o n c o g e n e fam i l y i so fo rm 1 V e s i c l e Tra f f i ck ing Vesic le membrane fusion I P I 0 0 6 9 4 1 9 8 . 1 R a s - r e l a t e d pro te in R a b - 5 C Ves i c l e Tra f f i ck ing Vesic le membrane fusion I P I 0 0 7 0 9 2 6 5 . 1 10 k D a pro te in (s im i la r to R a s - r e l a t e d pro te in R a b - 7 ) V e s i c l e Tra f f i ck ing Vesic le membrane fusion I P I 0 0 7 0 4 7 5 2 . 1 Hypo the t i ca l p ro te in M G C 1 2 7 5 9 7 (s im i la r to R a s - r e l a t e d pro te in R a b - 7 ) Ves i c l e Tra f f i ck ing Vesicle membrane fusion I P I 0 0 7 1 8 0 3 5 . 1 P R E D I C T E D : s im i l a r to cel l l ine N K 1 4 de r i ved t r a n s f o r m i n g o n c o g e n e (Rab 8 h o m o l o g ) Ves i c l e Tra f f i ck ing Vesic le membrane fusion I P I 0 0 6 9 3 6 3 6 . 2 P R E D I C T E D : s im i la r to R a s - r e l a t e d pro te in R a b -8 B V e s i c l e Tra f f i ck ing Vesic le membrane fusion I P I 0 0 7 2 6 1 3 1 . 1 P R E D I C T E D : s im i l a r to R a s - r e l a t e d pro te in R a b -9 A ( R a b - 9 ) i so fo rm 1 V e s i c l e Tra f f i ck ing Vesic le membrane fusion I P I 0 0 7 0 8 1 2 5 . 1 14 k D a pro te in ( P R E D I C T E D : s im i l a r to R a s -re la ted pro te in R a b - 1 0 ) V e s i c l e Tra f f i ck ing Vesic le membrane fusion I P I 0 0 7 1 8 1 5 2 . 1 R A B 1 1 A , m e m b e r R A S o n c o g e n e fam i l y V e s i c l e Tra f f i ck ing Vesic le membrane fusion I P I 0 0 7 1 8 2 9 1 . 2 P R E D I C T E D : s im i la r to R A B 1 4 , m e m b e r R A S o n c o g e n e fam i l y V e s i c l e Tra f f i ck ing Vesic le membrane fusion I P I 0 0 6 9 1 8 2 6 . 2 P R E D I C T E D : s im i l a r to R a s - r e l a t e d pro te in R a b -18 i so fo rm 4 V e s i c l e T ra f f i ck ing Vesic le membrane fusion I P I 0 0 7 1 0 7 8 0 . 1 19 k D a pro te in ( P R E D I C T E D : s im i l a r to R a s -re la ted pro te in R a b - 2 1 ) V e s i c l e Tra f f i ck ing Vesic le membrane fusion I P I 0 0 6 9 6 0 3 0 . 1 P R E D I C T E D : s im i l a r to R a s - r e l a t e d pro te in R a b -23 V e s i c l e Tra f f i ck ing Vesic le membrane fusion I P I 0 0 6 9 2 5 0 3 . 1 P R E D I C T E D : s im i l a r to R a s - r e l a t e d pro te in R a b -3 2 V e s i c l e T ra f f i ck ing Vesic le membrane fusion I P I 0 0 6 8 7 0 3 3 . 1 P R E D I C T E D : s im i l a r to R A B 3 3 B , m e m b e r R A S o n c o g e n e fam i l y V e s i c l e T ra f f i ck ing Vesic le membrane fusion I P I 0 0 7 1 3 9 5 3 . 2 P R E D I C T E D : s im i la r to R A B 3 5 , m e m b e r R A S o n c o g e n e fam i l y V e s i c l e T ra f f i ck ing Vesic le membrane fusion I P I 0 0 6 9 1 2 0 6 . 1 P R E D I C T E D : s im i l a r to R A B 3 9 B , m e m b e r R A S o n c o g e n e fam i l y V e s i c l e T ra f f i ck ing Vesic le membrane fusion I P I 0 0 6 9 8 5 9 4 . 1 Rab G D P d issoc ia t i on inh ib i tor a lpha Ves i c l e T ra f f i ck ing Regulation of Rab G T P a s e s I P I 0 0 7 1 3 7 6 0 . 2 Rab G D P d issoc ia t i on inh ib i tor beta Ves i c l e Tra f f i ck ing Regulation of Rab G T P a s e s I P I 0 0 6 9 0 9 6 2 . 1 S y n t a x i n - b i n d i n g pro te in 1 ( M u n c - 1 8 - 1 ) V e s i c l e Tra f f i ck ing Vesic le membrane fusion I P I 0 0 6 9 3 8 2 3 . 1 P R E D I C T E D : s im i l a r to S y n t a x i n - b i n d i n g pro te in 3 V e s i c l e Tra f f i ck ing Vesic le membrane fusion I P I 0 0 7 0 2 5 3 2 . 2 P R E D I C T E D : s im i l a r to S H 3 - d o m a i n G R B 2 - l i k e 2 (Endoph i l i n 1) Ves i c l e Tra f f i ck ing Vesic le formation I P I 0 0 7 1 5 7 7 0 . 1 A D P - r i b o s y l a t i o n fac to r 1 V e s i c l e Tra f f i ck ing Vesic le formation I P I 0 0 7 0 6 4 5 1 . 1 Hypo the t i ca l p ro te in M G C 1 2 8 0 5 9 ( A D P -R ibosy la t i on Fac to r 4) Ves i c l e Tra f f i ck ing Vesic le formation I P I 0 0 7 0 4 1 5 5 . 1 P R E D I C T E D : s im i l a r to A D P - r i b o s y l a t i o n fac to r -l ike 2 - l i ke 1 i so fo rm 1 i so fo rm 3 V e s i c l e Tra f f i ck ing Vesic le trafficking I P I 0 0 7 1 6 0 7 7 . 2 Hypo the t i ca l p ro te in M G C 1 3 3 6 5 1 (S im i l a r to A D P - r i b o s y l a t i o n fac to r - l i ke 3) V e s i c l e T ra f f i ck ing Vesic le trafficking I P I 0 0 7 0 9 5 3 3 . 1 18 k D a pro te in ( P R E D I C T E D : s im i la r to A D P -r ibosy la t ion fac to r - l i ke 8A) V e s i c l e T ra f f i ck ing Vesicle trafficking I P I 0 0 6 9 1 6 4 1 . 2 P R E D I C T E D : s im i l a r to A D P - r i b o s y l a t i o n fac to r -l ike 1 0 C V e s i c l e T ra f f i ck ing Vesic le trafficking I P I 0 0 6 8 8 8 1 6 . 1 C la th r in h e a v y cha in V e s i c l e T ra f f i ck ing Vesic le coat formation I P I 0 0 7 2 3 4 0 4 . 1 P R E D I C T E D : s im i l a r to phospha t i dy l i nos i t o l -b ind ing c la thr in a s s e m b l y p ro te in , par t ia l V e s i c l e T ra f f i ck ing Vesic le coat formation I P I 0 0 7 3 2 3 5 7 . 1 P R E D I C T E D : s im i l a r to a d a p t o r - r e l a t e d pro te in c o m p l e x 1 beta 1 subun i t i so fo rm b V e s i c l e T ra f f i ck ing Vesic le coat formation I P I 0 0 7 3 4 0 8 4 . 1 P R E D I C T E D : s im i l a r to C o a t o m e r a l pha subun i t ( A l p h a - c o a t pro te in) ( A l p h a - C O P ) ( H E P C O P ) V e s i c l e Tra f f i ck ing Vesicle coat formation 109 A c c e s s i o n # N a m e C a t e g o r y Function/Comments I P I 0 0 7 0 2 5 6 5 . I P I 0 0 7 2 6 3 2 2 . I P I 0 0 6 9 4 7 6 0 . I P I 0 0 7 0 2 9 2 8 . I P I 0 0 6 8 9 9 5 4 . I P I 0 0 7 0 2 3 1 4 . I P I 0 0 7 3 1 3 2 2 . 1 I P I 0 0 7 3 3 9 0 5 . I P I 0 0 7 3 4 2 7 3 , I P I 0 0 6 9 8 8 0 5 . I P I 0 0 7 1 8 3 4 5 . I P I 0 0 7 2 1 2 2 7 . I P I 0 0 7 1 4 0 4 8 . I P I 0 0 7 1 7 4 6 5 . I P I 0 0 7 1 4 0 0 4 . I P I 0 0 7 2 5 1 9 3 . I P I 0 0 7 1 7 8 2 8 . I P I 0 0 7 1 8 1 5 5 . I P I 0 0 7 2 3 9 1 5 . 1 I P I 0 0 6 9 9 8 8 7 . 1 I P I 0 0 7 0 0 2 3 5 . I P I 0 0 7 0 5 8 9 9 . I P I 0 0 7 0 4 3 5 3 . I P I 0 0 7 2 4 7 2 5 . I P I 0 0 6 9 7 1 0 7 . I P I 0 0 7 0 6 5 9 4 . I P I 0 0 7 2 1 6 9 9 . I P I 0 0 7 1 2 3 9 6 . I P I 0 0 7 2 4 3 6 0 . 1 I P I 0 0 7 2 7 9 5 5 . I P I 0 0 6 8 8 0 8 1 . I P I 0 0 6 9 2 0 7 9 . I P I 0 0 7 1 2 8 3 8 . I P I 0 0 7 0 8 7 6 7 . ( H E P - C O P ) i so fo rm 19 Hypo the t i ca l p ro te in ( P R E D I C T E D : s im i l a r to S E C 2 2 ves i c le t ra f f ick ing p ro te in - l i ke 1) P R E D I C T E D : s im i l a r to v e s i c l e - a s s o c i a t e d m e m b r a n e pro te in 2 / 3 ( V A M P - 2 / 3 ) P R E D I C T E D : s im i l a r to V e s i c l e - a s s o c i a t e d m e m b r a n e pro te in 5 S y n a p t o t a g m i n - 1 146 k D a pro te in ( S y n a p t o j a n i n - 1 ) G e n e r a l v e s i c u l a r t r anspo r t fac tor pi 15 P R E D I C T E D : s im i l a r to Dynac t i n -1 ( 150 k D a d y n e i n - a s s o c i a t e d po lypep t ide ) ( D P - 1 5 0 ) ( D A P -150) ( p l 5 0 - g l u e d ) i so fo rm 1 P R E D I C T E D : s im i l a r to D y n a m i n - 1 ( D 1 0 0 ) ( D y n a m i n , bra in) ( B - d y n a m i n ) P R E D I C T E D : s im i l a r to d y n a m i n 2 i so fo rm 24 33 k D a pro te in ( P R E D I C T E D : s im i l a r to s y n t a x i n 3 , par t ia l ) U n c - 1 1 9 h o m o l o g (Ret ina l prote in 4 ) P R E D I C T E D : s im i l a r to N - e t h y l m a l e i m i d e -sens i t i ve fac to r i so fo rm 4 Go lg in sub fam i l y A m e m b e r 7 Hypo the t i ca l p ro te in M G C 1 2 7 5 1 7 ( S A R I g e n e h o m o l o g A ) Hypo the t i ca l p ro te in (s im i la r to po lypos is locus pro te in 1- l ike 1) P R E D I C T E D : s im i l a r to Per ipher in (Ret ina l d e g e n e r a t i o n s low pro te in) Rod ou te r s e g m e n t m e m b r a n e pro te in 1 ( R o m -1) P R E D I C T E D : s im i la r to P romin in 1 p recu rso r P R E D I C T E D : s im i la r to P romin in 1 p recu rso r (P rom in i n - l i ke pro te in 1) (An t igen A C 1 3 3 ) ( C D 1 3 3 a n t i g e n ) , par t ia l O x y g e n - r e g u l a t e d pro te in 1 (Ret in i t is P i g m e n t o s a 1 pro te in) P R E D I C T E D : s im i l a r to ret in i t is p i g m e n t o s a 1-l ike 1 P R E D I C T E D : s im i l a r to t ubu l i n , a lpha 1 T u b u l i n , a l pha 1 P R E D I C T E D : s im i l a r to Tubu l i n a l p h a - 2 cha in (A lpha - tubu l i n 2) i so fo rm 15 Be ta tubu l in Tubu l i n b e t a - 2 C cha