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Regulation of budding yeast kinetochore proteins by SUMO modification Montpetit, Benjamen H. W. 2007

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Regulation of budding yeast kinetochore proteins by SUMO modification by Benjamen H.W. Montpetit B.Sc, Simon Fraser University, 2001 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in The Faculty of Graduate Studies (Biochemistry and Molecular Biology) THE UNIVERSITY OF BRITISH COLUMBIA March 2007 -© Benjamen H.W. Montpetit, 2007 A B S T R A C T Genome stability is a fundamental requirement for the stable propagation of eukaryotic cells, maintenance of phenotype, and cell viability. For example, aneuploidy (abnormal chromosome number), is a hallmark of most human cancers, and can be attributed to increased rates of chromosome instability in cancer cells. The multiprotein kinetochore complex contributes to faithful chromosome segregation by mediating the attachment of a specialized chromosomal region, the centromere, to the mitotic spindle. A dysfunctional kinetochore represents one possible source for chromosome instability and the generation of aneuploid cancer cells, due to a failure to properly mediate this attachment. To better understand the regulation of kinetochore proteins and their role in chromosome segregation, a series of genomic screens were performed with known kinetochore components in the budding yeast Saccharomyces. cerevisiae, to identify genetic/physical interactions and novel functions that are critical for proper chromosome segregation. This work lead to the study of two distinct relationships: (1) between the kinetochore and the nuclear envelope, and (2) between kinetochore proteins and the ubiquitin-like protein S U M O . In the first study, genes that are linked to chromosome stability were identified by performing genome-wide synthetic lethal screens using a series of novel temperature sensitive mutations in essential genes encoding a central (SPC24) and outer, (SPC34) kinetochore protein. B y performing these screens using different mutant alleles of each gene, we aimed to identify genetic interactions that revealed diverse pathways affecting chromosome stability. This study, which is the first example of genome-wide synthetic lethal screening with multiple alleles of a single gene, demonstrated that functionally i i distinct mutants uncover different cellular processes required for chromosome maintenance. Two of our screens identified APQ12, a gene that encodes a nuclear envelope protein required for proper nucleocytoplasmic transport of m R N A , which was subsequently characterized with respect to chromosome stability. We found that apql2 mutants are delayed in anaphase, re-replicate their D N A and re-bud prior to completion of cytokinesis, suggesting a defect in controlling mitotic progression. Overall, this analysis revealed a novel relationship between nucleocytoplasmic transport and chromosome stability. In the second study, functional genomics lead to the identification of the kinetochore proteins NdclO, B i r l , Ndc80, and Cep3 as being sumoylated substrates in budding yeast. This work demonstrated that Ndc lO, B i r l , and Cep3, but not Ndc80, are differentially modified upon exposure to nocodazole, indicating distinct roles for S U M O modification in modulating kinetochore protein function and providing a potential link between sumoylation of kinetochore proteins and mitotic checkpoint function. Specific lysine to arginine mutations, were shown to eliminate sumoylation of Ndc lO and to cause chromosome instability, mis-localization of Ndc lO from the mitotic spindle, and abnormal anaphase spindles, suggesting that sumoylation of Ndc lO and other kinetochore proteins plays a critical role during the mitotic process. These results support the recent findings that post-translational modifications by the ubiquitin-like protein S U M O is an important regulator of many cellular processes including genome integrity. i i i TABLE OF CONTENTS Abstract i i Table of Contents iv List of Tables v i i List of Figures vi i i List of Abbreviations x Acknowledgements x i i Dedication xi i i CHAPTER 1. Chromosome segregation in Saccharomyces cerevisiae 1 Introduction 2 Genomic Instability and Disease 2 The Cel l Cycle 5 Cel l Cycle Checkpoints 9 The Mitotic Spindle and Chromosome segregation.. 14 The Kinetochore of Budding Yeast 18 Scope of the Thesis • 23 CHAPTER 2. Genome-wide synthetic lethal screens identify an interaction between the nuclear envelope protein, Apql2, and the kinetochore in Saccharomyces cerevisiae 25 Introduction 26 Materials and Methods 29 Results 34 iv Isolation of Ts alleles in central and outer kinetochore proteins...34 Genome-wide S L screen with spc24 and spc34 alleles 37 A P Q 1 2 genetically interacts with the kinetochore 42 apql2 mutants have aberrant chromosome segregation 46 apql2 mutants have defects in exiting mitosis. 48 Discussion 52 CHAPTER 3. Sumoylation of the budding yeast kinetochore protein NdclO is required for NdclO spindle localization and regulation of anaphase spindle elongation 56 Introduction 57 Materials and Methods 63 Results : 69 Ndc lO interacts with multiple components of the sumoylation machinery 69 Ndc lO is sumoylated in vitro and in vivo 71 Dynamics of NdclO sumoylation 73 Loss of Ndc lO Sumoylation is linked to checkpoint activity 76 Kinetochore proteins Cep3 and Ndc80 are sumoylated 76 Identification of lysine residues required for Ndc lO , Ndc80, and Cep3 sumoylation 78 Ndc lO mitotic spindle localization is lost in the ndclO 4xK-^R mutant .83 K779 is the key sumoylation site 85 ndc!0-4xK^R mutants display chromosome instability 88 ndclO-4xK-+R mutants have mitotic spindle defects 88 B i r l and ndclO-4xK—+R physically interact 90 B i r l is sumoylated in an NdclO-dependent manner ..92 B i r l sumoylation is independent of C B F 3 function 92 Discussion 93 CHAPTER 4. Future Directions 101 Future directions in the field of kinetochore research 102 Spc24, Spc34 and A p q l 2 in chromosome segregation 103 Sumoylation: Future bouts with chromosome segregation 106 Conclusion I l l References 112 List of Tables C H A P T E R II: Table 2-1 List of yeast strains used in this chapter... 31 Table 2-2 Genetic interactions between spc24 and gene deletion mutants 41 Table 2-3 Chromosome loss events in spc24 and apql2 mutants 47 C H A P T E R III: Table 3-1 List of yeast strains used in this chapter 64 Table 3-2 List of plasmids used in this chapter ; 66 Table 3-3' Ndc lO two-hybrid interactions 70 Table 3-4 Potential sumoylation sites targeted by site directed mutagenesis 81 vii List of Figures CHAPTER 1: Figure 1-1 The budding yeast cell cycle 6 Figure 1-2 The budding yeast kinetochore 20 CHAPTER 2: Figure 2-1 Characterization of spc24 Ts mutants. ...36 Figure 2-2 Characterization of spc34 Ts mutants '. 38 Figure 2-3 spc24 and spc34 genomic S L screen results 40 Figure 2-4 Growth defects and benomyl sensitivity of spc24 and apql2 mutants 44 Figure 2-5 Spc24 protein levels and sub-cellular localization of Spc24 and O k p l in apql2 mutants. 45 Figure 2-6 apql2 mutants are delayed in anaphase and prematurely enter a new cell cycle 50 CHAPTER 3: Figure 3-1 S U M O modification cycle 60 Figure 3-2 Sumoylation of the kinetochore protein Ndc lO 72 Figure 3-3 Dynamics of NdclO sumoylation 75 Figure 3-4 Loss of Ndc lO Sumoylation is linked to checkpoint activity 77 Figure 3-5 Sumoylation of the kinetochore proteins Cep3 and Ndc80 79 Figure 3-6 Identification of lysine residues in Ndc lO and Ndc80 that affect sumoylation 82 vi i i Figure 3-7 Loss of Ndc lO spindle localization ." 84 Figure 3-8 ndclO 4xK-^R strains do not have defects related to cytokinesis 86 Figure 3-9 K779 is the key sumoylation site ...87 Figure 3-10 ndclO 4xK—*R strains have increased rates of chromosome mis-segregation and mitotic spindle defects 89 Figure 3-11 B i r l is sumoylated in an NdclO dependent manner independent of C B F 3 function 91 Figure 3-12 B i r l phosphorylation and the I A P domain are required for B i r l sumoylation 98 C H A P T E R 4: Figure 4-1 NdclO is hypersumoylated in response to loss of I p l l kinase activity. ..110 ix List of Abbreviations a F alpha factor A P C / C Anaphase Promoting Complex / Cyclosome bp base pairs C E N centromere C I N Chromosome Instability Cdk Cycl in Dependent Kinase C F Chromosome Fragment C F P Cyan Fluorescent Protein C T F Chromosome Transmission Fidelity C P P Chromosome passenger proteins D M S O Dimethylsulphoxide D N A deoxyribonucleic acid F A C S Fluorescence Activated Cel l Sorting G I Gap 1 (Growth before D N A replication phase of cell cycle) G2 Gap2 (Growth after D N A replication phase of cell cycle) G F P Green Fluorescent Protein G D P Guanine diphosphate G T P Guanine triphosphate IP Immunoprecipitation L O H Loss Of Heterozygosity M Mitosis M I N Microsatellite Instability M T O C Microtubule Organizing Center N Z Nocodazole S Synapsis ( D N A replication phase of cell cycle) S A C Spindle assembly checkpoint S C F Skp 1 / Cul l in / F-box protein SPB Spindle Pole Body S U M O Small Ubiquitin-like modifier U B C Ubiquitin Carrying Enzyme U L P Ubiquitin-like protease V F P "Venus" yellow fluorescent protein Y F P Yel low Fluorescent Protein Y P D Yeast Extract Peptone Dextrose A c k n o w l e d g e m e n t s I would like to thank the faculty, staff and fellow students of the University of British Columbia who have encouraged and inspired me during my thesis work. This includes my supervisor, Phil Hieter, for his advice and guidance when it was needed, and for giving me the freedom to follow my own path. I am appreciative of his generosity through the years that I have been a member of his lab. Within the Hieter lab many others have been generous with their time and I would like to thank them for their support. This includes all members past and present, but I would like to especially acknowledge Vivien Measday, who provided me with scientific opportunity, and through out the years always made time to discuss science and provide valuable input into published works that I have written. I would also like to thank my scientific colleagues that have contributed to this work including Tony Hazbun, Stan Fields, Xuewen Pan, Jef Boeke, Mark Flory, Rudi Aebersold, Jason Ptacek, M i k e Snyder and Erica Johnson. I would like to thank my committee members, Elizabeth Conibear, Lawrence Mcintosh, and Miche l Roberge, who provided guidance and advice through out my doctoral studies. During the course of this work I received funding from The Natural Sciences and Engineering Research Council of Canada and The Michael Smith Foundation for Health Research, and I would like to acknowledge their generous support. I would finally like to thank those people that are most important to me, my family. This includes my parents and siblings who have supported me through the many years of school, all in their own unique way. Most of all I would like to thank my wife, Rachel, who always believed in me, and continues to support me in all that I do. She has been and continues to be the most wonderful thing in my life. xi i This workjs dedicated to my Coving wife Rachel and our daughter Stella Chapter 1: Chromosome Segregation in Saccharomyces cerevisiae I n t r o d u c t i o n Maintenance of the genetic material is essential to an organism's health and survival. For this reason, cellular organisms have evolved an intricate program of events collectively referred to as the cell cycle, which in concert, ensure that each cell created by cell division has the correct complement of all cellular materials (e.g. chromosomes). In the case of the genetic material of eukaryotes, this includes making sure each chromosome is faithfully replicated, packaged, held together as sister chromatids, and then properly segregated to each daughter cell in the correct temporal order with high fidelity. Errors in any of these steps can result in aneuploidy, which has phenotypic consequences that include birth defects, such as Down Syndrome, and the development of cancers (Duesberg et al., 2006; Hassold and Hunt, 2001). Genomic Instability and Disease Genomic instability can be defined as an increase in the rate of change to the genetic material. This can occur at the level of single base pair changes (mutations) or can include the loss or gain of whole chromosomes or fragments thereof, both of which are associated with numerous diseases (Andrew and Peters, 2001). These two types of genomic instability are referred to as microsatellite instability (MIN) and chromosome instability (CIN), respectively. In colon cancer, M I N is the result of the inactivation of specific D N A repair genes that ultimately results in a mutator phenotype, where defects in repair leads to increased mutation rates throughout the genome (Lengauer et al., 1997). The mutator phenotype is also prevalent in numerous neurological diseases that are the result of repeat expansion events, and is associated with - 1 3 % of all solid cancers; however, the majority of solid cancers (>80%) contain aneuploid cells that are likely the 2 result of C I N (Nowak et al., 2002). C I N can be defined as the loss or gain of whole chromosomes or parts thereof, and may function to induce a disease state by loss of heterozygosity (LOH) and the uncovering of recessive alleles, or by altering gene dosage balance between genes that have opposing function (e.g. tumor suppressors and oncogenes). In specific instances, these changes caused by genomic instability can confer an advantage to a cell and become fixed in the population through selection and clonal expansion. This process can repeat itself multiple times leading to the selection and expansion of an ever increasing abnormal population of cells, with the M I N or C I N phenotypes driving this process at an accelerated rate compared to that in normal cells, ultimately resulting in a disease such as cancer. The genetic alterations responsible for M I N in colon cancers have been shown to result from mutations in the mismatch repair genes (e.g. hMLHl and hMSH2) (Fishel et al., 1993; Leach et al., 1993; Papadopoulos et al., 1994; Strand et al., 1993). In contrast, the cause of C I N in the vast majority of solid tumors is unknown, and due to the large number of genes that may give rise to a C I N phenotype, it is likely to be much more complicated than the case of M I N (Yuen et al., 2006). Current experimental evidence strongly supports the hypothesis that the C I N phenotype occurs early in the development of cancer, and represents an important step in the initiation and/or progression of the disease (Lengauer et al., 1998; Rajagopalan et al., 2004; Shih et al., 2001). For instance, the recent report that germline biallelic mutations in a spindle checkpoint gene, h B U B I B , is associated with inherited predispositions to cancer strongly supports a causal link between C I N and cancer development (Hanks et al., 2004); therefore, a major goal has been to determine the genetic basis of C I N in tumors. 3 One approach to identifying mutations responsible for C I N in cancer cells is to test for mutations in genes known to be important for chromosome segregation in human cells, or in human homologs of C I N genes discovered in model organisms, which serve as cross-species C I N candidate genes. For example, Vogelstein and colleagues initially showed that the h B U B l (identified originally in yeast as a mitotic spindle checkpoint mutant) is mutated in a small percentage of colorectal tumors (Cahill et al., 1998). Additional mutation testing of candidate genes associated with genomic instability has provided evidence for somatic mutation in another 6 C I N genes (hCDC4, hRod, hZWIO, hZwilch, h M R E l l , and Ding) that together account for less than 20% of the C I N mutational spectrum of colon cancer (Rajagopalan et al., 2004; Wang et al., 2004). Thus, the genetic basis for C I N in colon cancer, and in all types of cancer, is largely unknown. Candidate C I N genes encode proteins that function in all aspects of chromosome segregation, including proteins that function at kinetochores, telomeres, origins of replication, and in microtubule dynamics, sister chromatid cohesion, D N A replication, D N A repair, D N A condensation and cell cycle checkpoints. O f these, the kinetochore offers a logical choice for mutational testing since four out of seven C I N genes known to be mutated in C I N colon cancers encode kinetochore proteins. Furthermore, the -100 predicted human genes that encode kinetochore components comprise a large mutational target that could be mutable to a C I N phenotype (Fukagawa, 2004). For example, kinetochore proteins constitute a significant portion of the collection of chromosome transmission fidelity (ctf) mutants identified in a classical genetic screen in yeast (9 out of the 24 C T F genes cloned and characterized to date) (Spencer et al., 1990). Thus, continued identification and characterization of conserved kinetochore components in 4 budding yeast and other model organisms provides an important source of information pertaining to C I N candidate genes that may be mutated in cancers. The Cell Cycle Genome stability is a product of the control imposed on a growing and dividing cell by the cell division cycle^ regulatory machinery to ensure that each event during cell division take place with high fidelity and in the correct temporal order (Murray and Hunt, 1993). This includes controlling the frequency and timing of each event to ensure that a particular event occurs only after the preceding event has been completed (e.g. D N A segregation does not occur until D N A replication is complete), and that certain events occur only once, during the cell cycle (e.g. D N A replication). This involves passing checkpoints in the cell cycle, points at which cells wi l l either irreversibly commit to the next phase of the cell cycle, or arrest until the previous events are completed or mistakes are repaired before proceeding further. In budding yeast, morphological changes occur to a yeast cell as it progresses through a cell cycle, which can be correlated with other events in the cell cycle including the process of D N A replication and segregation (Figure 1-1). These distinct morphologies were used to isolate mutations in key components that are essential for cell cycle progression in budding yeast, which have been shown to be highly conserved in all other eukaryotes (Hartwell, 1980; Lee and Nurse, 1987; Wood and Hartwell, 1982). This work resulted in 1/3 of the Nobel Prize in Physiology and Medicine each being awarded to Leland Hartwell and Paul Nurse in 2001 for the discoveries of key regulators of the cell cycle and their conservation. The machinery controlling the cell cycle is highly conserved among all eukaryotes. This includes the Cycl in Dependent Kinases (Cdks), which serve as 5 C l n l , 2 , 3 • Kinetochore Spindle pole imiiiiiiiiiH jf-body microtubule Figure 1-1 The budding yeast cell Cycle. The cell cycle is divided into four stages [GI, S (DNA replication), G2 and M (mitosis)]. The size of the bud gives an approximate indication of cell cycle stage. During cell division, chromosomes undergo a replication and segregation cycle that is synchronized with the cell cycle. A specialized region of DNA, the centromere, onto which kinetochore proteins assemble (blue dot), is the site of attachment of chromosomes to microtubules of the mitotic spindle, emanating from the spindle pole body (green lines, red dot is the SPB). Each stage of the cell cycle is regulated by Cdk/Cyclin pairs as indicated (blue arrows). regulators of the cell cycle in concert with a variety of cyclin subunits. The function of the cyclin subunits is to activate Cdk kinase activity towards substrates appropriate to that stage of the cell cycle. Thus, the combination of a particular cyclin/Cdk pair can drive the cell cycle by phosphorylating the appropriate substrates, and the periodic accumulation and degradation of cyclins allows Cdk activity to be regulated in phase with a given stage of the cell cycle (Figure 1-1). This periodic expression of cyclins was the basis for their discovery by T i m Hunt as proteins that accumulated and then disappeared in a cell cycle dependent manner (Evans et al., 1983; Hadwiger et al., 1989; Murray et al., 1989; Nash et al., 1988). This work was also recognized by 1/3 of the Nobel Prize in Physiology and Medicine being awarded to T i m Hunt in 2001 for the discovery of key regulators of the cell cycle. Kinases, such as the Polo-like kinase(Plk)/Cdc5 and Aurora kinase/(Ipll) are also emerging as key regulators of the cell cycle in addition to the Cdks (Donaldson et al., 2001; Katayama et al., 2003; Nigg, 2001). In budding yeast, Cdc28 and Pho85 are the Cdks involved in cell cycle progression with Cdc28 being the essential regulator (Mendenhall and Hodge, 1998). Cdc28, which is expressed at a high level throughout the cell cycle, is controlled by the G I cyclins ( C l n l , Cln2 and Cln3) (Cross, 1990; Hadwiger et al., 1989; Richardson et al., 1989), S phase cyclins (Clb5, Clb6, Clb3, and Clb4) (Dahmann et al., 1995; Richardson et al., 1989), and mitotic cyclins ( C M and Clb2) (Fitch et al., 1992; Ghiara et al., 1991). The targets of each stage specific Cdk/cyclin include transcription factors that when phosphorylated by Cdc28 control the expression of genes specific to and required for that particular stage of the cell cycle (Wittenberg and Reed, 2005). Proteolysis of stage specific gene products by the proteasome is also 7 required for proper cell cycle regulation (Vodermaier, 2004). For example, S i c l is a Cdk kinase inhibitor that must be degraded during G I in order for the G l / S phase transition to occur (Bai et al., 1996; Donovan et al., 1994; Schwob et al., 1994). Proteolysis of target proteins is an effective means of controlling the cell cycle since it provides an irreversible directionality to events (i.e. once a protein is destroyed its activity is irreversibly lost unless new transcription and translation occurs). Ce l l cycle regulated proteolysis is accomplished by targeting proteins for degradation by the 26S proteasome (Chun et al., 1996; Hershko, 1997). Targeting is accomplished by attaching ubiquitin to lysine residue(s) of a target protein, and once attached internal lysine residues in ubiquitin can then serve as sites for ubiquitin addition. Multiple rounds of this process lead to the creation of a poly-ubiquitin chain on the target protein that serves as a marker for degradation by the 26S proteasome. Ubiquitin mediated proteolysis is a highly conserved process in eukaryotes as is the underlying ubiquitin machinery. The ubiquitin machinery includes E l ubiquitin activating enzymes that bind to ubiquitin and activate ubiquitin for subsequent binding to an E 2 protein, a ubiquitin conjugating enzyme. A n E3 complex, ubiquitin ligase, then brings substrate proteins and the E2-ubiquitin complex together to provide the specificity required to target select proteins for degradation.. E3 complexes that are important for cell cycle proteolysis include the Anaphase Promoting Complex / Cyclosome ( A P C / C ) and the S C F complex (Skpl / Cul l in / F-box protein) (Page and Hieter, 1999; Peters, 1998; Vodermaier, 2004). Each complex contains a core set of subunits required for E3 activity, which then rely upon substrate specificity factors that bind the core A P C / C or S C F complex to efficiently target proteins for degradation. A P C / C specificity factors include Cdc20 and C d h l (Visintin et al., 1997), 8 and a meiosis specific factor, A m a l (Cooper et al., 2000), while S C F specificity factors are recognizable as a set of F-box containing proteins that include Cdc4, G r r l , and Met30 (Skowyra et al., 1997). Each specificity factor contains protein-protein interaction domains such as W D - 4 0 repeats, which contribute to protein-protein binding and are responsible for bringing substrates together with the ubiquitin machinery. For instance, Cdc20 is responsible for targeting proteins that are important for the metaphase to anaphase transition (e.g. Pds l ) and passage through mitosis, whereas C d h l is responsible for proteolysis later in mitosis and during early G I (Pfleger et al., 2001; Visintin et al., 1997). The post-translational modification of A P C / C subunits is also critical for the proper regulation of E3 activity, and includes phosphorylation and sumoylation (Dieckhoff et al., 2004; Harper, 2002; Page and Hieter, 1999). Overall, regulation of the Cdk/cyclin and proteolysis machinery is key to controlling the cell division cycle, which make these complexes and their activities central targets for the action of cell cycle checkpoints. Cell Cycle Checkpoints A checkpoint is a mechanism to ensure that events in the cell cycle occur in the proper temporal order and that the next event in the cell cycle does not start before the previous event is complete with no detectable errors (Hartwell and Weinert, 1989). In the case of an error, the checkpoint wi l l retard progress through the cell cycle to allow cells time to repair the defect(s). Checkpoints therefore are critical to the proper execution of the cell cycle since upcoming events in the cell cycle rely on the previous stage to have established conditions that are necessary for proper execution of the current event (e.g. segregation of one genomic complement to each cell during mitosis requires that the 9 genome was duplicated in S phase). Well-characterized checkpoints that are critical for maintaining euploidy include the D N A damage, spindle assembly and spindle position (mitotic exit) checkpoints. The D N A damage checkpoint monitors D N A replication and mediates D N A repair upon damage in G I , S, or G 2 / M (Longhese et al., 2003; Nyberg et al., 2002), the spindle assembly checkpoint ensures that the mitotic spindle is properly formed and all replicated sister chromatids are correctly attached to the spindle apparatus (Amon, 1999; Lew and Burke, 2003; Musacchio and Hardwick, 2002), while the spindle position checkpoint monitors proper spindle orientation, migration and completion of