in s im i l a r to t ubu l i n , be ta 5 i so fo rm 4 Tubu l i n b e t a - 3 cha in P R E D I C T E D : s im i l a r to M i c r o t u b u l e - a s s o c i a t e d pro te in I A ( M A P I A ) (P ro l i f e ra t ion - re la ted pro te in p80 ) i so fo rm 4 P R E D I C T E D : s im i l a r to M i c r o t u b u l e - a s s o c i a t e d pro te in I B ( M A P I B ) i so fo rm 4 M i c r o t u b u l e - a s s o c i a t e d pro te in 4 M i c r o t u b u l e - a s s o c i a t e d pro te in 4 i so fo rm 5 ( F r a g m e n t ) |Act in , c y t o p l a s m i c 2 P R E D I C T E D : s im i l a r to act in re la ted prote in 2 / 3 Icomplex, subun i t 4 V e s i c l e T ra f f i ck ing V e s i c l e Tra f f i ck ing V e s i c l e Tra f f i ck ing V e s i c l e Tra f f i ck ing |Vesic le Tra f f i ck ing V e s i c l e Tra f f i ck ing V e s i c l e T ra f f i ck ing V e s i c l e Tra f f i ck ing V e s i c l e Tra f f i ck ing V e s i c l e Tra f f i ck ing V e s i c l e Tra f f i ck ing V e s i c l e Tra f f i ck ing V e s i c l e Tra f f i ck ing V e s i c l e Tra f f i ck ing Ves i c l e Tra f f i ck ing S t ruc tu ra l S t r uc tu ra l S t r uc tu ra l S t r uc tu ra l S t r uc tu ra l S t r uc tu ra l S t r uc tu ra l S t r uc tu ra l S t r uc tu ra l S t r uc tu ra l S t r uc tu ra l S t r uc tu ra l S t r uc tu ra l S t r uc tu ra l S t r uc tu ra l S t r uc tu ra l S t r uc tu ra l S t r uc tu ra l S t r uc tu ra l Vesic le membrane fusion Vesic le membrane fusion Vesic le membrane fusion Calcium-dependent vesicule membrane fusion Vesic le endocytosis Vesic le membrane fusion Dynein-dependent vesicular/organelle transport Vesic le formation Vesicle formation Vesicle membrane fusion Photoreceptor synaptic vesicle trafficking (proposed) Disassembly of S N A R E s Vesicle membrane fusion Vesic le formation Vesic le trafficking (proposed) R O S morphogenesis R O S morphogenesis R O S morphogenesis R O S morphogenesis Photoreceptor structure Photoreceptor Component of Component of Component of (Component of Component of Component of Component of structure microtubuels microtubuels microtubuels microtubuels microtubuels microtubuels microtubuels Stabilization of microtubules Stabilization of microtubules Stabilization of microtubules Stabilization of microtubules Component of microfilaments [Actin polymerization 110 A c c e s s i o n # N a m e C a t e g o r y Function/Comments I P I 0 0 7 3 3 5 4 9 . I P I 0 0 7 1 4 4 5 4 . I P I 0 0 7 0 6 8 1 5 . I P I 0 0 7 1 5 6 9 0 . 1 I P I 0 0 7 3 3 6 2 7 . I P I 0 0 7 2 5 8 1 0 , I P I 0 0 7 3 2 4 1 7 . I P I 0 0 6 9 7 8 5 1 . I P I 0 0 7 0 0 4 7 1 . I P I 0 0 7 3 2 2 0 5 . 1 I P I 0 0 7 3 3 6 0 9 . 1 I P I 0 0 7 1 6 1 8 6 . 1 I P I 0 0 7 3 4 3 1 7 . 1 I P I 0 0 7 1 4 9 1 4 . 1 I P I 0 0 6 9 9 7 0 0 . 1 I P I 0 0 6 9 4 1 0 7 . I P I 0 0 7 1 6 3 1 4 . I P I 0 0 7 3 3 2 0 8 . I P I 0 0 7 1 8 2 7 1 . I P I 0 0 7 0 7 5 8 1 . I P I 0 0 7 0 0 7 9 9 . I P I 0 0 7 0 9 2 1 9 . I P I 0 0 7 2 9 3 1 7 . I P I 0 0 6 9 1 2 7 1 . 1 I P I 0 0 6 8 8 9 9 8 . 1 I P I 0 0 6 9 7 6 9 1 . 1 I P I 0 0 7 3 4 4 3 6 . 1 I P I 0 0 6 9 4 6 4 1 . I P I 0 0 7 1 1 3 6 8 . I P I 0 0 7 1 8 6 4 0 . I P I 0 0 6 9 8 5 7 0 . I P I 0 0 7 2 9 1 7 3 . s im i l a r to ac t in in , a lpha 4 i so fo rm 8 P R E D I C T E D : s im i l a r to A l p h a adduc in Fasc in h o m o l o g 1, ac t i n -bund l i ng pro te in Hypo the t i ca l p ro te in M G C 1 2 8 3 6 3 (s im i la r to |catenin ( c a d h e r i n - a s s o c i a t e d p ro te in ) , a l pha 1, 1 0 2 k D a ) P R E D I C T E D : s im i l a r to ca ten in ( c a d h e r i n -a s s o c i a t e d p ro te in ) , be ta 1, 8 8 k D a i so fo rm 9 P R E D I C T E D : s im i l a r to t ype II kera t in K b 9 P R E D I C T E D : s im i l a r to kera t in 4 i so fo rm 9 K e r a t i n , t y p e II cy toske le ta l 5 59 k D a pro te in (s im i la r to cy toke ra t i n t ype II) P R E D I C T E D : s im i l a r to K e r a t i n , t y p e I c y toske le ta l 14 (Cy toke ra t i n 14) ( K 1 4 ) (CK 14) i so fo rm 5 P R E D I C T E D : s im i l a r to kera t in c o m p l e x 2 , bas i c , g e n e 6a i so fo rm 3 81 k D a pro te in ( P R E D I C T E D : s im i l a r to Ge lso l i n ) P R E D I C T E D : s im i l a r to Spec t r i n a l pha c h a i n , bra in ( S p e c t r i n , non -e ry th ro i d a l pha cha in ) (A lpha- I I spec t r in ) i so fo rm 9 2 7 2 k D a pro te in (Spec t r i n beta) Co f i l i n -1 Prof i l in -1 164 k D a pro te in ( P R E D I C T E D : s im i l a r to M y o s i n -5A ( M y o s i n V a ) (Di lu te m y o s i n h e a v y c h a i n , n o n - m u s c l e ) P R E D I C T E D : s im i l a r to m y o s i n , h e a v y po l ypep t i de 14 i so fo rm 3 m y o s i n , l ight pep t ide 6 , a l ka l i , s m o o t h musc le and n o n - m u s c l e P R E D I C T E D : s im i l a r to M y o s i n - 6 P R E D I C T E D : s im i l a r to M y o s i n - 6 (Myos in V I ) , par t ia l M y o s i n - 1 0 P R E D I C T E D : s im i l a r to d y n e i n , c y t o p l a s m i c , h e a v y po l ypep t i de 1 i so fo rm 1 2 0 3 k D a pro te in (K ines in fami l y m e m b e r I B ) 'T rans fo rm ing pro te in R h o A p r e c u r s o r P R E D I C T E D : s im i l a r to B a n d 4 .1 - l i ke pro te in 2 ( G e n e r a l l y e x p r e s s e d pro te in 4 .1 ) ( 4 . 1 G ) i so fo rm 1 P R E D I C T E D : s im i l a r to B a n d 4 .1 - l i ke pro te in 3 ( 4 . I B ) (Di f ferent ia l ly e x p r e s s e d in i a d e n o c a r c i n o m a of the lung pro te in 1) ( D A L - 1 ) ( D A L 1 P ) ( m D A L - 1 ) i so fo rm 7 Ezr in Hypo the t i ca l p ro te in ( P R E D I C T E D : s im i la r to r ad i x i n , par t ia l ) P R E D I C T E D : s im i la r to ta l in 2 P R E D I C T E D : s im i la r to c i l iary root let co i l ed -co i l , root le t in P R E D I C T E D : s im i la r to c i l iary root le t co i l ed -co i l , root le t in S t r u c t u r a S t r u c t u r a S t r u c t u r a S t r u c t u r a S t r u c t u r a S t r u c t u r a S t r u c t u r a S t r u c t u r a S t r u c t u r a S t r u c t u r a S t r u c t u r a S t r u c t u r a S t r u c t u r a S t r u c t u r a S t r u c t u r a S t r u c t u r a S t r u c t u r a S t r u c t u r a S t r u c t u r a S t r u c t u r a S t r u c t u r a S t r u c t u r a S t r u c t u r a S t r u c t u r a S t r u c t u r a S t r u c t u r a S t r u c t u r a S t r u c t u r a S t r u c t u r a S t r u c t u r a S t r u c t u r a S t r u c t u r a Actin polymerization Assembly of actin-spectrin network Actin polymerization Cell adhesion by association with [actin filaments and cadherins Component of cell adheren junction Component of intermediate filaments Component of intermediate filaments Component of intermediate filaments Component of intermediate filaments Component of intermediate filaments Component of intermediate filaments Regulation of actin cytoskeleton Movement of cytoskeleton at the membrane Movement of cytoskeleton at the membrane Regulation of actin cytoskeleton Regulation of actin cytoskeleton Actin-based motor protein Actin-based motor protein Actin-based motor protein Actin-based motor protein Actin-based motor protein Actin-based motor protein Microtubule-based motor protein Microtubule-based motor protein Regulation of myosin and actin [cytoskeleton Component of spectrin-based cytoskeleton Component of spectrin-based cytoskeleton Links actin cytoskeleton to P M Links actin cytoskeleton to P M Links actin cytoskeleton to integrin Component of rootlet Component of rootlet 111 A c c e s s i o n # N a m e C a t e g o r y Function/Comments I P I 0 0 7 2 9 0 6 7 . 1 P R E D I C T E D : s im i l a r to c i l ia ry root le t c o i l e d - c o i l , root le t in S t r uc tu ra l Component of rootlet I P I 0 0 7 0 5 2 6 5 . 2 P R E D I C T E D : s im i l a r to B a r d e t - B i e d l s y n d r o m e 1 S t ruc tu ra l R e s p o n s i b l e fo r Ba rde t -B ied l s y n d r o m e I P I 0 0 7 1 6 1 0 5 . 1 Hypo the t i ca l p ro te in ( B a r d e t - B i e d l s y n d r o m e 2) S t r uc tu ra l R e s p o n s i b l e for B a r d e t - B i e d l s y n d r o m e I P I 0 0 7 2 8 1 8 6 . 1 P R E D I C T E D : s im i l a r to A D P - r i b o s y l a t i o n fac to r -l ike pro te in 6 i so fo rm 1 ( B B S 3 ) S t r uc tu ra l R e s p o n s i b l e fo r Ba rde t -B ied l s y n d r o m e I P I 0 0 7 1 2 1 9 2 . 