mitosis. In the context of these studies of kinetochore function and regulation, the critical checkpoint to consider is the spindle assembly checkpoint. During mitosis, the mitotic spindle forms a polarized network of fibers that physically connects to sister chromatids and are used to pull replicated chromosomes apart. For proper segregation to occur every sister chromatid must be attached to this apparatus and sisters must be attached to opposite poles (bi-orientated) to allow equal segregation of the genetic material to each daughter cell. Monitoring of these attachments is accomplished by the spindle assembly checkpoint (SAC) , which in the case of a defect, wi l l arrest cells in G 2 / M by preventing the metaphase to anaphase transition until the defect is repaired. This checkpoint system is composed of a set of proteins that were first described in budding yeast but are largely conserved among eukaryotes (Amon, 1999; Smith, 2002). In budding yeast, five non-essential genes, BUB1, BUB3 (budding uninhibited by benzimidazole), MAD1, MAD2 and MAD3 (mitotic arrest-deficient) encode essential components of the S A C machinery (Hoyt et al., 1991; L i and Murray, 1991). Deletion of these genes leads only to a modest increase in chromosome loss rates 10 in unperturbed conditions suggesting that in a "normal" cell cycle errors that require the action of the checkpoint are rare (Li and Murray, 1991; Warren et al., 2002). This is in contrast to mammalian cells, where the components of the S A C are essential for viability (Basu et al., 1999; Dobles et al., 2000; Kalitsis et al., 2000). This most likely reflects the fact that in budding yeast kinetochore microtubule attachments are maintained throughout the cell cycle and are present in S phase, so there are few chances for errors and ample time to facilitate proper bi-polar attachment (Winey and O'Toole, 2001). Where as in mammalian cells, kinetochore microtubule attachment are established only after nuclear envelope breakdown in mitosis, which provides only a small window of time for attachment and alignment of sister chromatids on the metaphase plate before progression into anaphase. Therefore, it is likely that in every mammalian cell mitosis, the S A C is required to retard the cell cycle until attachment and congression has occurred after nuclear envelope breakdown. To function a checkpoint generally requires an ability to recognize the damage or defect and a method of transducing this signal to effector proteins, which ultimately arrest the cell cycle and mediate repair of the defect (Nyberg et al., 2002). The signal that is recognized by the S A C has been proposed to be either lack of tension exerted across sister chromatids or the lack of microtubule binding at the kinetochore(s) (Lew and Burke, 2003). The difficulty in distinguishing between these two possibilities is that both processes are intimately associated with one another. For example, unattached kinetochores are not under tension and it has been observed that tension can regulate microtubule attachment. In this regard, the Aurora kinase I p l l has been shown to destabilize kinetochore microtubule attachments that lack tension and signal this lack of 11 \ tension; therefore, it is possible that the transiently unattached kinetochore that is generated by.Ipl l activity may be the signal activating the checkpoint and not the lack of tension (Pinsky and Biggins, 2005; Tanaka et al., 2005). However, it is l ikely that both processes are important as spindle assembly checkpoint components have shown differences in sub-cellular localization dependent on the status of kinetochore microtubule attachment and tension (Lew and Burke, 2003), but exactly how a mechanical signal (lack of tension) is processed and turned into a biochemical signal is not known. Since the S A C is sensitive enough to detect even a single unattached kinetochore (Rieder et al., 1994), the signal that is detected by the checkpoint must lead to a potent and diffusible inhibitor of the cell cycle. This most likely occurs through a process of amplification through checkpoint transducers that include a number of kinases. In this regard, the recognition of an error by the S A C has been proposed to involve tension regulated phosphorylation (Li and Nicklas, 1997). The checkpoint machinery requires the action of several kinases including I p l l , M p s l , and B u b l ( L e w and Burke, 2003). B u b l is a kinase that is known to phosphorylate Bub3, while M p s l can phosphorylate M a d l , and might function to recruit other checkpoint components to the kinetochore (Abrieu et al., 2001; Hardwick et al., 1996; Winey and Huneycutt, 2002). MPS1 overexpression constitutively activates the checkpoint, and this activation requires the M a d and Bub components (Fraschini et al., 2001; Hardwick et al., 1996). Conversely, M p s l proteolysis is required to silence the S A C , and is targeted by the A P C / C for destruction in anaphase (Palframan et al., 2006). Loss of M p s l activity in anaphase is necessary since once cohesion between sister chromatids has been resolved at the metaphase to anaphase transition, microtubule forces 12 are no longer opposed by cohesion and tension ceases at the kinetochore. This lack of tension would then be recognized by the S A C and cell cycle progression halted inappropriately i f the checkpoint was not silenced. Upon activation of the S A C , it is proposed that Mad2 dynamically interacts with unattached kinetochores rendering Mad2 competent to interact with the A P C / C regulatory subunit Cdc20. Mad2 binding to Cdc20, leads to inhibition of A P C / C activity, and stabilization of Cdc20-dependent A P C / C substrates (Amon, 1999). Substrates stabilized by silencing the A P C / C include Pds l (Securin), which is a potent inhibitor of E s p l (Separase), a protease required to resolve sister chromatid cohesion. Stabilization of P d s l , therefore blocks the metaphase to anaphase transition by preventing dissolution of sister chromatid cohesion (De Antoni et al., 2005; Lew and Burke, 2003; Luo et al., 2000). (Laloraya et al., 2000). The activated form of Mad2 may be an oligomerized complex, and the kinetochore might act as the catalytic site for polymerization of Mad2 subunits. Alternatively, the activated form of Mad2 may consist of a Mad-Bub complex (Shah and Cleveland, 2000). In either case, phosphorylation events, complex formation between the different checkpoint components, conformational changes induced by differential binding of components and effectors, and localization of these complexes all seem to be important in checkpoint activity (Musacchio and Hardwick; 2002). In addition to the checkpoint components themselves, an intact kinetochore is also necessary for the checkpoint response as it provides a platform to allow detection of impaired kinetochore microtubule attachments and proper checkpoint signaling (Gardner ( et al., 2001; Goh and Kilmartin, 1993). Of the protein complexes at the kinetochore, the Cbf3 complex has a direct role in checkpoint function since one of its members, S k p l , 13 has been found to associate with B u b l , and this association is necessary for signaling kinetochore tension defects (Kitagawa et al., 2003). The Ndc80.complex also plays a role in checkpoint response by recruiting checkpoint components to the kinetochore and thereby regulating their activity (Janke et al., 2001; McCleland et al., 2003). The Mitotic Spindle and Chromosome Segregation The mitotic spindle is made up of individual microtubules (MTs) organized by two microtubule organizing centers (MTOCs) , which are assembled during mitosis in a polarized manner to physically pull apart sister chromatids during anaphase (Gadde and Heald, 2004). In budding yeast, the mitotic spindle consists of microtubules that emanate from spindle pole bodies (SPBs) and bind to kinetochores (kinetochore microtubules), and M T s that form the central spindle, where microtubules from opposite spindle pole bodies interact in an anti-parallel array (interpolar microtubules). The spindle pole body is also the site of nucleation for astral microtubules that project out into the cytoplasm. Unlike higher eukaryotes, the yeast nuclear membrane does not break down during mitosis and the SPB remains embedded within the membrane during the whole cell cycle (Hoyt and Geiser, 1996; Mcintosh et al., 2002). The SPB is made up of distinguishable layers, divided into three major plaques, which include the outer plaque (on the cytoplasmic face) and the inner plaque (on the nuclear face) that are linked by a central plaque embedded in the nuclear envelope (Francis and Davis, 2000; OToo le et al., 1999). Microtubules nucleated by the M T O C are composed of non-covalent hetero-dimer polymers of P-tubulin (Tub2 in budding yeast) and a-tubulin (Tubl and Tub3 in budding yeast) (Desai and Mitchison, 1997). The a-tubulin and P-tubulin subunits are arranged in a head to tail conformation giving the microtubule polarity. Microtubule plus 14 ends are more dynamic and form the growing head of the filament structures, which associate to form a hollow cylindrical tube of a microtubule (Kline-Smith and Walczak, 2004). As dynamic structures, microtubules are governed by the rate of polymerization, the rate of de-polymerization, the frequency of catastrophe (switch from growth to collapse), and the frequency of rescue (switch from collapse to growth) (Desai and Mitchison, 1997; Gadde and Heald, 2004; Kline-Smith and Walczak, 2004). During polymerization, G T P bound (3-tubulin is incorporated at the growing ends of microtubules, which is then converted to the G D P bound form of (3-tubulin by hydrolysis. Once in the G D P form, (3-tubulin does not exchange and thus forms the majority of the (3-tubulin in the microtubule. A delay between incorporating the G T P bound tubulin and G T P hydrolysis provides a G T P cap on growing ends of microtubules that is thought to provide some stability to the growing plus end. When catastrophes occur, de-polymerizing (3-tubulin rapidly exchanges G D P for G T P allowing tubulin subunits to be reused for polymerization i f rescue occurs. Beyond the intrinsic properties of microtubules, microtubule-associated factors and motor proteins are critical for controlling the dynamic instability of microtubules (Mcintosh and Hering, 1991; Mitchison and Kirschner, 1984), and allow these structures to be harnessed for work (e.g. chromosome segregation). Dynamic instability is thought to allow rapid re-organization of microtubules in response to various stimuli and allow a process such as "search and capture" to occur (Kline-Smith and Walczak, 2004). "Search and capture" is a term used to describe the mechanism by which microtubules find and bind to kinetochores. This consists of microtubules alternately growing and shrinking, probing the nuclear space for 15 kinetochores until attachment occurs. As a result of sister chromatid cohesion, sister kinetochores are oriented away from each other which promotes attachments of sister kinetochores to microtubules emanating from opposite spindle poles (Tanaka et al., 2000). In many organisms, centromeres reside close to the centrosome even before mitosis, which likely mediates rapid capture of microtubules by kinetochores. In budding yeast, it appears that kinetochores may be attached to the SPB for most of the cell cycle (Winey and O'Toole, 2001). Once the sister kinetochore of a mono-oriented chromosome connects to a microtubule emanating from the opposite pole, tension results due to the opposition of microtubule pulling by cohesion. This tension is thought to further stabilize these attachments (Kline-Smith et al., 2005; Pinsky and Biggins, 2005). Failure to form bi-orientated attachment results in the activation of regulating factors such as the Ipl l /Aurora kinase, which destabilize kinetochore microtubule attachments allowing microtubule capture to reoccur (Biggins and Murray, 2001; Dewar et al., 2004; Pinsky et al., 2003; Tanaka, 2002). It is worth noting that microtubules in the absence of spindle poles and kinetochores have also been shown to self assemble into spindles (Heald et al., 1996) and mechanisms in addition to "search and capture" may be at work in mitotic spindle formation (Kapoor et al., 2006; Rieder and Alexander, 1990; Tanaka et al., 2005). Although microtubules are highly dynamic, the exchange rate of tubulin subunits in kinetochore-associated microtubules is much slower than in unattached microtubules (Mcintosh et al., 2002). Kinetochores thus have a stabilizing effect on kinetochore microtubules up to the onset of anaphase, after which depolymerization occurs. This suggests that there are factors at kinetochores that have the ability to influence 16 microtubule stability, which are regulated during mitosis to provide the motive force for chromosome segregation. One such known factor is the D a m l outer kinetochore complex, which has been recently shown to form a collar around kinetochore microtubules functionally stabilizing kinetochore microtubules prior to anaphase. The D a m l collar can then slide along microtubules during anaphase and can be used to capture the force generated by depolymerization (Asbury et al., 2006; Westermann et al., 2006). In support of this model, the D a m l ring structure has been shown to interact with microtubules via the acidic C-tail of tubulin, rather than the microtubule lattice like most other microtubule-interacting factors. This allows passive one-dimensional diffusion of the D a m l ring on the surface of the microtubule. Importantly, this diffusion becomes unidirectional when the microtubule depolymerizes and effectively pushes the ring at the speed of microtubule shrinkage. Cryo-electron microscopy studies of depolymerizing microtubules provide evidence of protofilaments peeling from the ends of microtubules. Overall, this provides an elegant model of force generation where depolymerizing protofilaments push the D a m l ring and the associated kinetochore and sister chromatid in the minus end direction to facilitate chromosome segregation (Mandelkow et al., 1991; Nogales and Wang, 2006). Movement of sister chromatids away from each other at anaphase proceeds in two stages: (1) anaphase A , where chromosomes move closer to the poles, and (2) anaphase B , where the interpolar distance is increased by pushing apart of the SPBs. At the end of anaphase, kinetochores are held very close to SPBs by short microtubules, and the remaining spindle consists of a few interpolar microtubules that interdigitate at the spindle mid-zone, where a number of proteins also reside, such as the yeast kinetochore 17 protein Ndc lO and the Aurora kinase Ip l l (Buvelot et al., 2003). One of these proteins, A s e l , is degraded in an A P C / C dependent manner, linking cell cycle progression control to the spindle cycle (Winey and OToole , 2001). Finally, spindle disassembly occurs, which is necessary for cytokinesis, through the depolymerization of the interpolar microtubules from their plus ends (Winey and OToole , 2001). Two types of motors are thought to be involved in pole separation during anaphase B , the plus-end directed b i m C / K i n N kinesin-related motors, and the minus-end directed dynein. b i m C / K i n N motors are involved in spindle assembly and maintenance, and are thought to crosslink interpolar microtubules with subsequent sliding along two adjacent anti-parallel microtubules toward the plus'end providing a pushing force to separate poles (Hoyt and Geiser, 1996). The minus-end directed motor dynein, and its associated activator complex dynactin, mediates movement of the nucleus into the bud, spindle elongation, and spindle positioning. To accomplish these tasks dynein associates with the cell cortex and with SPBs in a microtubule-dependent manner. When associated with microtubules, dynein is thought to slide along microtubules while being anchored at the cortex providing a pulling force on the SPBs that is also important for pole separation (Yeh et al., 1995). Motors have also been shown to be localized to kinetochores where they may contribute to microtubule attachment and provide some of the motive force required to move chromosomes along microtubules during anaphase. The Kinetochore of Budding Yeast The kinetochore (which consists of centromere D N A and associated proteins) is a macromolecular complex that is critical to the process of chromosome segregation; functioning to mediate the attachment of sister chromatids to the spindle microtubules 18 and direct chromosome movement during mitosis and meiosis (Nasmyth, 2002; Nasmyth et al., 2000). Remarkably, the kinetochore also monitors the state of M T attachment and is critical for sensing the completion of metaphase (bi-polar attachment of all chromosomes) before allowing anaphase to begin (Lew and Burke, 2003; Tanaka, 2002). To fulfill these critical tasks, the kinetochore acts as a central hub where kinetochore proteins, centromeric chromatin, cohesin, spindle checkpoint and MT-associated proteins gather to coordinate chromosome segregation. The budding yeast centromere (CEN) is a "point" centromere and consists of three conserved elements ( C D E I, C D E II, and C D E III) that span 125 base pairs (Loidl , 2003). C D E I and III are conserved sequences while C D E I I is an A/T-r ich region of conserved length and base composition (Hyman and Sorger, 1995). C D E I is not essential for centromere function, whereas the lack of C D E I I and CDEII I or point mutations in CDEIII result in an inactive centromere (Hegemann et al., 1986; M c G r e w et al., 1986). How this small stretch of D N A facilitates assembly of a kinetochore is not known, but one model proposes that C D E I and III serve as sites of interaction with kinetochore proteins, while C D E I I wraps around a specialized centromeric nucleosome (Cheeseman et al., 2002b). However, recent data supports a different model in which yeast centromere D N A is nucleosome-free (Espelin et al., 2003). Proteins associated with budding yeast centromere D N A are assembled in a hierarchical manner in distinct .biochemical sub-complexes (De W u l f et al., 2003), which can be categorized based on their proximity to D N A (Figure 1-2). These include inner kinetochore proteins that directly contact C E N D N A (or are in a complex with proteins that directly interact with C E N D N A ) , central kinetochore proteins that serve as 19 CDE I CDE II CDE III 8bp 78-86bp (>90%AT) 25bp Inner Kinetochore Central Kinetochore Outer Kinetochore Spindle Assembly Checkpoint Other proteins at the kinetochore: Spcl05, Ydr352, Gle2, Slkl9, Cnnl, Plcl, Glc7, Stul, Stu2, Bikl, Biml, Pact, Kar9, Kar3, Kipl, Kip3, Cin8, Mpsl, Hirl and Cacl Figure 1-2. The budding yeast kinetochore. Organization of the budding yeast kinetochore with proteins grouped based upon biochemical purification. Black arrows indicate a dependence on localization and red arrow denotes a enzymatic activity. Figure adapted from (Chan et al., 2005). 20 scaffolding and link the inner kinetochore to the outer kinetochore proteins, and the outer kinetochore proteins that provide the interface required to interact with microtubules (Cheeseman et al., 2002b; Kitagawa and Hieter, 2001; M c A i n s h et al., 2003; Measday and Hieter, 2004). This general design of a kinetochore appears to be well conserved among other eukaryotes as defined by the continued discovery of conservation between individual kinetochore proteins and protein complexes of higher eukaryotes and yeast (Cheeseman et al., 2004; Hayashi et al., 2004; Kitajima et a l , 2004; Kl ine et al., 2006; Obuse et a l , 2004; Okada et al., 2006; Wei et al., 2005; Zhou et al., 2004). These data suggest that the basic building blocks and design of a kinetochore are conserved among eukaryotic organisms. The budding yeast inner kinetochore consists of the C B F 3 complex, the histone H3 variant Cse4, and C b f l (McAinsh et a l , 2003). The C B F 3 complex binds to the CDEIII region of the centromere and is composed of Ndc lO, C t f l 3 , Cep3 and S k p l , whereas C b f l binds to the C D E I region of the centromere. Cse4 is the conserved histone H3 variant that replaces H3 in the histone octamer in and around the centromere region (Cheeseman et al., 2002b). In agreement with its position in the kinetochore, the C B F 3 complex is required for the localization of all known central and outer kinetochore components (Cheeseman et al., 2002b; M c A i n s h et al., 2003). The central kinetochore consists of at least three different sub-complexes based upon their biochemical purification properties, which include the N D C 8 0 complex, the M I N D complex and the C O M A complex. Other proteins of the central kinetochore have been isolated, but have not yet been definitively placed into specific sub-complexes 21 (De Wul f et al., 2003; Measday et al., 2002; Pot et al., 2003). Many of the proteins of the central kinetochore are not essential for viability but the corresponding deletion mutants lose chromosomes at much higher rates than wi ld type strains. In the case of the N D C 8 0 complex, all four subunits (Ndc80, Nuf2, Spc24, and Spc25) are essential and conserved in human cells (DeLuca et al., 2002; He et al., 2001; Janke et al., 2001; McCleland et al., 2003; Wigge and Kilmartin, 2001). Mutations in Spc24 or Spc25 result in the inactivation of the mitotic checkpoint due to the failure of the M a d and Bub spindle checkpoint proteins to localize to the kinetochore, suggesting that the Ndc80 complex is required for their interaction with the kinetochore (He et al., 2001; Janke et al., 2001). The outer kinetochore consists of protein complexes that directly bind to microtubules. This includes the ten members of the D A M 1 complex, which has been shown to bind the mitotic spindle, and D a m l itself, which has been shown to bind microtubules in vitro (Cheeseman et al., 2002a; Kang et al., 2001; L i and Elledge, 2003). The localization of the D A M 1 complex to the kinetochore is dependent on an intact mitotic' spindle since the D A M 1 complex forms a collar around microtubules that is important for capturing the force generated by depolymerizing microtubules for sister chromatid segregation (Miranda et al., 2005; Westermann et al., 2005; Westermann et al., 2006). Other proteins that have been localized to the kinetochore with microtubule binding capabilities include Stu2, B i k l , B i m l and the kinesin related proteins Cin8, K i p l and Kip3 (Cheeseman et al., 2002a; He et al., 2001). Finally, there are a number of proteins that function at the kinetochore in a regulatory manner, and not in a structural capacity. These proteins tend to localize at the kinetochore transiently. This includes: (1) the I p l l / B i r l / S l i l 5 / kinase-complex, which 22 monitors tension across sister chromatids and (2) components of the mitotic spindle assembly checkpoint, which in budding yeast consists of M p s l , M a d l , Mad2, Mad3, B u b l and Bub3 (Lew and Burke, 2003; Pinsky and Biggins, 2005). A subset of kinetochore proteins also re-localize to the mitotic spindle in anaphase and to the spindle mid-zone prior to cytokinesis, reflecting the localization of chromosome passenger proteins in higher eukaryotes (Adams et al., 2001a; Vagnarelli and Earnshaw, 2004). In yeast, the set of chromosome passenger like proteins includes the Aurora kinase I p l l , the I A P repeat protein B i r l , and the inner kinetochore proteins Cep3 and Ndc lO (Biggins et al., 1999; Bouck and Bloom, 2005; Buvelot et al., 2003; Goh and Kilmartin, 1993; Huh et al., 2003; Montpetit et al., 2006; Widlund et al., 2006). The localization of these proteins to the mitotic spindle is thought to be important for regulating mitotic spindle dynamics and for cytokinesis (Adams et al., 2001a; Buvelot et al., 2003; Montpetit et al., 2006). Scope of the Thesis In the years preceding this work, application of technologies such as mass spectrometry on purified protein complexes led to the identification of more than 65 proteins that localize to the budding yeast kinetochore. The speed at which the new kinetochore subunits were identified far outpaced the detailed characterization that is needed to understand the function of each individual protein and how these functions are regulated. For this reason, I have focused on investigating a few essential budding yeast kinetochore proteins from the inner (NdclO), central (Spc24), and outer kinetochore (Spc34). The general approach of this work focused oh the genetic and physical interactions that these individual genes and gene products make, with the goal being to further our basic knowledge of these specific proteins. These basic studies of genes 23 required for chromosome transmission fidelity and their regulation are directly relevant to human biology and disease. In particular, the elucidation of the genetic basis of CIN in model organisms provides a mechanistic basis for understanding CIN in human cancers, and provides candidate genes for those CIN genes mutated in cancer. 24 Chapter 2 Genome-wide synthetic lethal screens identify an interaction between the nuclear envelope protein, Apql2, and the kinetochore in Saccharomyces cerevisiae This work is reprinted (with modifications) from the journal of Genetics (Montpetit B , Thorne K , Barrett I, Andrews K , Jadusingh R, Hieter P, Measday V . Genome-wide synthetic lethal screens identify an interaction between the nuclear envelope protein, A p q l 2 , and the kinetochore in Saccharomyces cerevisiae. Genetics. 2005 Oct; 171(2):489-501) with permission from The Genetics Society of America. Data presented in this chapter are part of a collaboration with Dr .Viv ien Measday (University of British Columbia), and as such, acknowledgements of other's work is given in each figure legend. 