2 P R E D I C T E D : s im i l a r to B a r d e t - B i e d l s y n d r o m e 4 pro te in S t r uc tu ra l R e s p o n s i b l e fo r Ba rde t -B ied l s y n d r o m e I P I 0 0 7 1 0 1 2 9 . 1 P R E D I C T E D : s im i la r to B a r d e t - B i e d l s y n d r o m e 5 i so fo rm 1 S t ruc tu ra l R e s p o n s i b l e fo r B a r d e t - B i e d l s y n d r o m e I P I 0 0 6 9 6 6 4 8 . 1 P R E D I C T E D : s im i l a r to B a r d e t - B i e d l s y n d r o m e 7 p ro te in i so fo rm a , par t ia l S t ruc tu ra l R e s p o n s i b l e for B a r d e t - B i e d l s y n d r o m e I P I 0 0 6 9 7 5 7 3 . 2 P R E D I C T E D : s im i l a r to pa ra thy ro id h o r m o n e -r e s p o n s i v e B l g e n e i so fo rm 2 ( B B S 9 ) S t ruc tu ra l R e s p o n s i b l e for B a r d e t - B i e d l s y n d r o m e I P I 0 0 6 9 0 7 5 1 . 2 P R E D I C T E D : s im i l a r to anky r i n repea t d o m a i n 3 3 P R E D I C T E D : s im i l a r to S o d i u m / p o t a s s i u m -S t ruc tu ra l Unknown function I P I 0 0 7 0 5 1 5 9 . 2 t r anspo r t i ng A T P a s e a lpha -1 cha in p r e c u r s o r ( S o d i u m p u m p 1) ( N a + / K + A T P a s e 1) , par t ia l T r a n s p o r t Ion transport across P M I P I 0 0 7 3 3 5 0 4 . 1 P R E D I C T E D : s im i l a r to N a + / K + - A T P a s e a lpha 3 subun i t i so fo rm 6 T r a n s p o r t Ion transport across P M I P I 0 0 7 0 4 0 8 2 . 1 S o d i u m / p o t a s s i u m - t r a n s p o r t i n g A T P a s e b e t a - 2 cha in T r a n s p o r t Ion transport across P M I P I 0 0 7 1 4 6 5 6 . 1 S o l u t e ca r r i e r f am i l y 2 , fac i l i ta ted g l ucose t r a n s p o r t e r m e m b e r 1 T r a n s p o r t Glucose transport across P M I P I 0 0 6 8 5 1 9 6 . 1 S o l u t e ca r r i e r f am i l y 3 (Ac t i va to rs of d i bas i c a n d neut ra l a m i n o ac id t r anspo r t ) , m e m b e r 2 T r a n s p o r t Neutral amino acid transporter I P I 0 0 7 1 5 8 5 3 . 1 S o l u t e ca r r i e r f am i l y 16 m e m b e r 1 T r a n s p o r t Monocarboxylic acid transporter I P I 0 0 7 3 0 2 1 9 . 1 s im i l a r to so lu te ca r r i e r f a m i l y ' 2 5 (m i tochondr ia l ca r r ie r , A r a l a r ) , m e m b e r 12 i so fo rm 4 T r a n s p o r t Calcium-dependent mitochondrial solute carrier I P I 0 0 7 4 4 3 8 0 . 1 73 k D a pro te in (so lu te car r ie r fami l y 4 4 , m e m b e r 1) T r a n s p o r t Choline transporter (proposed) I P I 0 0 7 1 1 4 7 1 . 1 Sp l i ce I so fo rm 2 of V a c u o l a r p ro ton t rans loca t i ng A T P a s e 116 k D a subun i t a i so fo rm 1 T r a n s p o r t Acidification of intracellular compartments I P I 0 0 6 8 6 0 7 4 . 1 A T P a s e , H+ t r a n s p o r t i n g , l y s o s o m a l (Vacuo la r p ro ton p u m p ) , a lpha po l ypep t i de , 7 0 k D , i so fo rm 1 T r a n s p o r t Acidification of intracellular compartments I P I 0 0 6 8 8 5 2 2 . 1 V a c u o l a r A T P s y n t h a s e subun i t B, bra in i so fo rm T r a n s p o r t Acidifcation of intracellular compartments I P I 0 0 7 1 0 1 0 1 . 1 V a c u o l a r A T P s y n t h a s e subun i t E T r a n s p o r t Acidifcation of intracellular compartments I P I 0 0 7 1 3 7 5 6 . 1 P l a s m a m e m b r a n e c a l c i u m - t r a n s p o r t i n g A T P a s e T r a n s p o r t Ion transport across P M I P I 0 0 7 1 1 4 4 1 . 1 s im i l a r to Neu t ra l a m i n o ac id t r anspo r te r A T r a n s p o r t Neutral amino acid transporter I P I 0 0 6 8 6 3 3 1 . 1 Exc i t a to ry a m i n o ac id t r anspo r te r 1 T r a n s p o r t Sodium-dependent glutamate/aspartate transporter I P I 0 0 6 9 6 2 5 7 . 1 P R E D I C T E D : s im i l a r to g a m m a - a m i n o b u t y r i c ac id ( G A B A - A ) t r a n s p o r t e r 4 , par t ia l T r a n s p o r t Neurotransmitter transport I P I 0 0 7 0 3 1 2 9 . 2 V o l t a g e - d e p e n d e n t an ion -se lec t i ve channe l p ro te in 1 T r a n s p o r t Ion transport I P I 0 0 6 8 7 2 4 6 . 2 V o l t a g e - d e p e n d e n t an ion -se lec t i ve channe l p ro te in 2 T r a n s p o r t Ion transport I P I 0 0 7 0 8 5 8 2 . 1 V o l t a g e - d e p e n d e n t a n i o n - s e l e c t i v e channe l p ro te in 3 T r a n s p o r t Ion transport I P I 0 0 7 1 5 2 1 1 . 2 P R E D I C T E D : s im i l a r to S y n a p t i c ves ic le m e m b r a n e pro te in V A T - 1 h o m o l o g T r a n s p o r t Vesic le amine transport I P I 0 0 6 8 9 7 8 9 . 1 P R E D I C T E D : s im i l a r to ves i c le a m i n e t r anspo r t p ro te in 1 i so fo rm 1 T r a n s p o r t Vesic le amine transport I P I 0 0 7 0 8 9 8 6 . 2 P R E D I C T E D : s im i l a r to ion t r a n s p o r t e r p ro te in T r a n s p o r t Ion transport A c c e s s i o n # N a m e C a t e g o r y Function/Comments I P I 0 0 7 1 2 8 5 8 . 1 A D P / A T P t r a n s l o c a s e 2 T r a n s p o r t Nucleotide transport I P I 0 0 7 0 5 3 7 8 . 1 A D P / A T P t r ans l ocase 3 P R E D I C T E D : s im i l a r to A T P a s e , T r a n s p o r t Nucleotide transport I P I 0 0 7 2 1 0 9 1 . 1 a m i n o p h o s p h o l i p i d t r anspo r t e r - l i ke , c l ass I, t y p e 8 A , m e m b e r 2 , par t ia l T r a n s p o r t Phospholipid transport (proposed) I P I 0 0 7 0 9 6 8 9 . 2 P R E D I C T E D : s im i l a r to Potent ia l p h o s p h o l i p i d -t r anspo r t i ng A T P a s e IB T r a n s p o r t Phospholipid transport (proposed) I P I 0 0 6 8 6 9 4 3 . 1 P R E D I C T E D : s im i la r to C D W 9 2 an t i gen i so fo rm 2 , par t ia l T r a n s p o r t Choline transporter (proposed) I P I 0 0 7 1 3 4 9 6 . 1 P R E D I C T E D : s im i la r to A T P - b i n d i n g c a s s e t t e , s u b - f a m i l y A , m e m b e r 7 i so fo rm a i so fo rm 1 T r a n s p o r t Unknown function I P I 0 0 6 9 7 2 9 0 . 1 1 4 - 3 - 3 p ro te in b e t a / a l p h a H o u s e - k e e p i n g Signal transduction I P I 0 0 6 9 6 4 3 5 . 1 1 4 - 3 - 3 pro te in eps i lon H o u s e - k e e p i n g Signal transduction I P I 0 0 7 0 3 1 1 0 . 1 1 4 - 3 - 3 pro te in z e t a / d e l t a H o u s e - k e e p i n g Signal transduction I P I 0 0 6 9 5 4 4 8 . 1 1 4 - 3 - 3 pro te in e ta H o u s e - k e e p i n g Signal transduction I P I 0 0 6 9 3 1 0 6 . 1 1 4 - 3 - 3 pro te in g a m m a H o u s e - k e e p i n g Signal transduction I P I 0 0 7 1 1 1 7 2 . 2 P R E D I C T E D : s im i l a r to 1 4 - 3 - 3 prote in the ta H o u s e - k e e p i n g Signal transduction I P I 0 0 7 1 8 2 6 1 . 1 Ret ina l rod r h o d o p s i n - s e n s i t i v e c G M P 3 ' , 5 ' - cyc l i c p h o s p h o d i e s t e r a s e de l t a - subun i t H o u s e - k e e p i n g Regulation of prenylated proteins I P I 0 0 7 3 2 7 0 3 . 1 P R E D I C T E D : s im i l a r to S K B 1 h o m o l o g i so fo rm 11 H o u s e - k e e p i n g s n R N P assembly I P I 0 0 6 9 0 3 6 7 . 1 Hypo the t i ca l p ro te in M G C 1 2 6 9 5 1 (Ras re la ted v - H o u s e - k e e p i n g Signal transduction ral s i m i a n l e u k e m i a v i ra l o n c o g e n e h o m o l o g A ) I P I 0 0 7 2 2 8 1 1 . 1 P R E D I C T E D : s im i l a r to R a s - r e l a t e d prote in R a l -B, par t ia l H o u s e - k e e p i n g Signal transduction I P I 0 0 7 0 9 1 1 7 . 2 P R E D I C T E D : s im i l a r to R a s - r e l a t e d prote in M -R a s H o u s e - k e e p i n g Signal transduction I P I 0 0 7 0 0 4 5 3 . 1 P R E D I C T E D : s im i l a r to c - K - r a s 2 pro te in i so fo rm b H o u s e - k e e p i n g Signal transduction I P I 0 0 7 0 3 6 6 5 . 1 R a s - r e l a t e d pro te in R a p - I A p recu rso r H o u s e - k e e p i n g Signal transduction I P I 0 0 6 9 5 7 7 6 . 1 R a s - r e l a t e d pro te in R a p - l b p r e c u r s o r H o u s e - k e e p i n g Signal transduction I P I 0 0 6 9 9 2 9 6 . 1 7 5 k D a pro te in (s im i la r to R a s G A P - a c t i v a t i n g -l ike pro te in 1) P R E D I C T E D : s im i l a r to Ras G T P a s e - a c t i v a t i n g H o u s e - k e e p i n g Signal transduction I P I 0 0 7 2 8 1 3 3 . 1 pro te in 4 ( R a s G A P - a c t i v a t i n g - l i k e pro te in 2) i so fo rm 6 H o u s e - k e e p i n g Signal transduction I P I 0 0 7 2 0 7 6 1 . 1 P R E D I C T E D : s im i l a r to G T P a s e N R a s ( T r a n s f o r m i n g pro te in N -Ras ) i so fo rm 3 H o u s e - k e e p i n g Signal transduction I P I 0 0 6 8 9 2 2 9 . 1 R a s - r e l a t e d C 3 bo tu l i num tox in subs t ra te 1 p r e c u r s o r ( R a c l ) H o u s e - k e e p i n g Membrane fusion, apotosis (proposed) I P I 0 0 7 1 3 8 1 5 . 