25 I n t r o d u c t i o n Budding yeast undergo a "closed mitosis" in which the nuclear envelope (NE) does not breakdown during the course of mitosis, and the spindle pole bodies remain anchored in the nuclear membrane (Hurt et al., 1992). In contrast, mammalian cells undergo an "open mitosis" in which the N E breaks down and chromosomes and centrosomes become part of the cytoplasm. Although the N E does not break down in yeast there is evidence that suggests that proteins associated with the N E have an active role in controlling mitotic progression and chromosome segregation (Kerscher et al., 2001; Makhnevych et al., 2003; Ouspenski et al., 1999). This includes recently identified links between the nuclear pore complex (NPC) and the kinetochore providing a precedent for communication between the N E and the spindle checkpoint machinery (Loiodice et al., 2004; Rabut et al., 2004; Stukenberg and Macara, 2003). For example, the M a d l and Mad2 spindle checkpoint proteins localize to the N P C in both yeast and mammalian cells (Campbell et al., 2001; Iouk et al., 2002). In budding yeast, upon activation of the spindle checkpoint, Mad2 and a portion of M a d l re-localize to the kinetochore (Gillett et al., 2004; Iouk et al., 2002), while in human cells, checkpoint proteins localize to the N P C and upon N E breakdown re-localize to the kinetochore until M T attachment has occurred (Campbell et al., 2001). Studies in multiple eukaryotic systems have shown that the Ndc80 complex is required to localize spindle checkpoint proteins (Mads and Bubs) to the kinetochore and that cells carrying mutations in Ndc80 complex components are defective in checkpoint signaling (Gillett et al., 2004; Maiato et al., 2004). In addition to checkpoint proteins, other components have also been identified that interact with both the kinetochore and N E including Mps2, which localize to both the SPB and N E , are 26 required for SPB insertion into the N E (Munoz-Centeno et al., 1999; Winey et al., 1991). Interestingly, Mps2 interacts in vivo with Spc24, a component of the Ndc80 complex, supporting this kinetochore-NE connection (Le Masson et al., 2002). N E associated proteins have also been identified that are required for chromosome stability, but do not appear to be components of the kinetochore. For example, the nucleoporin Nup l70 was identified in a genetic screen for mutants that exhibit an increased rate of chromosome loss and was subsequently shown to be required for kinetochore integrity, despite the fact that N u p l 7 0 does not interact with centromere D N A (Kerscher et al., 2001; Spencer et al., 1990). Also in support of a kinetochore-NE connection, centromeres cluster near the S P B / N E in interphase and co-localize with SPBs during late anaphase providing the opportunity for a physical interaction between these two macromolecular protein complexes (Goshima and Yanagida, 2000; He et al., 2000; Jin et al., 2000; Pearson et a l , 2001).-Our current understanding of chromosome segregation has benefited from many studies focused on protein purification and mass spectrometry analysis that have been instrumental in identifying structural components of the kinetochore based on the stoichiometric interaction of novel proteins with known kinetochore proteins (Biggins and Walczak, 2003; M c A i n s h et al., 2003). This includes the inner and central kinetochore complexes, which are assembled in a hierarchical manner onto CEN D N A and serve as a link to the D a m l outer kinetochore complex that encircles M T s (Biggins and Walczak, 2003; M c A i n s h et a l , 2003; Miranda et al., 2005; Westermann et al., 2005), and the Ndc80 complex, a highly conserved central kinetochore complex that is required for the D a m l mediated kinetochore microtubule attachment (He et al., 2001; Janke et al., 27 2001; Le Masson et al., 2002; Wigge and Kilmartin, 2001). However, the identification of proteins or pathways that affect chromosome segregation via transient or indirect interactions (e.g. nuclear pore proteins) has remained elusive. Genetic studies, however, have the ability to identify interactions that do not rely on direct protein-protein interaction yet still impact chromosome segregation. For this reason, synthetic genetic arrays (SGA) were used to identify nonessential mutants from the haploid yeast gene deletion set that have a role in chromosome stability (Tong et al., 2001). S G A is a method to automate the isolation of synthetically lethal (SL) or synthetically sick (SS) interactions that occur when the combination of two viable non-allelic mutations results in cell lethality or slower growth than either individual mutant. Two mutants that have a SL/SS interaction often function in the same or parallel biological pathways (Hartman et al., 2001). A s a starting point for S G A analysis, three independent Ts alleles were created in members of both the Ndc80 and D a m l kinetochore complexes. Genome-wide S L screens were performed using S G A methodology with all six Ts mutants as query strains. This study represents the first series of comparative genome-wide S L screens performed on different mutant alleles of the same gene. Using this approach, a novel role for the nuclear envelope protein A p q l 2 was uncovered in maintaining chromosome stability and proper cell cycle progression through anaphase, providing a novel link between chromosome segregation and the nuclear envelope. 28 M a t e r i a l s a n d M e t h o d s Creation of Ts mutants and integration into yeast strains The SPC24 and SPC34 ORFs (642bp and 888bp, respectively) including ~250bp of upstream sequence were amplified by P C R and cloned into pRS316 (Sikorski and Hieter, 1989) to create pRS316-SPC24 (BVM93a) and pRS316-SPC34 (BVM95c) . B V M 9 3 a and B V M 9 5 c were both sequenced to ensure that they carried wi ld type SPC24 and SPC34 sequence, respectively. SPC24 and SPC34 were P C R amplified from B V M 9 3 a and B V M 9 5 c using mutagenic conditions [lOOng template, Taq polymerase ( B R L ) , 2 0 0 u M of dNTPs with either limiting d A T P (40uM) or d G T P (40uM), 2 m M MgCl2 and 25pmol primers]. Next, mutagenized SPC24 and SPC34 were cloned into pRS315 using homologous recombination in strains Y V M 5 0 3 and Y V M 5 0 9 that contained deletions of SPC24 and SPC34 covered by the £//?A5-marked plasmids carrying wi ld type versions of SPC24 (BVM93a) and SPC34 ( B V M 9 5 c ) (Muhlrad et al., 1992). W i l d type plasmids were removed from Y V M 5 0 3 and Y V M 5 0 9 by successive incubation on media containing 5-Fluoroorotic acid. Colonies now carrying mutagenized pRS315-SPC24 or pRS315-SPC34 as the sole source of either gene were incubated at 37° to identify Ts mutants, and plasmids rescued from these strains were re transformed to confirm the Ts phenotype. F A C S analysis was performed on each Ts mutant after incubation at 37° for 2 to 6 hours. Mutants representing different F A C S profiles at 37° -three spc24 (spc24-8, spc24-9, spc24-10) and three spc34 (spc34-5, spc34-6, spc34-7) mutants - were chosen for further analysis. Mutants were next integrated in the genome replacing the wi ld type SPC24 or SPC34 loci in both the S G A starting strain (Y2454) and our lab S288C background strain (YPH499) as described (Tong et al., 2001). Mutants 29 were sequenced and the corresponding amino acids changes are illustrated in Figure 2-1 and 2-2. A l l yeast strains used in this study are listed in Table 2-1. S L screen using S G A methodology The deletion mutant array was manipulated via robotics and the S L screens were performed as described (Tong et al., 2001). Each S L screen was performed twice and double mutants were detected visually for SS /SL interactions. For each query gene, all deletion mutants isolated in the first and second screen were condensed onto a mini-array and a third S L screen was performed. S S / S L interactions that were scored at least twice were first confirmed by random spore analysis (Tong et al., 2004) and then subsequently by tetrad analysis on Y P D medium at 25°. Two-dimensional (2D) hierarchical cluster analysis 2D hierarchical clustering was performed as described (Tong et al., 2001; Tong et al.,2004). Chromosome fragment loss assay Quantitative half-sector analysis was performed as described (Hyland et al., 1999; Koshland and Ffieter, 1987). Homozygous diploid strains containing a single chromosome fragment were plated to isolate single colonies on solid media containing limiting adenine (Spencer et al., 1990). Colonies were grown at either 30° or 35° (see Table 2-3) for three days before incubation at 4° for red pigment development. Chromosome loss or 1:0 events were scored as colonies that were half red and half pink, nondisjunction or 2:0 events were scored as colonies that were half red and half white and over-replication or 2:1 events were scored as colonies that were half-white and half-pink. 30 TABLE 2-1. Yeast strain list Strain Genotype ; Reference DBY1385 MATa his4 ura3-52 tub2-104 D. Botstein DBY2501 MATaura3-52ade2-101 tubl-1 D. Botstein IPY1986 MATa ura3-52, lys2-801, ade2-101, his3A200, leu2Al, trplA63 OKP1- P. Hieter VFP::kanMX6 SPC29-CFP::hphMX4 Y2454 MATa mfal A::MFAlpr-HlS3 canlA ura3AO leu2AO his3Al lys2AO (Tong et al., 2001) YIB329 MATa mfal A:\MFAlpr-HIS3.canl A ura3A0 leu2A0 his3Al lys2A0 spc24- This study 9::URA3 YIB331 MATa mfal A::MFAlpr-HlS3 canlA ura3A0 leu2A0 his3A\ lys2A0 spc24- This study 10::URA3 YIB338 MATa mfal A:\MFAlpx-HlS3 can!A ura3AO leu2A0 his3Ai lys2A0 spc24- This study 8::URA3 YIB343 MATa mfal A:\MFAlpv-HlS3 canlA ura3A0 leu2A0 his3Al lys2A0 spc34- This study 5::URA3 YIB351 MATa mfalA::MFAlpx-HlS3 canlA ura3A0 leu2AO his3A\ lys2A0 spc34- This study 6::URA3 YIB355 MATa mfal A:\MFAlpr-HlS3 canlA ura3A0 leu2A0 his3Al lys2A0 spc34- This study 7::URA3 YM20 MATa ura3-52, lys2-801, ade2-101, his3A200, leu2Al, trplA63 OKP1- This study VFP::kanMX6SPC29-CFP::hphMX4apql2ATRPl YM40 MATa ura3-52, lys2-801, ade2-101, his3A200, leu2Al, trplA63 spc34- This study 5::kanMX6 YM41 MATa ura3-52, lys2-801, ade2-101, his3A200, leu2Al, trplA63 spc34- This study 7::kanMX6 YM61 MATa ura3-52, lys2-801, ade2-101, his3A200, leu2Al, trplA63 This study SPC34::kanMX6 YM192 MATa/aura3-52 ura3-52/, lys2-801/lys2-801, ade2-101/ade2-101, This study his3A200his3A200/, leu2Al/leu2Al, trplA63/trplA63 spc24-8::kanMX61/ spc24-8::kanMX6 CFI1I CEN3.d URA3 SUP11 YM194 MATa/aura3-52 ura3-52/;iys2-801/lys2-801, ade2-101/ade2-101, This study his3A200his3A200/, leu2Al/leu2Al, trplA63/trplA63 spc24-10::kanMX61/ spc24-10::kanMX6 CF1I1 CEN3.d URA3 SUPU YM196 MATa/a ura3-52 ura3-52/, lys2-801/lys2-801, ade2-101/ade2-101, This study his3A200his3A200/tleu2Al/leu2Al,trplA63/trplA63 SPC24::kanMX61/ SPC24::kanMX6 CFIII CEN3.d URA3 SUP11 YM160 MATa/aura3-52 ura3-52/Iys2-801/lys2-801, ade2-101/ade2-101, This study rlis3A200his3A200/,leu2Al/leu2Al,trplA63/trplA63 apql2ATRPl/apql2ATRPl CFVI1 RAD2.d URA3SUP11 YPH272 MATa/aura3-52 ura3-52/ Iys2-801/lys2-801, ade2-101/ade2-101, P. Hieter his3A200his3A200/ leu2Al/leu2Al, trplA63/trplA63 CFVII RAD2.d URA3 SUP11 YPH499 MATa ura3-52, lys2-801, ade2-101, his3A200, leu2Al, trplA63 P. Hieter YPH982 MATa/aura3-52 ura3-52/, Iys2-801/lys2-801, ade2-101/ade2-101, P. Hieter his3A200 his3A200/, leu2Al/ leu2Al, trplA63'/trpl A63 CFIII CEN3.L URA3 SUP11 YVM503 MATa ura3-52, lys2-801, ade2-101, his3A200, leu2Al, trplA63, This study spc24AHlS3, pBVM93a YVM509 MATa ura3-52, lys2-801, ade2-101, his3A200, leu2Al, trplA63, This study spc34AHIS3, pBVM95c 31 YVM1363 YVM1370 YVM1380 YVM1448 YVM1579 YVM1585 YVM1764 YVM1864 YVM1892 YVM1893 YVM1902 YVM1904 YVM1906 YVM1918 YVM1919 adel 101, his3A100, leulAl, trplA63 spc!4-adel 101, his3A100, leulAl, trplA63 adel 101, his3A100, leulAl, trplA63 spc!4-ade2 101, his3A200, leulAl, trplA63 spc!4-adel 101, his3A100, leulAl, trplA63 SPC14-adel 101, his3A100, leulAl, trplA63 spc!4-8 adel 101, his3A100, leulAl, trplA63 adel 101, his3A100, leulAl, trplA63 spc34-10::kanMX6 MATa ura3-51, lysl-8L SPC14::kanMX6 MATa ura3-51, lysl-8l 9::kanMX6 MATa ura3-51, lysl-8( 8::kanMX6 MATa ura3-52, lysl-8L GFP::TRPl::kanMX6 MATa ura3-51, lysl-8L GFP::TRPl::kanMX6 MATa ura3-51, lysl-81. apql2::TRPl 6::kanMX6 MATa/aura3-51 ura3-51/, lysl-801/lysl-801, adel-101/adel-101, his3A100his3A200/, leulAl/leulAl, trplA63/trplA63 spc!4-9::kanMX61/ spc!4-9::kanMX6 CFIII CEN3.d URA3 SUP11 MATa/aura3-51 ura3-51/, lysl-801/lys2-801, adel-101/adel-101, his3A100his3A100/ leulAl/ leulAl, trplA63'/trplA63 apqllATRPl/apqllATRPl CFIII CEN3.L URA3 SUP11 MATa ura3-51, lysl-801, adel-101, his3A100, leulAl, trplA63 apqllr.TRPl SPC14::kanMX6 MATa ura3-51, lysl-801, adel-101, his3A100, leulAl, trplA63 apqllr.TRPl spc!4-10::kanMX6 MATa ura3-51, lysl-801, adel-101, his3A100, leulAl, trplA63 apqllr.TRPl spcl4-8::kanMX6 MATa ura3-51, lysl-801, adel-101, his3A100, leulAl, trplA63 SPC14-GFP::TRPl::kanMX6 apqllr.TRPl MATa ura3-51, 'lysl-801, adel-101, his3A200, leulAl, trplA63 spc!4-8-GFP::TRPl::kanMX6 apqllr.TRPl This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study 32 Preparation of yeast cell lysate and immunoblot analysis: Cells were grown to log phase and then lysed by bead beating (Tyers et al., 1992). 