1 Hypo the t i ca l p ro te in ( P R E D I C T E D : s im i l a r to r hoB gene ) H o u s e - k e e p i n g Intracellular protein transport, cell adhesion I P I 0 0 7 0 5 9 4 1 . 1 R A N , m e m b e r R A S o n c o g e n e fami l y H o u s e - k e e p i n g Nuclear protein export, cell cycle regulation I P I 0 0 7 0 6 0 0 2 . 1 A n n e x i n A 2 H o u s e - k e e p i n g Membrane fusion, links cytoskeleton to P M I P I 0 0 6 8 6 9 8 1 . 1 A n n e x i n A 4 H o u s e - k e e p i n g Membrane fusion, exocytosis I P I 0 0 6 8 8 6 4 9 . 2 P R E D I C T E D : s im i l a r to A n n e x i n A 6 H o u s e - k e e p i n g Signal transduction, retinoid-binding I P I 0 0 7 3 3 7 6 9 . 1 P R E D I C T E D : s im i l a r to a n n e x i n VII i so fo rm 2 i so fo rm 3 H o u s e - k e e p i n g Membrane fusion, exocytosis I P I 0 0 6 8 9 4 3 6 . 2 P R E D I C T E D : s im i l a r to es te rase D / fo rmy lg l u ta th i one hyd ro lase i so fo rm 1 H o u s e - k e e p i n g Esterase I P I 0 0 6 9 6 7 9 3 . 2 P R E D I C T E D : s im i la r to Th io redox in - l i ke pro te in 1 H o u s e - k e e p i n g Protein disulfide oxidoreductase (proposed) P R E D I C T E D : s im i l a r to t h i o redox in - l i ke 6 H o u s e - k e e p i n g Protein disulfide oxidoreductase I P I 0 0 7 1 3 1 1 5 . 1 (proposed) I P I 0 0 6 8 7 1 3 0 . 2 P R E D I C T E D : s im i l a r to A P 2 a s s o c i a t e d k i nase 1 H o u s e - k e e p i n g Regulation of clathrin-mediated endocytosis 113 A c c e s s i o n # N a m e C a t e g o r y Function/Comments I P I 0 0 6 9 5 2 0 8 . 1 Pro te in k i n a s e C a lpha t y p e H o u s e k e e p i n g Signal transduction I P I 0 0 6 9 1 4 0 7 . 1 Pro te in k i n a s e C be ta t ype House- k e e p i n g Signal transduction I P I 0 0 7 3 3 0 3 8 . 1 P R E D I C T E D : s im i l a r to pro te in k i nase C , de l ta i so fo rm 3 House- k e e p i n g Signal transduction I P I 0 0 7 1 3 1 8 5 . 1 3 2 k D a pro te in ( P R E D I C T E D : s im i l a r to Prote in k i n a s e C , eps i l on t ype ) H o u s e k e e p i n g Signal transduction I P I 0 0 6 8 8 0 5 4 . 1 81 k D a pro te in (s im i la r to Prote in k i nase C , nu t y p e ( n P K C - n u ) (Pro te in k inase E P K 2 ) ) H o u s e k e e p i n g Signal transduction I P I 0 0 6 9 9 7 0 8 . 1 6 5 k D a pro te in (Pro te in k i nase C a lpha t ype ) H o u s e k e e p i n g Signal transduction I P I 0 0 7 1 3 6 7 6 . 2 P R E D I C T E D : s im i l a r to Prote in k i nase C and case in k i n a s e subs t ra te in n e u r o n s pro te in 1 H o u s e k e e p i n g Vesic le formation and trafficking (proposed) I P I 0 0 7 1 7 6 5 5 . 1 2 ' , 3 ' - c yc l i c - nuc l eo t i de 3 ' - p h o s p h o d i e s t e r a s e H o u s e k e e p i n g Myelin-associated protein, microtubule assembly I P I 0 0 7 1 3 5 0 5 . 1 Prepro c o m p l e m e n t c o m p o n e n t C 3 p recu rso r P R E D I C T E D : s im i l a r to P r o t e a s o m e subun i t H o u s e k e e p i n g Immune response I P I 0 0 7 3 2 5 6 6 . 1 a l pha t ype 6 ( P r o t e a s o m e iota cha in ) (Mac ropa in iota cha in ) (Mu l t i ca ta ly t i c e n d o p e p t i d a s e c o m p l e x iota cha in) i so fo rm 3 H o u s e k e e p i n g Component of proteasome I P I 0 0 6 9 5 0 7 8 . 1 Hypo the t i ca l p ro te in M G C 1 2 6 9 2 9 (S im i l a r to P r o t e a s o m e subun i t a l pha t ype 7) H o u s e k e e p i n g Component of proteasome I P I 0 0 7 1 3 9 2 3 . 1 P R E D I C T E D : s im i l a r to p r e - B - c e l l l e u k e m i a H o u s e k e e p i n g Unknown function t ransc r ip t i on fac to r in te rac t ing pro te in 1 I P I 0 0 7 0 0 1 1 2 . 1 Fat ty ac i d -b i nd ing p ro te in , bra in H o u s e keep ing C N S development I P I 0 0 6 8 9 6 4 6 . 1 ce l lu la r re t ino ic ac id b ind ing pro te in 1 H o u s e - keep ing Retinoid carrier I P I 0 0 6 8 6 8 0 3 . 1 Hypo the t i ca l p ro te in M G C 1 2 7 2 5 7 (S im i l a r to r i bonuc lease U K 1 1 4 ) H o u s e - keep ing Translation regulation I P I 0 0 6 9 6 0 1 1 . 1 M i t o g e n - a c t i v a t e d p ro te in -b ind ing p ro te in -in te rac t ing pro te in H o u s e - keep ing Signal transduction I P I 0 0 7 0 4 2 3 2 . 1 Leuc ine z i p p e r t ransc r ip t i on fac to r - l i ke 1 H o u s e - keep ing Unknown function I P I 0 0 3 7 7 3 5 1 . 2 Apo l i pop ro te i n A - I V p recu rso r H o u s e - keep ing Lipid carrier (proposed) I P I 0 0 7 1 3 7 8 0 . 3 Hypo the t i ca l p ro te in ( P R E D I C T E D : s im i l a r to Apo l i pop ro te i n D p recu rso r ( A p o - D ) ) H o u s e - keep ing Lipid carrier (proposed) I P I 0 0 6 9 7 1 2 9 . 1 10 k D a pro te in ( A c y l - C o A - b i n d i n g pro te in) H o u s e - keep ing Acy l -CoA carrier, regulation of neurotransmission (proposed) I P I 0 0 7 1 6 7 1 6 . 1 P R E D I C T E D : s im i l a r to A l a n y l - t R N A s y n t h e t a s e H o u s e - keep ing Translation I P I 0 0 6 9 3 0 1 8 . 1 G l y c y l - t r a n s y n t h e t a s e (F ragmen t ) H o u s e - keep ing Translation I P I 0 0 7 0 4 3 2 5 . 1 W A R S pro te in ( t r y p t o p h a n y l - t R N A s y n t h e t a s e ) H o u s e - keep ing Translation I P I 0 0 7 1 1 4 5 5 . 1 Hypo the t i ca l p ro te in M G C 1 2 8 1 4 7 (s im i la r to t h r e o n y l - t R N A s y n t h e t a s e ) H o u s e - keep ing Translation I P I 0 0 6 8 6 0 9 2 . 1 P e r o x i r e d o x i n - 1 H o u s e - keep ing Cellular redox regulation I P I 0 0 7 1 3 1 1 2 . 1 P e r o x i r e d o x i n - 2 H o u s e - keep ing Cellular redox regulation I P I 0 0 6 8 9 8 5 7 . 1 P e r o x i r e d o x i n - 6 M G C 1 2 8 5 7 6 pro te in ( P R E D I C T E D : s im i la r to H o u s e - keep ing Cellular redox regulation I P I 0 0 6 8 8 6 5 1 . 3 C a l c i u m / c a l m o d u l i n - d e p e n d e n t pro te in k i nase t ype II de l ta cha in ( C a M - k i n a s e II de l ta cha in ) H o u s e - keep ing Oxidative stress response, neuronal plasticity ( C a M k i n a s e II de l ta subun i t ) ( C a M K - I I de l ta subun i t ) ) I P I 0 0 7 1 6 1 2 1 . 1 P i g m e n t e p i t h e l i u m - d e r i v e d fac to r p recu rso r H o u s e - keep ing Neuron differentiation I P I 0 0 6 9 2 9 1 1 . 2 P R E D I C T E D : s im i l a r to ac id ic ( leuc ine- r i ch ) nuc lea r p h o s p h o p r o t e i n 3 2 fam i l y , m e m b e r A Hypo the t i ca l p ro te in M G C 1 2 8 0 7 4 (Ac id ic H o u s e - keep ing Multifunctional intracellular signaling I P I 0 0 6 9 9 0 0 2 . 1 Leuc ine - r i ch Nuc lea r Phosphop ro te i n 32 Fami l y M e m b e r B) H o u s e - keep ing Cell cycle progression I P I 0 0 7 0 1 0 2 3 . 2 P R E D I C T E D : s im i l a r to ac id ic ( leuc ine- r i ch ) nuc lea r phosphop ro te i n 32 fam i l y , m e m b e r E H o u s e - keep ing Protein phosphatase 2A regulation I P I 0 0 7 0 2 8 5 8 . 1 10 k D a heat s h o c k p ro te in , m i tochondr ia l H o u s e - keep ing Chaperone I P I 0 0 7 0 4 8 3 6 . 1 Hea t shock 2 7 k D a pro te in 1 H o u s e - keep ing Chaperone, actin/microtubules organization 114 A c c e s s i o n # N a m e C a t e g o r y Function/Comments H o u s e - k e e p i n g Chaperone H o u s e - k e e p i n g Chaperone H o u s e - k e e p i n g Chaperone H o u s e - k e e p i n g Chaperone H o u s e - k e e p i n g Chaperone H o u s e - k e e p i n g Chaperone H o u s e - k e e p i n g Chaperone H o u s e - k e e p i n g Chaperone H o u s e - k e e p i n g Chaperone H o u s e - k e e p i n g Chaperone H o u s e - k e e p i n g Chaperone H o u s e - k e e p i n g Regulation of Hsp chaperones H o u s e - k e e p i n g Assembly of Hsp chaperones H o u s e - k e e p i n g Chaperone H o u s e - k e e p i n g Chaperone H o u s e - k e e p i n g Chaperone H o u s e - k e e p i n g Chaperone H o u s e - k e e p i n g Chaperone H o u s e - k e e p i n g Chaperone H o u s e - k e e p i n g Chaperone H o u s e - k e e p i n g Chaperone H o u s e - k e e p i n g Oxidative phosphorylation H o u s e - k e e p i n g Oxidative phosphorylation H o u s e - k e e p i n g Oxidative phosphorylation H o u s e - k e e p i n g Oxidative phosphorylation H o u s e - k e e p i n g Oxidative phosphorylation H o u s e - k e e p i n g Oxidative phosphorylation H o u s e - k e e p i n g Neuronal calcium sensor H o u s e - k e e p i n g Protease inhibitor H o u s e - k e e p i n g Protease inhibitor H o u s e - k e e p i n g Vesicular trafficking from T E R I P I 0 0 6 9 1 8 5 0 . 