50|ig of protein was loaded per lane, and western blots were performed with a - G F P monoclonal antibodies (Roche) and with a-Cdc28 antibodies (gift from Raymond Deshaies). Cell cycle synchronization To assess the arrest phenotype of spc24 and spc34 Ts mutants (Figures 2-1 and 2-2), strains were grown to an O D 6 0 o of 0.2 at 25° in Y P D media, a-factor (aF) was added (5u,g/uL) and cultures were incubated for 2 hours then released into Y P D at 37°. Samples were taken every hour for F A C S and immunofluorescence microscopy. For apql2A aF and nocodazole (NZ) arrest-release experiments (Figure 2-6), strains were grown to an OD600 of 0.3 at 30°. a F or N Z was added to a final concentration of 5 u M and 20u.g/mL, respectively. Cultures were incubated for 2 hours, and then released into Y P D . Samples were taken every 20 minutes for F A C S and fluorescence microscopy. FACS analysis F A C S analysis was performed as described (Haase and Lew, 1997). Microscopy Genes carrying C-terminal epitope tags were designed according to (Longtine et al., 1998). For immunofluorescence microscopy, cells were fixed in growth media by addition of 37% formaldehyde to 3.7% final concentration, washed and spheroplasted. Y o l 1-34 rat anti-tubulin antibody (Serotec, Oxford, U K ) was used with a fluorescein-conjugated goat anti-rat secondary antibody to visualize M T s in combination with D A P I to stain D N A . Cells were imaged using a Zeiss Axioplan 2 microscope equipped with a 33 C o o l S N A P H Q camera (Photometries) and Metamorph (Universal Imaging) software. Cells used for fluorescence microscopy were fixed by adding an equal volume of 4% paraformaldehyde and incubating for 10 minutes at 25°. Typically, a stack of 10 images was taken at 0.3|j,m spacing and then the images were displayed as maximum projections for analysis. SPB to SPB distances were determined using Metamorph software by measuring the straight-line distance between the brightest Spc29-CFP pixels. Results Isolation of Ts alleles in central and outer kinetochore proteins In order to identify pathways that are important for proper chromosome segregation, a series of S L screens were performed using mutants in essential kinetochore components as query strains. One member of the Ndc80 complex, Spc24, which has previously been shown to have a role in MT-attachment and spindle checkpoint control, and one member of the outer kinetochore D a m l complex, Spc34, which is required for MT-attachment, spindle stability and prevention of monopolar attachment were selected as queries (Janke et al., 2001; Janke et al., 2002; Le Masson et al., 2002; Wigge and Kilmartin, 2001). Genome-wide screening using only a single mutant of spc24 and spc34 might limit the results, depending on the nature of the mutation and the resulting defect in protein function. Thus, a series of Ts mutations in both genes were created and selected for mutants that displayed different arrest phenotypes upon shift to non-permissive temperature. Three alleles were isolated in both SPC24 (spc24-8, spc24-9, spc24-10) and SPC34 (spc34-5, spc34-6, spc34-7) and their effects on D N A content and cell morphology at 37°C was assessed. Strains were arrested in G I phase using the mating 34 pheromone aF , then released to 37°C, and monitored over a period of four hours. Multiple spc24 Ts mutants have been created yet only one arrest phenotype has been published (Janke et al., 2001; Le Masson et al., 2002; Wigge and Kilmartin, 2001). Unexpectedly, all three of the spc24 alleles displayed different arrest profiles (Figures 2-1A and B) . The spc24-10 mutant, which carries four point mutations (Figure 2-1C), duplicates its chromosomes, and elongates its spindles but D N A remains in the mother cells, suggesting a lack of MT-attachment (Figures 2-1A and B) . In addition, D N A re-replicates to 4 N due to continuation of the cell cycle despite the defect in chromosome segregation (Figure 2-1A). Previous groups have described the spc24-10 mutant arrest phenotype and have shown that the failure to arrest at the metaphase to anaphase transition is due to a defect in activation of the spindle checkpoint (Janke et al., 2001; Le Masson et al., 2002; Wigge and Kilmartin, 2001). The other two Ts mutants of spc24 exhibited previously undescribed spc24 arrest phenotypes. spc24-8 carries one mutation in the second predicted coiled-coil domain of Spc24 (Figure 2-1C) and arrests with a short spindle and D N A at the bud neck, indicative of an active checkpoint arrest. The third allele, spc24-9, carries one mutation in the C-terminus of Spc24 (Figure 2-1C) and displays a pleiotropic arrest phenotype. Three hours after shift to non-permissive temperature, -30% of spc24-9 cells have discontinuous spindles, which extend into the mother and break (Figure 2-1B). D A P I staining revealed that three hours after the temperature shift, unequal amounts of D N A have segregated, suggesting that partial M T attachment has occurred. The spindle defect phenotypes of spc24-9 mutants are reminiscent of the effects of some mutations in the D a m l complex (see below). 35 L i I n 2n 1 ii 2n 1 n S 4n I n S 4n l i i k tn 2n In 2n In 2n In 2n i l ^ jL^ji Lti i I A spc24-10 In 2n In 2n In 2n In 2n 4n ln2n ln2n In 2n 4n In 2n 4n aF 2h 3h 4h 37" aF 2h 3h 4h B s/?c24-5 spc24-P jpc24-9 spc24-J0 m I T u b u l i n D A P I 2h 3h 2h 3h 3h c I 51 61 87 122 213 r— .. . . . . . ; I L110P i i V179D 11 I i 16E L38P E66V K.123E t t 1 1 1 SPC24 spc24-8 spc24-9 spc24-10 Figure 2-1. Characterization of spc24 Ts mutants. ( A ) W i l d t y p e (SPC24), spc24-8, spc24-9 a n d spc24-10 s t r a i n s w e r e a r r e s t e d w i t h t h e m a t i n g p h e r o m o n e c c F a t 2 5 ° , a n d t h e n r e l e a s e d t o 3 7 ° a n d s a m p l e s t a k e n a t 2 h o u r s ( h ) , 3 h a n d 4 h . D N A c o n t e n t w a s a n a l y z e d b y flow c y t o m e t r y f o r I N , 2 N o r 4 N D N A c o n t e n t . ( B ) I m m u n o f l u o r e s c e n c e m i c r o s c o p y p e r f o r m e d o n c e l l s i n c u b a t e d a t 3 7 ° f o r 2 h o r 3 h p o s t a F r e l e a s e , a s d e s c r i b e d i n ( A ) . C e l l s w e r e s t a i n e d f o r T u b u l i n a n d D N A ( D A P I ) . ( C ) S c h e m a t i c r e p r e s e n t a t i o n o f a m i n o a c i d ( a a ) s u b s t i t u t i o n s i n spc24 m u t a n t s . G r e y s h a d i n g i d e n t i f i e s a a r e g i o n s o f S p c 2 4 p r e d i c t e d t o b e c o i l e d - c o i l d o m a i n s ( a a 5 1 - a a 6 1 a n d a a 8 7 - a a l 2 2 ) b a s e d o n M u l t i c o i l a n a l y s i s h t t p : / / m u l t i c o i l . l c s . m i t . e d u / c g i - b i n / m u l t i c o i l . D a t a i n t h i s f i g u r e w a s g e n e r a t e d b y V i v i e n M e a s d a y . 36 The three spc34 mutants that were generated fall into two phenotypic classes. spc34-6 and spc34-7 mutants have a metaphase arrest phenotype at restrictive temperature with a short spindle and D N A at the bud neck as described for spc24-8 (Figure 2-2A and B). In addition to having a similar arrest phenotype, spc34-6 and spc34-7 have a common amino acid mutation (S18P). However, they do not display similar genetic interactions (see below), possibly due to the K198E mutation in spc34-6, which is directly adjacent to an Ipll phosphorylation site (T199) (Cheeseman et al., 2002a). spc34-5 mutants have a mixed cell population after 3 hours at non-permissive temperature that have either a discontinuous spindle in the mother that progresses into the daughter cell and breaks (Figure 2-2B, first three panels from the left), or a short spindle and a M T projection (Figure 2-2B, fourth panel from the left). The spc34-5 mutant phenotypes are similar to those caused by the spc34-3 allele described by Janke et al., 2002. Other members of the Daml complex have phenotypes similar to all three of the spc34 mutants described here (Cheeseman et al., 2001a; Cheeseman et al., 2001b; Enquist-Newman et al., 2001; Hofmann et al., 1998; Janke et al., 2002; Jones et al., 1999). Genome-wide SL screen with spc24 and spc34 alleles The spc24 and spc34 mutations were introduced into the SGA query strain and reconfirmed by sequencing and checking the arrest phenotypes at restrictive temperature (data not shown). Genome-wide SL screens were performed twice using SGA methodology by mating each query strain to the yeast deletion set and selecting for double mutants (Tong et al., 2001). Since some of the spc24 and spc34 mutants are inviable at temperatures above 32°CV the SGA screens were performed at 25°C. Any nonessential mutants that were either inviable or slow growing in combination with spc24 37 A spc:34-6 In 2n In 2n In 2n In 2n In 2n In 2n In 2n In 2n 1n 2n I n 2n I n 2n 1 n 2n B C F30L i S18P In 2n In2n In 2n In 2n aF 2h 3h 4h 37" aF 2h 3h 4h spc34-5 spc34-6 spc34-7 T u b u l i n D A P I 295 K 1 9 8 E SI8P N124D j i SPC34 spc34-5 spc34-6 spc34-7 Figure 2-2. Characterization of spc34 Ts mutants. (A) Wild type (SPC34), spc34-5, spc34-6 and spc34-7 strains were arrested with the mating pheromone aF at 25°, and then released to 37° and samples taken at 2h, 3h and 4h. D N A content was analyzed by flow cytometry for IN, 2N or 4N D N A content. (B) Immunofluorescence microscopy performed on cells incubated at 37° at 3h post aF release, as described in (A). Cells were stained for Tubulin and D N A (DAPI). (C) Schematic representation of aa substitutions in spc34 mutants. Data in this figure was generated by Vivien Measday. 38 or spc34 mutants were placed on a miniarray and rescreened against the query strains. From these screens, positive genetic interactors were chosen when identified in at least two screens. Random spore analysis and then tetrad dissection were then performed to confirm double mutant phenotypes. Next, the data was organized by 2D hierarchical clustering, which orders both query genes and array genes based on the number of common interactions (Figure 2-3A). The profile of genetic interactions varied considerably depending on the allele screened. Three central kinetochore mutants (ctf3, ctfl9 and mcm21) were identified in both spc24-9 and spc34-6 S L screens while two additional kinetochore mutants (iml3 and chl4) were identified in the spc24-9 screen alone. To determine i f this is an allele specific interaction with spc24-9 and spc34-6 mutants, all three spc24 mutants were tested directly for genetic interactions via tetrad analysis. Neither spc24-8 nor spc24-10 displayed an S L or SS interaction with the chl4, ctf3, ctfl9 or mcm21 central kinetochore mutant or the madl spindle checkpoint mutant at 25°, suggesting that the interactions with spc24-9 are indeed allele specific at this temperature (Table 2-2). Thus, the defect in the spc24-9 mutant is more sensitive to loss of central kinetochore proteins than the other spc24 mutants. The 2D clustering analysis revealed that the spc24-9 and spc34-6 mutants have the most genetic interactions in common (Figure 2-3B). Many of the genes have known roles in chromosome segregation, chromatin structure, M T dynamics and spindle checkpoint function. Three members of the GimC/Prefoldin complex were identified (gim3, gim.4 and yke2), which is a molecular chaperone that promotes M T and actin 39 t i l l < u i i l l E E s i i i l Array Genes i i 2 5 £ > 1^ V) c u a 3 a B 1 /m/J ct/8 arp6 htzl Igel top3 gim3 spc24-9 O Ism 7 O ira2 O pde2 O /jpz/ O ml06 O ydr290w O ygl088w O ylr235C O c/*2 O leol O rttl03 O subl hcml O ynl296W spc24-9 spc34-6 spc24-8 spc34-7 spc24-10 spc34-5 spc34-6 spc24-9 and spc34-6 • tiki • mat/3 O rim 21 O yml095C-A • c#3 • c//79 • mem 21 • A// / ; / • g/ml • y£e2 • madl O r//7 O apql2 O 55/2 • Chromosome segregatior O Chromatin structure • Microtubule dynamics • Spindle checkpoint O RNA Pol II associated O Other Figure 2-3. spc24 and spc34 genomic SL screen results. (A) 2 D hierarchical cluster analysis of mutants identified in spc24 and spc34 SL screens. Rows are the spc24 and spc34 query genes and columns are the mutants, or array genes, identified in the SL screens. Blue represents SL genetic interactions whereas red represents SS genetic interactions. (B) Unique and common genes identified in the spc24-9 and spc34-6 screens. Genes are color coded based on their cellular roles. Data in this figure was generated by Vivien Measday. 40 TABLE 2-2. Genetic interactions between spc24 a n d gene deletion mutants Query Strain ORF SGD name Function spc24-9 spc24-8 spc24-10 YIL040W APQ12 mRNA export S L a YDR254W CHL4 Central kinetochore SL YLR381W CTF3 Central kinetochore SS YPL018W CTF19 Central kinetochore SL YGL086W MAD1 Spindle checkpoint SS -YDR318W MCM21 Central kinetochore SL a A l l interactions were determined at 25° on YPD media, Synthetic lethal (SL), Synthetic < sick (SS), No phenotype (-). Data in this table was generated by Ken Thorne. 