1 I P I 0 0 7 1 1 0 3 1 . I P I 0 0 7 3 2 8 0 7 . I P I 0 0 7 0 8 3 4 2 . 1 I P I 0 0 7 3 1 9 5 6 . 1 I P I 0 0 6 9 2 2 4 7 . I P I 0 0 7 0 8 5 2 6 . I P I 0 0 0 3 7 0 7 0 . I P I 0 0 6 9 9 6 2 2 . I P I 0 0 7 0 9 4 3 5 . I P I 0 0 7 0 5 4 7 7 . 1 I P I 0 0 7 3 4 2 9 3 . 1 I P I 0 0 7 1 7 6 8 5 . 1 I P I 0 0 6 9 8 5 6 8 . I P I 0 0 7 1 8 3 8 3 , I P I 0 0 7 3 3 2 1 5 . 1 I P I 0 0 6 8 9 3 7 7 . 1 I P I 0 0 6 8 6 5 4 6 . I P I 0 0 7 1 0 7 2 6 . I P I 0 0 7 1 2 1 3 4 . I P I 0 0 7 1 4 6 2 4 . I P I 0 0 6 9 4 2 9 5 . I P I 0 0 7 1 7 8 8 4 . I P I 0 0 7 1 2 2 5 2 . I P I 0 0 7 2 7 3 1 4 . I P I 0 0 7 0 3 4 6 8 . I P I 0 0 6 9 1 8 2 7 . I P I 0 0 7 0 3 5 9 2 . I P I 0 0 7 0 0 6 2 2 , I P I 0 0 7 2 7 7 1 2 . I P I 0 0 7 1 0 7 2 7 , P R E D I C T E D : s im i l a r to 6 0 k D a hea t shock p ro te i n , m i t ochond r i a l p recu rso r ( H s p 6 0 ) (60 k D a chape ron in ) ( C P N 6 0 ) (Heat s h o c k pro te in 60 ) ( H S P - 6 0 ) (M i tochondr ia l ma t r i x p ro te in P I ) (P60 l y m p h o c y t e pro te in) ( H u C H A 6 0 ) i so fo rm 1 Hea t s h o c k 7 0 k D a pro te in I A P R E D I C T E D : s im i l a r to heat shock 7 0 k D a pro te in 4 i so fo rm a i so fo rm 1 P R E D I C T E D : s im i l a r to hea t shock 7 0 k D a pro te in 6 P R E D I C T E D : s im i l a r to Heat s h o c k 70 k D a pro te in 4 L ( O s m o t i c s t ress prote in 9 4 ) (Hea t s h o c k 7 0 - r e l a t e d pro te in A P G - 1 ) i so fo rm 4 Hea t shock 7 0 k D a pro te in 9 B Hea t shock c o g n a t e 71 kDa pro te in I so fo rm 2 of Hea t shock c o g n a t e 71 k D a prote in 9 0 - k D a hea t s h o c k pro te in a lpha 9 0 - k D a hea t s h o c k prote in beta P R E D I C T E d : s im i l a r to H e a t - s h o c k prote in 105 k D a (hea t shock H O k D a pro te in) (An t igen NY-C O - 2 5 ) i so fo rm 1 P R E D I C T E D : s im i l a r to heat s h o c k 7 0 k D pro te in b ind ing pro te in Hypo the t i ca l p ro te in M G C 1 2 8 1 1 0 (s im i la r to S t r e s s - i n d u c e d - p h o s p h o p r o t e i n 1 (STI1 ) ( H s c 7 0 / H s p 9 0 - o r g a n i z i n g pro te in) (Hop) ( T r a n s f o r m a t i o n - s e n s i t i v e prote in IEF S S P 3 5 2 1 ) ) P R E D I C T E D : s im i l a r to T B C 1 d o m a i n f am i l y , m e m b e r 10A Hypo the t i ca l p ro te in (s im i la r to T - c o m p l e x pro te in 1, a l pha subun i t ) P R E D I C T E D : s im i l a r to T - c o m p l e x pro te in 1, e ta subun i t ( T C P - l - e t a ) ( C C T - e t a ) (H IV -1 Nef in te rac t ing pro te in) i so fo rm 15 Hypo the t i ca l p ro te in M G C 1 2 8 0 9 6 (s im i la r to T-c o m p l e x pro te in 1, ze ta subun i t ( T C P - l - z e t a ) ( C C T - z e t a ) ( C C T - z e t a - 1 ) ) Hypo the t i ca l p ro te in M G C 1 2 8 8 5 2 (s im i la r to c h a p e r o n i n con ta in ing T C P 1 , subun i t 2) C h a p e r o n i n con ta in ing T C P 1 , subun i t 8 Hypo the t i ca l p ro te in ( chaperon in con ta in ing T C P 1 , subun i t 4 (del ta)) C C T 3 pro te in |ATP s y n t h a s e a lpha cha in hear t i s o f o r m , m i tochond r i a l p recu rso r |ATP s y n t h a s e beta c h a i n , m i tochondr ia l p r e c u r s o r |ATP s y n t h a s e D c h a i n , m i tochondr ia l P R E D I C T E D : s im i l a r to A T P s y n t h a s e a lpha c h a i n , m i t ochond r i a l p recu rso r i so fo rm 1 8 k D a pro te in (ATP s y n t h a s e e c h a i n , m i tochondr ia l ) S p l i c e I so fo rm L ive r of A T P s y n t h a s e g a m m a c h a i n , m i t ochond r i a l p recu rso r P R E D I C T E D : s im i l a r to h ippoca lc in - l i ke 1 i so fo rm 1 E n d o p i n - 1 p recu rso r P R E D I C T E D : s im i l a r to A l p h a - 1 -a n t i c h y m o t r y p s i n p r e c u r s o r Hypo the t i ca l p ro te in M G C 1 2 8 1 4 2 (Trans i t iona l e n d o p l a s m i c re t i cu lum A T P a s e ) 115 A c c e s s i o n # N a m e C a t e g o r y Function/Comments P R E D I C T E D : s im i l a r to 5 - a m i n o i m i d a z o l e - 4 -I P I 0 0 7 3 2 4 1 0 . 1 c a r b o x a m i d e r ibonuc leo t ide f o r m y l t r a n s f e r a s e / I M P c y c l o h y d r o l a s e , par t ia l House- k e e p i n g Purine biosynthesis I P I 0 0 7 3 3 2 5 6 . 1 P R E D I C T E D : s im i l a r to P u r o m y c i n - s e n s i t i v e a m i n o p e p t i d a s e ( P S A ) i so fo rm 2 H o u s e k e e p i n g Proteolysis, cell growth I P I 0 0 6 9 6 8 2 4 . 1 T h i o r e d o x i n - d e p e n d e n t pe rox ide r e d u c t a s e , m i tochond r i a l p recu rso r H o u s e k e e p i n g Cellular redox regulation I P I 0 0 6 9 6 0 2 1 . 1 Sp l i ce I so fo rm C S P 1 of DnaJ h o m o l o g sub fam i l y C m e m b e r 5 H o u s e k e e p i n g Synaptic transmission I P I 0 0 7 1 7 7 0 4 . 1 H i s t o n e - b i n d i n g pro te in R B B P 4 H o u s e k e e p i n g Transcription regulation I P I 0 0 7 0 9 4 0 3 . 1 H i s t o n e - b i n d i n g pro te in R B B P 7 ( re t i nob las toma b ind ing pro te in 7) P R E D I C T E D : s im i l a r to C h a r g e d mu l t i ves i cu la r H o u s e k e e p i n g Transcription regulation I P I 0 0 7 3 3 0 5 5 . 1 body pro te in 4b ( C h r o m a t i n mod i f y ing pro te in 4b ) ( C H M P 4 b ) i so fo rm 1 H o u s e k e e p i n g Component of ESCRT-III complex I P I 0 0 7 0 5 9 3 3 . 1 P R E D I C T E D : s im i la r to ch roma t i n mod i f y i ng pro te in 6 H o u s e k e e p i n g Component of ESCRT-III complex I P I 0 0 0 1 7 3 7 3 . 1 Rep l i ca t ion prote in A 14 k D a subun i t H o u s e k e e p i n g DNA replication/repair I P I 0 0 7 1 3 6 3 2 . 1 Hypo the t i ca l p ro te in (s im i la r to Ub iqu i t in - l i ke p ro te in 3 ( H C G - 1 pro te in) ) H o u s e keep ing Unknown function I P I 0 0 7 1 0 7 8 3 . 1 H e m o g l o b i n a lpha subun i t H o u s e •keep ing Oxygen carrier I P I 0 0 7 1 6 4 5 5 . 1 H e m o g l o b i n be ta subun i t H o u s e •keep ing Oxygen carrier I P I 0 0 7 1 7 6 3 8 . 1 Ferr i t in l ight cha in H o u s e •keep ing Iron homeostasis I P I 0 0 6 8 9 3 6 2 . 1 T rans thy re t i n p r e c u r s o r H o u s e •keep ing Thyroxine carrier I P I 0 0 0 1 9 5 6 8 . 1 P ro th romb in p r e c u r s o r (F ragmen t ) H o u s e •keep ing Peptidolysis, blood coagulation I P I 0 0 7 0 8 3 9 8 . 1 S e r u m a l b u m i n p recu rso r H o u s e •keep ing Osmotic pressure regulation I P I 0 0 7 1 6 1 1 6 . 1 R i b o s o m a l pro te in S 2 8 H o u s e - keep ing Component of ribosomes I P I 0 0 6 9 0 0 7 3 . 1 2 3 k D a pro te in ( 4 0 S r i bosoma l prote in S 3 ) H o u s e - keep ing Component of ribosomes I P I 0 0 6 9 4 2 4 3 . 2 s im i l a r to 4 0 S r i bosoma l pro te in S 7 H o u s e - keep ing Component of ribosomes I P I 0 0 7 3 0 2 6 9 . 1 s im i l a r to 4 0 S r i bosoma l pro te in S 4 , X i so fo rm H o u s e - keep ing Component of ribosomes I P I 0 0 7 0 3 2 1 7 . 1 21 k D a pro te in ( 6 0 S r i bosoma l prote in L9) H o u s e - keep ing Component of ribosomes I P I 0 0 7 2 4 0 2 4 . 1 s im i l a r to 6 0 S r i bosoma l pro te in L 2 3 a H o u s e - keep ing Component of ribosomes I P I 0 0 7 0 2 4 5 6 . 1 6 0 S ac id ic r i b o s o m a l pro te in P2 Hypo the t i ca l p ro te in ( P R E D I C T E D : s im i l a r to H o u s e - keep ing Component of ribosomes I P I 0 0 7 1 4 6 8 3 . 1 D ihyd rop te r i d i ne r educ tase ( H D H P R ) (Qu ino id d i hyd rop te r i d i ne reduc tase ) ) Hypo the t i ca l p ro te in M G C 1 2 7 3 2 5 ( P R E D I C T E D : H o u s e - keep ing Tetrahydrobiopterin biosynthesis I P I 0 0 6 9 4 0 8 2 . 1 s im i l a r to C N D P d ipep t i dase 2 (me ta l l opep t i dase M 2 0 fam i l y ) H o u s e - keep ing Nonspecif ic peptidolysis I P I 0 0 7 3 3 8 2 2 . 1 s im i l a r to h e m e b ind ing prote in 1 H o u s e - keep ing Removal of toxic compounds (proposed) I P I 0 0 7 3 1 2 0 2 . 1 s im i l a r to T I P 1 2 0 pro te in i so fo rm 1 H o u s e - keep ing Transscription regulation I P I 0 0 6 9 8 5 2 9 . 1 Bre fe ld in A - i nh ib i t ed g u a n i n e nuc leo t i de -e x c h a n g e pro te in 1 ( A R F G E P 1) H o u s e - keep ing Regulation of Arf G T P a s e s I P I 0 0 6 9 2 2 9 5 . 1 P la te le t -ac t i va t ing fac to r a c e t y l h y d r o l a s e IB beta subun i t H o u s e - keep ing Neuronal migration (proposed) I P I 0 0 6 9 4 0 4 0 . 2 P R E D I C T E D : s im i l a r to p r o g r a m m e d cel l dea th 6 in te rac t ing pro te in i so fo rm 1 H o u s e - keep ing Intracellular protein transport, apoptosis I P I 0 0 7 0 3 5 4 7 . 1 P R E D I C T E D : s im i l a r to Ca lb ind in H o u s e - keep ing Calcium-dependent signaling I P I 0 0 7 0 0 4 3 1 . 1 Hypo the t i ca l p ro te in M G C 1 2 8 3 1 8 (Ca l re t in in ) H o u s e - keep ing Calcium-dependent signaling I P I 0 0 7 1 2 7 3 9 . 2 Pro te in S 1 0 0 - B H o u s e - keep ing Calcium-dependent signaling I P I 0 0 7 0 8 1 8 9 . 2 P R E D I C T E D : s im i l a r to H is tone H I . 2 H o u s e - keep ing Transcription regulation I P I 0 0 7 3 0 9 5 0 . 1 P R E D I C T E D : s im i l a r to g e r m i n a l h i s tone H4 g e n e P R E D I C T E D : s im i l a r to ubiqui t in C i so fo rm 1 H o u s e - keep ing Transcription regulation I P I 0 0 7 3 3 7 3 4 . 1 H o u s e - keep ing Ubiquitin-dependent protein turnover I P I 0 0 7 0 5 8 1 5 . 1 Bra in ac id so lub le pro te in 1 H o u s e - keep ing Unknown, axonal membrane-associated I P I 0 0 6 9 8 6 7 3 . 1 5 ' - nuc l eo t i dase p recu rso r H o u s e - keep ing Nucleotide metabolism 116 A c c e s s i o n # N a m e C a t e g o r y Function/Comments P R E D I C T E D : s im i l a r to g u a n i n e nuc leo t i de -I P I 0 0 7 1 2 8 1 9 . 2 b ind ing p ro te in , be ta 2 i so fo rm 1 (T ransduc in be ta 2) P R E D I C T E D : s im i l a r to G u a n i n e nuc leo t i de -House - k e e p i n g Signal transduction I P I 0 0 7 1 1 5 3 7 . 1 b ind ing pro te in G ( i ) , a l p h a - 2 subun i t ( A d e n y l a t e cyc lase - i nh ib i t i ng G a lpha prote in) i so fo rm 1 House - k e e p i n g Signal transduction I P I 0 0 6 9 4 1 0 6 . 1 G u a n i n e nuc leo t i de -b ind ing prote in G ( I ) / G ( S ) / G ( 0 ) g a m m a - 7 subun i t p r e c u r s o r House - k e e p i n g Signal transduction I P I 0 0 7 1 8 4 8 0 . 2 P R E D I C T E D : s im i l a r to G u a n i n e nuc leo t i de -b ind ing pro te in G ( z ) , a lpha subun i t House - k e e p i n g Signal transduction I P I 0 0 7 1 1 4 7 9 . 1 T a r g e t of m y b l House - k e e p i n g Intracellular protein transport I P I 0 0 7 1 6 3 0 4 . 2 P R E D I C T E D : s im i l a r to ta rge t of m y b l - l i k e 2 House - k e e p i n g Intra-golgi protein transport I P I 0 0 6 5 6 3 2 9 . 1 K n g l p ro te in (K in i nogen 1) House - k e e p i n g Protease inhibitor, inflammation response I P I 0 0 7 0 6 0 9 4 . 1 A l p h a - S l - c a s e i n p recu rso r House - k e e p i n g Calcium phosphate carrier in milk I P I 0 0 6 9 8 8 4 3 . 1 A l p h a - S 2 - c a s e i n p recu rso r House - k e e p i n g Calc ium phosphate carrier in milk I P I 0 0 7 0 9 4 0 7 . 2 P R E D I C T E D : s im i l a r to e n d o p l a s m i c re t i cu lum pro te in 2 9 p recu rso r House - k e e p i n g Chaperone (proposed) I P I 0 0 7 0 9 4 6 5 . 1 Pro te in d i s u l f i d e - i s o m e r a s e p recu rso r House - k e e p i n g Disulfide bonds arrangement I P I 0 0 6 8 9 3 2 5 . 1 Pro te in d i s u l f i d e - i s o m e r a s e A 3 p recu rso r P R E D I C T E D : s im i l a r to Prote in d isu l f i de -House - k e e p i n g Disulfide bonds arrangement I P I 0 0 7 2 4 1 8 9 . 1 i s o m e r a s e A 4 p r e c u r s o r (Prote in E R p - 7 2 ) ( E R p 7 2 ) i so fo rm 2 House - k e e p i n g Disulfide bonds arrangement I P I 0 0 7 1 7 2 3 4 . 2 P R E D I C T E D : s im i l a r to 78 k D a g l u c o s e -regu la ted p ro te in p recu rso r House- k e e p i n g Chaperone I P I 0 0 7 2 5 9 3 0 . 1 P R E D I C T E D : s im i l a r to 78 k D a g l u c o s e -regu la ted pro te in p recu rso r House- keep ing Chaperone I P I 0 0 6 9 2 8 6 5 . 1 E n d o p l a s m i n p recu rso r House- k e e p i n g Chaperone I P I 0 0 7 0 9 6 6 5 . 1 P R E D I C T E D : s im i l a r to D - d o p a c h r o m e t a u t o m e r a s e House- k e e p i n g Cytokine I P I 0 0 6 9 4 1 4 2 . 2 M a c r o p h a g e m ig ra t i on inh ib i to ry fac to r House- k e e p i n g Cytokine I P I 0 0 7 1 0 5 1 5 . 1 Neura l cel l a d h e s i o n mo lecu le 1, 140 k D a i so fo rm p r e c u r s o r House- k e e p i n g Cell adhesion I P I 0 0 5 5 5 6 2 8 . 1 Neura l cel l a d h e s i o n mo lecu le 1, 120 k D a i so fo rm va r i an t ( F r a g m e n t ) House- k e e p i n g Cell adhesion I P I 0 0 6 9 6 3 2 5 . 1 Hypo the t i ca l p ro te in ( P R E D I C T E D : s im i l a r to bas ig in i so fo rm 2 i so fo rm 1 ) H o u s e k e e p i n g Neuronal development/maturation (proposed) I P I 0 0 7 1 1 2 1 9 . 1 P R E D I C T E D : s im i l a r to s t roma l cel l de r i ved fac to r recep to r 1 i so fo rm b i so fo rm 2 H o u s e k e e p i n g Chemokine receptor C X C R 4 I P I 0 0 7 3 2 7 4 7 . 1 P R E D I C T E D : s im i l a r to C H - T O G pro te in H o u s e •keep ing Cell cycle, spindle pole organization I P I 0 0 6 8 6 7 3 3 . 1 D P P 3 pro te in (d ipep t idy l pep t i dase III) P R E D I C T E D : s im i la r to Nuc leos ide d i p h o s p h a t e H o u s e k e e p i n g Proteolysis I P I 0 0 7 3 2 0 7 7 . 1 k i n a s e B ( N D K B) ( N D P k inase B) ( n m 2 3 - H 2 ) ( C - m y c pu r i ne -b i nd i ng t ranscr ip t ion fac to r PUF) i so fo rm 2 H o u s e k e e p i n g Nucleoside triphosphate biosynthesis, transcription regulation I P I 0 0 7 1 1 3 8 6 . 1 Nuc leos ide d i p h o s p h a t e k i nase N B R - B H o u s e - keep ing Nucleoside triphosphate biosynthesis, transcription regulation I P I 0 0 7 3 1 1 4 8 . 1 P R E D I C T E D : s im i l a r to X - l i n k e d j uven i l e re t inosch is i s p ro te in , par t ia l H o u s e - keep ing Cell adhesion I P I 0 0 7 0 8 7 6 1 . 1 C a r b o n y l r educ tase 1 H o u s e - keep ing Oxidoreductase I P I 0 0 2 9 5 3 8 6 . 6 C a r b o n y l r educ tase [ N A D P H ] 1 H o u s e - keep ing Oxidoreductase I P I 0 0 6 8 9 4 4 0 . 2 N u c l e a s e sens i t i ve e l e m e n t - b i n d i n g pro te in 1 H o u s e - keep ing Splicing, translation regulation I P I 0 0 7 1 8 1 9 7 . 1 F -box a n d leuc ine - r i ch repea t pro te in 20 H o u s e - keep ing Ubiquitin-dependent protein turnover I P I 0 0 7 1 3 5 4 7 . 1 S u p e r o x i d e d i s m u t a s e H o u s e - keep ing Oxidative stress response I P I 0 0 7 1 5 2 1 8 . 1 Hypo the t i ca l p ro te in ( L S M 3 h o m o l o g , U6 sma l l nuc lea r R N A a s s o c i a t e d ) H o u s e - keep ing m R N A processing I P I 0 0 6 9 9 8 6 6 . 2 P R E D I C T E D : s im i la r to G a l e c t i n - 5 H o u s e - keep ing Heterophilic cell adhesion 117 A c c e s s i o n # N a m e C a t e g o r y Function/Comments I P I 0 0 7 1 0 1 1 4 1 High mob i l i t y g r o u p pro te in B l ( ampho te r i n ) House - k e e p i n g Cytokine, immune response I P I 0 0 7 3 2 0 8 1 1 P R E D I C T E D : s im i l a r to nuc leo l i n - re la ted pro te in i so fo rm 8 House - k e e p i n g Unknown I P I 0 0 6 8 7 6 2 5 2 P R E D I C T E D : s im i l a r to h e t e r o g e n e o u s nuc lea r r i bonuc leop ro te in AO House - k e e p i n g R N A processing I P I 0 0 7 0 0 5 2 1 1 51 k D a pro te in ( h e t e r o g e n e o u s nuc lea r r i bonuc leop ro te in K) House - k e e p i n g R N A processing I P I 0 0 7 2 6 3 4 3 1 P R E D I C T E D : s im i l a r to h e t e r o g e n e o u s nuc lea r r i bonuc leop ro te in D i so fo rm c i so fo rm 8 House -k e e p i n g R N A processing I P I 0 0 7 3 3 8 4 1 1 S i m i l a r to h e t e r o g e n e o u s nuc lea r r i bonuc leop ro te in A 2 / B 1 i so fo rm 2 M G C 1 2 8 2 9 7 pro te in ( P R E D I C T E D : s im i l a r to H e t e r o g e n e o u s nuc lea r r i bonuc leopro te in A l House - k e e p i n g R N A processing I P I 0 0 6 9 2 2 3 5 1 (He l i x -des tab i l i z i ng pro te in) ( h n R N P co re pro te in A l ) ( H D P - 1 ) (Topo i somerase - i nh i b i t o r s u p p r e s s e d ) ) L O C 5 1 3 4 1 0 pro te in ( P R E D I C T E D : s im i l a r to House - k e e p i n g R N A processing I P I 0 0 6 9 1 0 6 8 3 h e t e r o g e n e o u s nuc lea r r i bonuc leopro te in A B i so fo rm a , par t ia l ) House - k e e p i n g R N A processing I P I 0 0 7 1 4 8 6 8 2 P R E D I C T E D : s im i l a r to Pro te in F A M 3 C p r e c u r s o r (Pro te in G S 3 7 8 6 ) i so fo rm 2 House- k e e p i n g Cytokine I P I 0 0 6 9 0 1 4 1 1 S i m i l a r to H L A - B assoc ia ted t ranscr ip t 1 House- k e e p i n g