41 filament folding (Geissler et al., 1998; reviewed in Haiti and Hayer-Hartl, 2002; Siegers et al., 1999; Vainberg et al., 1998). Genome-wide SL screens have been performed using members of the GimC/Prefoldin complex as query strains and have identified genetic interactors representing a wide variety of cellular processes (Tong et al., 2004). Two members of the SWR1 chromatin remodeling complex (arp6, swc6) were also identified, which function to incorporate the histone H2A variant, Htz l (which was also found in this screen) into nucleosomes (Korber and Horz, 2004). Other genes identified include, two components of the Pafl elongation complex, rtfl and leol, plus three additional mutants that interact with R N A polymerase II (ctk2, rtt!03 and subl) (Henry et al., 1996; Kim et al., 2004; Knaus et al., 1996; Mueller and Jaehning, 2002; Sterner et al., 1995). Interestingly, the apq!2 mutant was identified in both the spc24-9 and spc34-6 screens. apql2 mutants are known to have defects in nuclear transport (Baker et al., 2004), and recent evidence suggests that nuclear transport is specifically regulated during mitosis (Makhnevych et al., 2003). Thus, this potential link between nuclear transport and kinetochore function was investigated further. APQ12 genetically interacts with the kinetochore APQ12 encodes a small 16.5kD protein with a predicted transmembrane domain that localizes to the NE (Baker et al., 2004; Huh et al., 2003). Recently, it was shown that apql2 deletion mutants produce hyperadenylated mRNAs and accumulate poly(A) + R N A in the nucleus suggesting a defect in mRNA export (Baker et al., 2004). Unlike most mRNA transport mutants however, apq!2 mutants also have an aberrant cell morphology suggesting that Apql2 has additional cellular functions (Baker et a l , 2004; Saito et al., 2004). apq!2 deletion mutants are specifically SS/SL in combination with 42 either spc24-9 or spc34-6 mutants but do not display a phenotype when combined with the other spc24 or spc34 mutants at 25° (Table 2-2 and data not shown). However, when tested for genetic interactions between apq!2A and the other spc24 alleles at temperatures higher than that used in the genome-wide screens (25°C), it was found that spc24-10 was SS in combination with apql2A at 30°C and the spc24-8 apql2A double mutant was SS at 33°C (Figure 2-4A). Thus all three of the spc24 mutants exhibit an unexpected genetic interaction with a mutant that has m R N A export defects. Conceivably, A p q l 2 could contribute to the nuclear export of SPC24 m R N A and thereby affect Spc24 cellular protein levels. Combining an SPC24 m R N A export defect with a spc24 Ts mutation could result in low levels of the Spc24 protein, which is essential, and may explain the lethality of apql2 spc24 Ts double mutants. To determine i f Spc24 protein levels are perturbed in apql2A strains, the abundance and localization of GFP-tagged Spc24 in a log phase apql2A cell population was analyzed. Both Spc24-GFP and scp24-8-GFP protein levels and localization are not noticeably different between a wi ld type and apq!2A strain (Figure 2-5A). spc24-8-GFP still remains Ts and the strain arrests with the same F A C s profile as in Figure I A suggesting that the G F P tag does not alter the behaviour of the spc24-8 mutant (data not shown). Spc24-GFP localizes to punctate foci that are close to each other in small budded cells and separated in the mother and daughter in large budded cells (Figure 2-5B wild type panel). Spc24-GFP still localized to distinct foci in an apql2 mutant suggesting that kinetochore clustering and the structure of the Ndc80 complex is not significantly altered in apql2A strains (Figure 2-5B, apql2A panel). Thus, the Spc24 protein stability and localization data suggests that the Spc24 protein is not severely affected in apq!2A strains. Therefore, apq!2 mutants. 43 SPC24 apq!2A IB # # #,l* SPC24 apql2A spc24-8 spc24-8 apql2A • # ; spc24-10 spc24-10 apql2A 25° 30° 33° B SPC24 [ I T 0 9 spc24-8 # # ;.> • spc24-9 iiSti&Bk uSSSSi, A »Ma *S® spc24-10 apql2A V Wr V 9 * tubl-1 mm*** •ft tub2-104 DMSO 15u,g/ml benomyl 20u.g/ml benomyl Figure 2-4. Growth defects and benomyl sensitivity of spc24 and apqll mutants. (A) W i l d type (SPC24), apql2A , SPC24 apql2A, spc24-8, spc24-8 apq!2A, spc24-10 and spc24-10 apq!2A strains were grown to log phase, diluted to an OD600 of 0.1 and sequential five-fold dilutions were done. Cells were spotted onto Y P D plates and incubated at 25°, 30° and 33° for two days. (B) W i l d type (SPC24), spc24-8, spc24-9, spc24-10, apql2A, tubl-1 and tub2-104 strains were grown to log phase, diluted to an OD6oo of 0.1, sequential five-fold dilutions were performed and spotted onto D M S O control, 15u.g/ml and 20u,g/ml benomyl plates and incubated at 30° for 2 days. Data in this figure was generated by Ken Thorne and Ben Montpetit. 44 CL CL & •s. ±< 1 x O L I r i i Ou 5 I Op <] 4 a <N "2. C/5 Q B Spc24p-GFP4-DIC <-spc24-8p-GFP N<-Cdc28p G F P DIC G F P 1 Spc24p-GFP wild type Spc24p-GFP a/Hjl2A DIC V F P C F P Overlay O k p l p - V F P Spc29p-CFP wild type O k p l p - V F P Spc29p-CFP apq!2A Figure 2-5. Spc24 protein levels and sub-cellular localization of Spc24 and Okpl in apt/12 mutants. (A) Spc24-GFP and spc24-8-GFP protein levels in an apql2A strain. Spc24-GFP, Spc24-GFP apql2A, untagged, spc24-8-GFP and spc24-8-GFP apql2A strains were grown to log phase at 30° and lysates were prepared. Western blots were performed and immunostained with an anti-GFP antibody (upper panel). As a loading control, the anti-GFP blot was re-probed with an anti-Cdc28 antibody (lower panel). (B) Spc24-GFP sub-cellular localization in an apql2A strain. DIC and fluorescent images of Spc24-GFP localization in wild type and apql2A strains. GFP signal appears green. (C) Okpl-VFP Spc29-CFP subcellular localization in an apql2A strain. DIC and fluorescent images of Okpl-VFP Spc29-CFP localization in wild type and apql2A strains. VFP signal appears green and CFP signal appears red. Data in this figure was generated by Ben Montpetit. 45 are unlikely to have a specific defect in SPC24 m R N A export To determine i f apql2 mutants have a defect in overall kinetochore structure, the localization of the central kinetochore protein, O k p l - V F P , was analyzed in relation to the SPB protein Spc29-CFP in apql2A cells. Kinetochore proteins localize to the nuclear side of each SPB in cells with short spindles and co-localize with SPBs in cells with long spindles (Goshima and Yanagida, 2000; He et al., 2000; Pearson et al., 2001). O k p l - V F P Spc29-CFP localization was unperturbed in apql2A cells suggesting that kinetochore structure and dynamics are not greatly altered in apql2 mutants (Figure 2-5C). apql2 mutants have aberrant chromosome segregation To determine i f apql2A strains have a chromosome segregation defect, a colony color based half-sector assay was used to monitor the maintenance of a nonessential chromosome fragment (CF) (Koshland and Hieter, 1987). C F missegregation was also assayed in the three spc24 Ts mutants to compare C F loss phenotypes between the different mutants. As expected, the spc24 mutants showed an increased rate of chromosome loss (1:0 events) compared to wi ld type (Table 2-3). Interestingly, spc24-9 had a much higher rate of chromosome loss (380 fold increase) compared to spc24-8 and spc24-10 (33 and 14 fold increase respectively) at 30°C. apq!2 mutants did not display a significant increase in chromosome loss compared to the wi ld type strain but did show a dramatic increase (58.8 fold over wild type) in 2:1 segregation events when plated at 35°C. For 2:1 segregation to occur in the first mitotic division (which gives rise to half-sectored colonies), the parental cell must over-replicate the C F to three or more copies and segregate two or more copies to one cell and one copy to the other cell. It is also possible that there might be a selective advantage for apq!2 deletion strains to acquire 46 TABLE 2-3. Chromosome loss events in spc24 and apqll mutants Strain Number Genotype (a/a) Tem P (°C) Total Colonies Chromosome Loss (1:0 events) Nondisjunction (2:0 events) Over-Replication or Nondisjunction (2:1 events) YM196 SPC24::kanMX6/ SPC24::kanMX6 CFIII CEN3.d URA3 30° 24,976 2 x IO"4 (1.0) 2.8 x IO"4 (1.0) 0 YM192 spc24-8::kanMX6/ spc24-8::kanMX6 CFIII CEN3.d URA3 30° 25,488 6.6 x 10"3 (33) 0 0 YVM1892 spc24-9::kanMX6/ spc24-9::kanMX6 CFIII CEN3.d URA3 30° 7,606 7.6 x 10"2 (380) 1.3 x IO"4 (0.5) 0 YM194 spc24-10::kanMX6/ spc24-10::kanMX6 CFIII CEN3.d URA3 30° 23,824 2.8 x 10"3 (14) 0 0 YPH982 wild type CFIII CEN3.L URA3 35° 17,440 4.6 x IO"4 (1.0) 4.1 x IO"3 (1-0) 1.7 x IO"4 (1.0) YVM1893 apql2A/apql2A CFIII CEN3.L URA3 35° 47,612 6.5 X IO"4 (1-4). 2.0 X IO"3 (0.5) 1.0 x IO-2 (58.8) YPH272 wild type CFVII RAD2.d URA 35° 29,224 1.7 x 10"4(1.0) 1.4 x 10"5 (1-0) 1.7 x IO"4 (1.0) YM160 apql2A/apql2A CFVII RAD2.d URA3 35° 35,759 8.1 x IO"4 (4.8) 0 7.4 x 10"3 (43.5) Data in this table was generated by Irene Barrett. 47 an extra copy of the CF (CFIII CEN3.L) that contains part of chromosome III, even though the wild type copy of APQ12 is located on chromosome IX and thus not on the CF. Therefore, the screen was also performed using a different CF (CFVII RAD2.d) and there was still a significant increase (43.5 fold over wild type) in 2:1 segregation events for apql2A strains (Table 2-3). Mutants that have defects in chromosome segregation, including kinetochore mutants, are often sensitive to the microtubule depolymerizing drug benomyl. Although the precise mechanism of benomyl action is not known, evidence suggests that it may bind to the a and ^-tubulin hetero-dimers thereby inhibiting M T formation (Richards et al., 2000). apql2A mutant strains and the three spc24 Ts mutants, as well as tubulin mutant control strains were spotted on plates to test their sensitivity to benomyl. These tests surprisingly showed that apql2A strains are resistant to benomyl and grow nearly as well as a tub2-104 resistant allele on 20ug/ml benomyl plates at 30°C (Figure 2-4B). In contrast, the spc24-9 and spc24-10 mutants are sensitive to 15ug/ml and 20ug/ml benomyl (Figure 2-4B). Resistance to benomyl suggests that apql2 mutants may have stabilized MTs or high levels of tubulin. Anti-tubulin immunofluorescence was performed on fixed apql2 log phase cells but no noticeable differences in M T formation were apparent (data not shown), although the resolution may not be sufficient to detect minor changes in M T levels or structure. Thus, apql2A strains may have stabilized MTs or be resistant to benomyl due to an indirect mechanism. apql2 mutants have defects in exiting mitosis The 2:1 CF segregation phenotype and benomyl resistance of apql2A mutants are indicative of problems during mitosis. Thus, the progression of a wild type and apql2A 48 strain through the cell cycle was monitored by synchronizing cells in G I with the mating pheromone a F and releasing them into the cell cycle. Each strain carried a kinetochore and a SPB marker ( O k p l - V F P and Spc29-CFP, respectively). Samples were taken every 20 minutes for D N A profiling by F A C S analysis and for kinetochore-SPB localization analysis by fluorescence microscopy. After release from the a F arrest, wi ld type and apql2A strains showed similar timing of bud emergence and S P B duplication by analyzing fixed cells (data not shown). F A C S analysis indicated that the timing of D N A replication was also similar (Figure 2-6A, compare 40 minute time points). However, apq!2A cells showed a delay in the reapperance of I N cells by -20 minutes (Figure 2-6A, compare 100 minute time points), and that the population of I N cells remained small compared to wi ld type throughout the time course (Figure 2-6A). W i l d type cells showed a transient appearance of 4 N D N A which disappeared by 160 minutes, whereas the apq!2A cells had a 4 N population of cells from 40 minutes onwards suggesting that a percentage of apql2 mutants are re-replicating D N A prior to exiting mitosis (Figure 2-6A). apql2A cells also had an increased proportion of 2 N cells when arrested with aF , suggesting that a percentage of apq!2A G I cells are carrying an extra copy of all chromosomes or that a percentage of apql2A cells do not respond to mating pheromone (Figure 2-6A, 0 minutes, apq!2A panel). These observations are consistent with a delay for apql2 mutants in progression through mitosis and a failure to complete mitosis prior to initiating replication. To define the stage of mitosis that is delayed in apql2A mutants, a similar experiment using the MT-depolymerizing drug N Z was performed, which arrests cells in metaphase. apql2 mutants arrest primarily with a 2 N population of cells after two hours of exposure to N Z and with a peak of 4 N cells that persists throughout the 49 T i m e (m inu tes ) Figure 2-6. apql2 mutants are delayed in anaphase and prematurely enter a new cell cycle. WT and apq!2A cells were released from aF or NZ arrest and sampled every 20 minutes. (A) aF and (B) NZ treated cells were assessed for D N A content by FACS analysis. (C) Average SPB to SPB distances in cells sampled from each NZ arrest time point. SPBs were marked with Spc29-CFP and the distance was quantified from immunofluorescent images. (D) Distribution of SPB to SPB distances at 60 minutes after NZ release. (E) Percentage of multi-budded cells at each time point after NZ release. (F) DIC image of multi-budded cells at 60 minutes post NZ release in an apq!2A mutant. For each time point (C-E) 100 cells were analyzed. Experiments were performed in duplicate with representative data from one experiment shown. Data in this figure was generated by Ben Montpetit. 