Splicing, nuclear export of m R N A I P I 0 0 6 9 5 1 8 4 1 M i tochondr ia f i ss ion 1 prote in House- k e e p i n g Apoptosis I P I 0 0 6 9 3 9 0 0 2 Tol l in te rac t ing prote in House- k e e p i n g Component of IL-1 signaling I P I 0 0 7 1 3 6 7 2 2 M i t o g e n - a c t i v a t e d pro te in k i nase 1 House- k e e p i n g Signal transduction I P I 0 0 7 0 7 9 6 6 2 P R E D I C T E D : s im i l a r to e m b i g i n h o m o l o g , par t ia l H o u s e k e e p i n g Regulation of ce l l /ECM interaction I P I 0 0 7 2 3 8 0 3 1 P R E D I C T E D : s im i l a r to ca lnex in House- k e e p i n g Chaperone I P I 0 0 7 4 2 6 2 3 1 Hypo the t i ca l p ro te in (Cy toso l a m i n o p e p t i d a s e ) H o u s e k e e p i n g Protein turnover I P I 0 0 6 8 6 9 4 8 1 S e r p i n B6 H o u s e k e e p i n g Protease inhibitor I P I 0 0 7 0 5 6 0 3 2 P R E D I C T E D : s im i l a r to Ret icu lon 4 H o u s e k e e p i n g Neurite growth regulation I P I 0 0 7 0 4 0 0 6 1 3 2 k D a pro te in (s im i la r to L imb ic s y s t e m -a s s o c i a t e d m e m b r a n e pro te in p recu rso r ) H o u s e k e e p i n g Neuronal growth I P I 0 0 6 8 9 6 3 8 1 Hypo the t i ca l p ro te in (Sp l i ce I so fo rm Long of Lac tadhe r i n p recu rso r ) H o u s e k e e p i n g Cell adhesion I P I 0 0 7 0 9 1 6 2 1 G l y c i n a m i d e r ibonuc leo t ide f o r m y l t r a n s f e r a s e , i so fo rm 1 H o u s e k e e p i n g Purine biosynthesis I P I 0 0 2 9 0 2 7 9 1 I so fo rm Long of A d e n o s i n e k inase H o u s e k e e p i n g Nucleoside salvage I P I 0 0 6 8 8 0 0 6 1 Proh ib i t in H o u s e - keep ing Transcription regulation I P I 0 0 7 2 2 0 4 4 1 P R E D I C T E D : s im i l a r to B-cel l r ecep to r -a s s o c i a t e d pro te in 37 i so fo rm 1 (Proh ib i t in -2 ) H o u s e - keep ing Transcription regulation I P I 0 0 6 8 7 5 6 0 . 1 P o l y ( R C ) b ind ing pro te in 1 H o u s e - keep ing m R N A stabilization I P I 0 0 7 0 9 5 7 3 . 2 Hypo the t i ca l p ro te in M G C 1 2 7 5 9 6 ( F K 5 0 6 -B I N D I N G P R O T E I N 4 ) H o u s e - keep ing C h a p e r o n e I P I 0 0 7 0 3 7 3 1 . 1 4 0 k D a pep t idy l -p ro l y l c i s - t r ans i s o m e r a s e H o u s e - keep ing Chaperone I P I 0 0 7 3 1 6 4 5 . 1 18 k D a pro te in (Pep t idy l -p ro ly l c i s - t r ans H o u s e - keep ing Chaperone i s o m e r a s e A ) I P I 0 0 7 0 2 0 9 8 . 1 Pep t idy lp ro ly l i s o m e r a s e B H o u s e k e e p i n g Chaperone I P I 0 0 6 9 6 2 0 3 1 c A M P - d e p e n d e n t pro te in k i n a s e , a l pha -ca ta l y t i c subun i t H o u s e - keep ing Signal transduction I P I 0 0 6 9 6 5 3 9 1 c A M P - d e p e n d e n t pro te in k i n a s e , b e t a - 2 - c a t a l y t i c subun i t H o u s e - keep ing Signal transduction I P I 0 0 6 9 9 7 2 3 1 Hypo the t i ca l p ro te in ( R A N b ind ing prote in 6) H o u s e - keep ing Nuclear protein import (proposed) I P I 0 0 6 8 7 3 3 4 2 M G C 1 3 3 8 3 0 pro te in (s im i la r to inner m e m b r a n e p ro te in , m i t o c h o n d r i a l , par t ia l ) (mi tof i l in) H o u s e - keep ing Mitochondrial cristae morphology I P I 0 0 6 9 7 4 8 6 2 A d e n y l a t e k i nase 1 H o u s e - keep ing Cel l growth and maintenance I P I 0 0 7 1 7 2 0 7 2 P R E D I C T E D : s im i l a r to cy t i dy la te k i nase H o u s e - keep ing Nucleotide metabolism I P I 0 0 7 1 6 3 9 9 1 G u a n y l a t e k i n a s e H o u s e - keep ing Nucleotide metabolism 118 A c c e s s i o n # N a m e C a t e g o r y Function/Comments H o u s e - k e e p i n g Signal transduction H o u s e - k e e p i n g Ubiquitin-dependent protein turnover H o u s e - k e e p i n g Ubiquitin-dependent protein turnover H o u s e - k e e p i n g Antiapoptotic factor H o u s e - k e e p i n g Nuclear protein import H o u s e - k e e p i n g Oxidoreductase (proposed) H o u s e - k e e p i n g Cell division, membrane trafficking (proposed) H o u s e - k e e p i n g Complex N-glycans biosynthesis H o u s e - k e e p i n g Cel l cycle regulation, cytoskeleton regulation H o u s e - k e e p i n g Metabolism (proposed) H o u s e - k e e p i n g Metabolism (proposed) H o u s e - k e e p i n g Ubiquitin-dependent protein turnover H o u s e - k e e p i n g Cytokinesis H o u s e - k e e p i n g Cel l polarity H o u s e - k e e p i n g Protease H o u s e - k e e p i n g H o u s e - k e e p i n g H o u s e - k e e p i n g H o u s e - k e e p i n g H o u s e - k e e p i n g Transcription regulation DNA helicase Immune defense Neural cell adhesion whey protein H o u s e - k e e p i n g Immune response H o u s e - k e e p i n g Immune response H o u s e - k e e p i n g Immune response H o u s e - k e e p i n g Immune response H o u s e - k e e p i n g Immune response H o u s e - k e e p i n g Signal transduction H o u s e - k e e p i n g Signal transduction H o u s e - k e e p i n g Signal transduction H o u s e - k e e p i n g Signal transduction H o u s e - k e e p i n g Signal transduction H o u s e - k e e p i n g Calcium-depndent signal transduction I P I 0 0 7 0 3 9 9 2 . 1 I P I 0 0 7 1 6 1 3 0 . 1 I P I 0 0 7 0 3 4 7 0 . 1 I P I 0 0 7 1 4 9 1 0 . 2 I P I 0 0 7 2 7 6 4 5 . 1 I P I 0 0 7 1 2 5 9 0 . 1 I P I 0 0 7 1 0 1 7 5 . 1 I P I 0 0 7 1 0 4 5 0 . 2 I P I 0 0 6 9 8 0 4 3 . 2 I P I 0 0 7 1 6 1 8 3 . 1 I P I 0 0 7 0 2 7 6 8 . 1 I P I 0 0 7 1 0 9 0 4 . I P I 0 0 7 0 5 3 3 4 . I P I 0 0 7 0 4 2 5 7 . I P I 0 0 7 2 8 7 1 0 . I P I 0 0 6 9 9 3 9 9 , I P I 0 0 2 2 0 8 3 4 , I P I 0 0 7 1 4 7 6 5 , I P I 0 0 7 1 0 3 2 6 . I P I 0 0 6 9 9 6 9 8 , I P I 0 0 3 3 1 2 8 6 . 2 I P I 0 0 4 0 7 2 7 3 . I P I 0 0 7 1 4 4 7 6 . I P I 0 0 7 0 6 3 1 7 . I P I 0 0 6 9 9 0 1 1 . 3 I P I 0 0 6 9 8 1 0 2 . I P I 0 0 4 1 3 7 3 1 . I P I 0 0 7 0 2 1 7 5 . 1 I P I 0 0 7 0 4 3 1 4 . I P I 0 0 7 3 3 2 8 0 . I P I 0 0 7 1 1 9 0 0 . P R E D I C T E D : s im i l a r to g u a n i n e nuc leo t i de -b ind ing p ro te in , b e t a - 3 subun i t P R E D I C T E D : s im i l a r to ub iqu i t i n -ac t i va t ing e n z y m e E l i so fo rm 2 P R E D I C T E D : s im i l a r to ub iqu i t i n -con juga t ing e n z y m e E 2 N i so fo rm 1 Hypo the t i ca l p ro te in ( P R E D I C T E D : s im i l a r to a p o p t o s i s inh ib i tor 5 , par t ia l ) P R E D I C T E D : s im i la r to k a r y o p h e r i n beta 1 i so fo rm 5 D e h y d r o g e n a s e / r e d u c t a s e S D R fami l y m e m b e r 11 p recu rso r P R E D I C T E D : s im i l a r to C o p i n e - 1 (Cop ine I) i so fo rm 3 P R E D I C T E D : s im i l a r to A l p h a - m a n n o s i d a s e II P R E D I C T E D : s im i l a r to N A D - d e p e n d e n t d e a c e t y l a s e s i r tu in -2 4 3 k D a pro te in (s im i la r to ha loac id d e h a l o g e n a s e - l i k e hyd ro lase d o m a i n con ta in ing 2) A l ka l i ne p h o s p h a t a s e , t i s sue -nonspec i f i c i s o z y m e p recu rso r P R E D I C T E D : s im i l a r to S - p h a s e k i n a s e -a s s o c i a t e d pro te in I A i so fo rm b S e p t i n - 7 M G C 1 2 8 6 1 2 pro te in ( P R E D I C T E D : s im i l a r to cel l d i v i s ion cyc le 4 2 , par t ia l ) P R E D I C T E D : s im i l a r to C a t h e p s i n D p recu rso r i so fo rm 4 P R E D I C T E D : s im i l a r to H is tone d e a c e t y l a s e 11 | A T P - d e p e n d e n t D N A he l i case 2 subun i t 2 P R E D I C T E D : s im i la r to L a n C - l i k e pro te in 1 Neu ro t r im in Be ta - l ac tog lobu l i n p recu rso r B o n e m a r r o w m a c r o p h a g e c D N A , R I K E N fu l l -leng th en r i c hed l ib rary , c l o n e : I 8 3 0 1 2 9 N 0 7 p r o d u c t : b e t a - 2 m ic rog lobu l i n , ful l inser t s e q u e n c e P R E D I C T E D : s im i l a r to Ig k a p p a cha in V reg ion E V 1 5 p recu rso r Hypo the t i ca l p ro te in M G C 1 3 4 1 4 7 ( I m m u n o g l o b u l i n supe r f am i l y , m e m b e r 4) P R E D I C T E D : s im i l a r to i m m u n o g l o b u l i n s u p e r f a m i l y , m e m b e r 4 D , par t ia l Hypo the t i ca l p ro te in ( P R E D I C T E D : s im i l a r to I m m u n o g l o b u l i n l a m b d a - l i k e po l ypep t i de 1 p r e c u r s o r ( I m m u n o g l o b u l i n - r e l a t e d 14.1 pro te in) ( I m m u n o g l o b u l i n o m e g a po lypep t ide ) ( L a m b d a 5) ( C D 1 7 9 b an t igen) ) S e r i n e / t h r e o n i n e k inase recep to r a s s o c i a t e d p ro te in Pro te in p h o s p h a t a s e 3 P R E D I C T E D : s im i l a r to pro te in p h o s p h a t a s e 3 ( fo rmer l y 2 B ) , ca ta ly t i c subun i t , g a m m a i so fo rm (ca lc ineur in A g a m m a ) , par t ia l Sp l i ce I so fo rm 1 of S e r i n e / t h r e o n i n e - p r o t e i n p h o s p h a t a s e 2 B ca ta ly t i c subun i t a lpha i so fo rm P R E D I C T E D : s im i l a r to pro te in p h o s p h a t a s e 3 , ca ta ly t i c subun i t , be ta i so fo rm i so fo rm 8 Ca l c i neu r i n subun i t B i so fo rm 1 119 A c c e s s i o n # N a m e C a t e g o r y Function/Comments I P I 0 0 7 3 1 6 0 4 . 1 P R E D I C T E D : s im i l a r to a l p h a - 2 - m a c r o g l o b u l i n p recu rso r i so fo rm 5 L O C 5 2 0 1 7 0 pro te in ( P R E D I C T E D : s im i la r to H o u s e - k e e p i n g Protease inhibitor I P I 0 0 7 0 5 7 4 9 . 4 Ub iqu i t in c a r b o x y l - t e r m i n a l hyd ro lase i s o z y m e L3 ( U C H - L 3 ) (Ub iqu i t in t h i o l es te rase L3)) H o u s e - k e e p i n g Ubiquitin-dependent protein turnover I P I 0 0 7 0 2 9 5 0 . 1 S i m i l a r to g l u t a t h i o n e - S - t r a n s f e r a s e , m u 5 P R E D I C T E D : s im i l a r to G lu ta th i one S -H o u s e - k e e p i n g Glutathione transfer I P I 0 0 6 9 6 7 6 6 . 1 t r a n s f e r a s e Mu 1 ( G S T M 1 - 1 ) ( G S T c l a s s - m u 1) ( G S T M l a - l a ) ( G S T M l b - l b ) (HB subun i t 4) ( G T H 4 ) i so fo rm 1 H o u s e - k e e p i n g Glutathione transfer I P I 0 0 7 0 4 2 0 0 . 1 P R E D I C T E D : s im i l a r to g lu ta th ione S - t r a n s f e r a s e M l i so fo rm 2 i so fo rm 3 H o u s e - k e e p i n g Glutathione transfer I P I 0 0 6 8 6 1 7 3 . 1 G lu ta th i one S - t r a n s f e r a s e P H o u s e - k e e p i n g Glutathione transfer I P I 0 0 7 1 4 2 9 4 . 1 S - a d e n o s y l h o m o c y s t e i n e hyd ro lase H o u s e - k e e p i n g Activated methyl cycle I P I 0 0 7 0 7 7 5 1 . 2 E longa t ion fac to r 2 H o u s e - k e e p i n g Translation I P I 0 0 6 4 2 9 7 1 . 2 Euka ryo t i c t rans la t i on e longa t i on fac to r 1 de l ta i so fo rm 1 H o u s e - k e e p i n g Translation I P I 0 0 6 8 7 1 3 5 . 1 Euka ryo t i c t rans la t i on e longa t ion fac tor 1 beta 2 H o u s e - k e e p i n g Translation I P I 0 0 7 1 2 7 7 5 . 1 E longa t ion fac to r 1 -a lpha 1 H o u s e - k e e p i n g Translation I P I 0 0 7 0 9 8 0 5 . 1 S i m i l a r to Mu-c rys ta l l i n h o m o l o g H o u s e - k e e p i n g NADPH-regulated thyroid hormone-binding protein I P I 0 0 7 0 2 4 6 8 . 1 Ze ta -c r ys ta l l i n H o u s e - k e e p i n g Quinone oxidoreductase I P I 0 0 7 1 1 4 0 4 . 3 T e t r a s p a n i n - 1 8 H o u s e - k e e p i n g Unknown function P R E D I C T E D : s im i l a r to D i h y d r o p y r i m i d i n a s e -H o u s e - k e e p i n g Nucleic acid metabolism, nervous I P I 0 0 7 3 1 3 9 1 . 1 re la ted pro te in 1 ( D R P - 1 ) (Co l laps in r esponse system development Nucleic acid metabolism, signal m e d i a t o r p ro te in 1) ( C R M P - 1 ) , par t ia l I P I 0 0 6 9 8 3 3 8 . 3 D i h y d r o p y r i m i d i n a s e - r e l a t e d pro te in 2 H o u s e - k e e p i n g transduction, nervous system development P R E D I C T E D : s im i l a r to D i h y d r o p y r i m i d i n a s e -Nucleic acid metabolism, signal I P I 0 0 6 8 7 5 3 9 . 1 H o u s e - k e e p i n g transduction, nervous system re la ted pro te in 3 P R E D I C T E D : s im i l a r to a m y o t r o p h i c la tera l development I P I 0 0 6 9 3 5 0 6 . 1 sc le ros is 2 ( juven i le ) c h r o m o s o m e reg ion , c a n d i d a t e 4 H o u s e - k e e p i n g U n k n o w n func t ion I P I 0 0 7 0 2 7 6 5 . 1 D J - 1 pro te in H o u s e - k e e p i n g Neuroprotection, transcription regulation I P I 0 0 6 9 1 7 4 7 . 3 B e t a - s y n u c l e i n H o u s e - k e e p i n g Neuronal plasticity (proposed) I P I 0 0 6 9 8 0 6 9 . 1 Neu roca l c i n de l ta H o u s e - k e e p i n g Calcium sensor, signal transduction I P I 0 0 7 3 3 9 7 0 . 1 P R E D I C T E D : s im i l a r to c a l u m e n i n p r e c u r s o r i so fo rm 3 H o u s e - k e e p i n g Chaperone I P I 0 0 7 2 8 5 7 6 . 1 P R E D I C T E D : s im i l a r to t r a n s m e m b r a n e pro te in 3 0 A H o u s e - k e e p i n g Unknown function I P I 0 0 6 9 2 7 3 3 . 1 P R E D I C T E D : s im i l a r to t r a n s m e m b r a n e pro te in 3 0 A , par t ia l H o u s e - k e e p i n g Unknown function I P I 0 0 7 2 5 5 7 3 . 1 P R E D I C T E D : s im i l a r to t r a n s m e m b r a n e pro te in 6 3 B i so fo rm 3 H o u s e - k e e p i n g Unknown function I P I 0 0 4 1 4 6 8 4 . 6 I so fo rm 2 of S e m e n o g e l i n - 1 p recu rso r H o u s e - k e e p i n g Unknown function in retina I P I 0 0 0 2 5 4 1 5 . 1 S e m e n o g e l i n - 2 p recu rso r H o u s e - k e e p i n g Unknown function in retina I P I 0 0 7 1 3 9 8 9 . 1 P R E D I C T E D : s im i l a r to T u m o r d i f ferent ia l ly e x p r e s s e d pro te in 1 i so fo rm 1 H o u s e - k e e p i n g Cellular transformation I P I 0 0 6 9 6 2 8 6 . 1 G l y c e r a l d e h y d e - 3 - p h o s p h a t e d e h y d r o g e n a s e l i ke -17 pro te in ( F r a g m e n t ) H o u s e - k e e p i n g Unknown function I P I 0 0 7 2 1 4 6 3 . 1 P R E D I C T E D : s im i l a r to P h o s p h a t i d y l e t h a n o l a m i n e - b i n d i n g prote in P R E D I C T E D : s im i la r to p lecks t r in h o m o l o g y H o u s e - k e e p i n g cell differentiation, signal transduction I P I 0 0 6 9 2 1 3 2 . 2 d o m a i n c on t a i n i ng , f am i l y B (evec t ins ) m e m b e r 1 i so fo rm 1 H o u s e - k e e p i n g Unknown function I P I 0 0 7 1 6 8 4 3 . 1 G u a n i n e nuc leo t i de -b ind ing prote in G ( o ) , a lpha subun i t 1 H o u s e - k e e p i n g Unknown function 120 A c c e s s i o n # N a m e C a t e g o r y Function/Comments I P I 0 0 6 9 7 4 8 8 . 1 Hypo the t i ca l p ro te in M G C 1 2 8 1 5 8 (S im i l a r to Op t i neu r i n ) H o u s e - k e e p i n g Neuroprotection, membrane trafficking (proposed) I P I 0 0 7 0 8 0 3 0 . 2 Hypo the t i ca l p ro te in ( A c y l p h o s p h a t a s e 1) H o u s e - k e e p i n g Unknown function I P I 0 0 7 1 6 9 1 6 . 1 14 k D a pro te in U n k n o w n Uncharacterized protein I P I 0 0 6 9 0 5 0 8 . 3 M G C 1 3 7 3 3 6 pro te in U n k n o w n Uncharacterized protein I P I 0 0 6 9 6 6 5 3 . 2 P R E D I C T E D : s im i l a r to C 0 3 A 3 . 3 U n k n o w n Uncharacterized protein I P I 0 0 7 0 5 2 6 9 . 2 S i m i l a r to Y 5 5 F 3 A M . 9 U n k n o w n Uncharacterized protein I P I 0 0 7 0 7 5 9 3 . 1 4 2 k D a pro te in U n k n o w n Uncharacterized protein I P I 0 0 7 2 1 7 3 1 . 1 Hypo the t i ca l p ro te in F U 3 6 8 1 2 U n k n o w n Uncharacterized protein I P I 0 0 6 8 9 4 3 0 . 1 P R E D I C T E D : s im i l a r to C G 3 1 3 2 - P A U n k n o w n Uncharacterized protein I P I 0 0 7 3 0 6 9 1 . 2 Hypo the t i ca l p ro te in U n k n o w n Uncharacterized protein I P I 0 0 7 3 4 5 2 8 . 1 P R E D I C T E D : s im i l a r to C G 1 5 3 2 - P A i so fo rm 3 U n k n o w n Uncharacterized protein I P I 0 0 6 9 9 9 9 5 . 1 P R E D I C T E D : s im i l a r to C G 8 7 6 8 - P A i so fo rm 1 U n k n o w n Uncharacterized protein I P I 0 0 7 2 9 5 8 0 . 1 P R E D I C T E D : s im i l a r to C G 8 7 6 8 - P A i so fo rm 2 U n k n o w n Uncharacterized protein I P I 0 0 7 0 7 1 1 4 . 1 P R E D I C T E D : s im i l a r to C G 1 0 2 3 7 - P B , i so fo rm B U n k n o w n Uncharacterized protein I P I 0 0 4 1 2 5 9 2 . 4 c h r o m o s o m e 9 o p e n read ing f r a m e 77 i so fo rm 2 U n k n o w n Uncharacterized protein I P I 0 0 6 8 7 1 4 4 . 1 Hypo the t i ca l p ro te in U n k n o w n Uncharacterized protein I P I 0 0 7 1 8 1 8 5 . 1 12 k D a pro te in U n k n o w n Uncharacterized protein I P I 0 0 7 1 5 2 1 0 . 1 7 0 k D a pro te in U n k n o w n Uncharacterized protein I P I 0 0 7 1 1 8 2 6 . 1 57 k D a pro te in U n k n o w n Uncharacterized protein I P I 0 0 6 9 0 9 9 3 . 1 Hypo the t i ca l p ro te in (Pro te in C 1 1 0 R F 6 7 h o m o l o g ) U n k n o w n Uncharacterized protein I P I 0 0 6 9 0 6 2 3 . 1 119 k D a pro te in U n k n o w n Uncharacterized protein I P I 0 0 7 0 8 6 9 1 . 1 Hypo the t i ca l p ro te in M G C 1 2 7 3 9 9 U n k n o w n Uncharacterized protein 121 

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