50 apql2A time course (Figure 2-6B). Although a small peak of 4 N cells is detectable in wild type cells responding to N Z , a greater proportion of apql2A cells contain a 4 N quantity of D N A in the N Z imposed G2 arrest (Figure 2-6B). The D N A profile of apql2A cells released from N Z arrest showed a~20 minute delay in the reappearance of I N cells compared to wi ld type cells as was seen in the G I synchronization experiment (Figure 2-6B, compare 20 and 40 minute time points). The distance between two Spc29-C F P foci was also monitored, as an indicator of spindle length, and it was found that wi ld type and apql2A cells entered anaphase with similar kinetics as judged by spindle elongation. However, anaphase spindles persisted longer in the apq!2A cell population suggesting that apq!2A cells are delayed during mitosis (Figure 2-6C). More specifically, at 60 minutes post N Z release, 70% of wi ld type cells had a spindle length of 1 to 1.99|im suggesting that they had progressed from anaphase to G I (Figure 2-6D). In contrast, only 19% of the apq!2A cells had a 1 to 1.99u,m spindle and 28% of the cells had a spindle between 8 and lOum suggesting that the cells were still in anaphase (Figure 2-6D). In addition, 12% of apq!2 mutants had spindle lengths greater than 10u,m whereas none of the wi ld type spindles reached this length. Finally, the apq!2 mutant population contained multi-budded cells throughout the time course, even immediately after release from N Z exposure, suggesting that some cells might be breaking through the mitotic checkpoint arrest (Figure 2-6E). The appearance of apq!2A cells with multiple buds and 4 N D N A content suggests that cells are attempting to re-enter the cell cycle prior to completing cytokinesis and that the spindle checkpoint is not sensing the defect causing apql2A cells to spend extended time in anaphase. 51 Discussion A genome-wide S L screens using novel mutations in kinetochore proteins was employed to uncover novel pathways important for chromosome stability when kinetochore function is compromised. A component of the N E , A p q l 2 , was identified that had not been previously linked to chromosome segregation. The data demonstrates that A p q l 2 has a role in the timely execution of anaphase and maintenance of chromosome stability and provides evidence that the N E is intimately linked with chromosome segregation. The results of the S L screens highlight the importance of using multiple alleles of essential genes as queries in S G A analysis. Moreover, allele specific interactions provide information about functional domains of the query protein. For instance, the S L data suggests that Spc24 can be divided into distinct functional domains. spc24-9, which carries a mutation in the C-terminus of Spc24, was SS or S L with the chl4, ctf3, ctfl9, iml3 and mcm21 central kinetochore mutants. spc24-8 and spc24-10, which carry mutations in the N-terminal region of Spc24 that contains two coiled-coil domains, did o not display genetic interactions with central kinetochore mutants at 25 C . spc24-9 mutants also have a much higher rate of chromosome loss than spc24-8 and spc24-10 mutants (Table 2-3). Thus, it is likely that the C-terminal mutation in spc24-9 affects a different Spc24 function or protein-protein interaction than the spc24-8 and spc24-10 mutants. This data is consistent with a recently published structural analysis of the Ndc80 complex which demonstrates that the C-terminus of Spc24 is a globular domain that likely interacts with the kinetochore (Wei et al., 2005). In addition to identifying numerous central kinetochore mutants, two negative 52 regulators of the c A M P pathway were also identified, ira.2 and pde2 in the spc24-9 genome-wide S L screen (Figure 2-3). Interestingly, PDE2 was recently identified as a high copy suppressor of D a m l kinetochore complex mutants (L i et al., 2005). Five negative regulators of the c A M P pathway, including ira2 and pde2, were also identified as benomyl sensitive mutants in genome-wide screens (Pan et al., 2004). Thus, upregulation of the c A M P pathway by mutation of its negative regulators appears to have a deleterious affect on kinetochore function. The apql2 mutant, which has defects in m R N A nucleocytoplasmic transport (Baker et al., 2004), was identified in both the spc24-9 and spc34-6 screens. Given-the role of A p q l 2 in m R N A transport, one possibility is that A p q l 2 could direct the export of specific m R N A s expressing kinetochore proteins. In this hypothesis, mutation of APQ12 could cause nuclear retention of these m R N A s and improper expression of their protein products. However, Spc24 protein levels and localization are not altered in apql2 mutants suggesting that Spc24 protein expression is not affected . In addition, the O k p l central kinetochore protein displayed a typical kinetochore localization pattern in apql2A cells suggesting that the kinetochore is intact. Finally, apql2 mutants are resistant to benomyl, whereas cells carrying mutations in kinetochore components or spindle checkpoint proteins are often benomyl sensitive, suggesting that apql2 mutants do not contain reduced levels of kinetochore and spindle checkpoint proteins. Since apql2 mutants appear to have normal levels of kinetochore proteins, A p q l 2 could have a direct role in chromosome segregation and cell cycle regulation by coordinating the localization of specific protein components to the N E . Recently identified links between the N P C and the kinetochore give precedent for communication 53 between the N E and the spindle checkpoint machinery (Loiodice et al., 2004; Rabut et al., 2004; Stukenberg and Macara, 2003). For example, the M a d l and Mad2 spindle checkpoint proteins localize to the N P C in both yeast and mammalian cells (Campbell et al.," ,2001; Iouk et al., 2002). The localization patterns of M a d l and Mad2 were analyzed in apql2 mutants during both normal cell growth and in response to the spindle checkpoint induced by N Z ; however, no changes in M a d l or Mad2 localization in apql2A strains were detected compared to a wi ld type strain suggesting that A p q l 2 is not required to sequester spindle checkpoint proteins in the N E (data not shown). Thus, A p q l 2 has an alternative role at the N E , perhaps by sequestering or trafficking other chromosome segregation proteins via the N E and N P C . apql2 mutants are delayed during mitosis and accumulate in anaphase, suggesting a defect in mitotic exit. Using two methods of cell synchrony, it was found that a small percentage of apql2 mutants re-replicate their D N A and rebud prior to completing cytokinesis. During mitosis, the transition from metaphase to anaphase is marked by degradation of the anaphase inhibitor protein Pds l (Cohen-Fix et al., 1996; Yamamoto et al., 1996a; Yamamoto et al., 1996b). Stabilization of P d s l is a hallmark of cells that are actively responding to the spindle checkpoint pathway, thus pdsl mutants are defective in the spindle checkpoint response. Recent genetic studies identified an S L interaction between pdsl and apql2 and between mad2 and apql2 (Sarin et al., 2004). Therefore, the mitotic defects of apql2A mutants render the spindle checkpoint pathway essential during normal cell growth. A p q l 2 is one of a growing member of N E associated proteins that have a role in chromosome stability and mitotic progression. For example, Sac3 is a nuclear pore 54 associated protein that connects transcription elongation with m R N A export (Fischer et al., 2002). sac3 deletion mutants accumulate in mitosis as large budded cells with extended M T s , are resistant to benomyl and have an increased rate of chromosome loss compared to wi ld type strains (Bauer and Rol l ing, 1996; Jones et al., 2000). In a previous genome-wide S L screen, a genetic interaction between sac3 and cep3-2 (an inner kinetochore protein) was also identified, further supporting a role for Sac3 in chromosome segregation (Measday et al., 2005b). A component of the SPB called Mps3 is another example of the connection between the N E and chromosome. Mps3 interacts with the Ctf7 cohesin protein and is required to maintain wi ld type levels of cohesion between chromosomes (Antoniacci et al., 2004). In mammalian cells, where the N E disassembles, multiple proteins located at the N P C re-localize to the kinetochore upon N E breakdown (Stukenberg and Macara, 2003). Ran is a small GTPase that regulates the interaction of cargo proteins with nucleoporins. The Ran GTPase activating protein, R a n G A P l and its associated nucleoporin RanBP2 are targeted to kinetochores in a M T and Ndc80 complex dependent fashion (Joseph et al., 2004). In addition, R a n G A P l and RanBP2 are required for the kinetochore localization of both spindle checkpoint and kinetochore proteins and for maintaining kinetochore-MT interactions (Joseph et al., 2004). Although no nucleoporin has been shown to re-localize to the kinetochore in yeast, the Nnf 1 kinetochore protein was originally identified from a purification of N E proteins and yeast cells depleted of N n f l accumulate poly(A)+ R N A (Shan et al., 1997) . The molecular mechanism by which N E proteins, such as A p q l 2 and kinetochore proteins interact may be a conserved cellular process that functions to promote proper chromosome segregation and mitotic progression. 55 Chapter 3: Sumoylation of the budding yeast kinetochore protein NdclO is required for NdclO spindle localization and regulation of anaphase spindle elongation The work presented in this chapter is reprinted (with modifications) from the Journal of Cell Biology (Montpetit B , Hazbun T, Fields S, Hieter P. Requirement for sumoylation of the budding yeast kinetochore protein NdclO for NdclO spindle localization and proper anaphase spindle elongation. J. Cel l B i o l : 2006 Sept; 174: 653-663.) with permission from The Rockefeller University Press, copyright 2006. I am solely responsible for all data presented in this chapter, data analysis, and manuscript writing with the exception of Table 3-3, which was generated by Tony Hazbun (Purdue University) and Stan Fields (University of Washington). 56 Introduction Proper completion of mitosis depends on the proper co-ordination of chromosome (e.g. chromosome segregation) and cytoskeletal events (e.g. cytokinesis). The factors important for regulating chromosome related events are well known, and include the S A C , which monitors the attachment of sister chromatids to the spindle apparatus (Lew and Burke, 2003). However, less is known about the mechanism that coordinate the chromosome cycle with the mitotic spindle and cytokinesis. Candidates for regulating these events include chromosome passenger proteins (CPPs), which are proteins that localize to centromeres during early mitosis and then move to the spindle mid-zone during anaphase (Adams et al., 2001a; Vagnarelli and Earnshaw, 2004). This temporal re-localization provides an excellent mechanism to coordinate events at these different sub-cellular locations, in that CPPs tasks at the spindle mid-zone could not be completed until the CPPs are released from the centromere upon completion of the centromere related tasks. In yeast, the set of chromosome passenger like proteins includes the Aurora kinase Ip l l and the I A P repeat protein B i r l , and the inner kinetochore protein Ndc lO (Biggins et al., 1999; Bouck and Bloom, 2005; Buvelot et al., 2003; Goh and Kilmart in, 1993; Huh et al., 2003; Widlund et al., 2006). The localization of these proteins to the mitotic spindle is thought to be important for regulating mitotic spindle dynamics and for cytokinesis (Adams et al., 2001b; Bouck and Bloom, 2005; Buvelot et al., 2003; Widlund et al., 2006). Indeed, temperature-sensitive alleles of ndclO-1 or birl-33 cause cytokinesis defects at the restrictive temperature (Bouck and Bloom, 2005; Gi l l i s et al., 2005). However, mutations in B i r l that result in loss of NdclO from the mitotic spindle cause defects only in proper spindle elongation, suggesting that the function of the spindle 57 bound pool of NdclO is related to spindle function, not cytokinesis (Widlund et al., 2006). Ndc lO and Cep3 are both members of the C B F 3 inner kinetochore complex that is required for the localization of all other kinetochore proteins (He et al., 2001), with loss of function mutations disrupting microtubule kinetochore attachments and spindle assembly checkpoint function (Fraschini et al., 2001; Goh and Kilmartin, 1993). Recently, it was shown that the whole C B F 3 complex is transported to the spindle mid-zone where it localizes with the plus-ends of microtubules. Furthermore, Ndc lO was shown to localize to M T plus-ends during spindle disassembly and along dynamic M T s in G I (Bouck and Bloom, 2005). This pattern of localization highlights three possible roles the C B F 3 complex, and NdclO, could be playing when associated with M T s : 1.) GBF3 could contribute to spindle stability; 2.) mid-zone C B F 3 could act as a signal to coordinate spindle disassembly with completion of chromosome segregation; and 3.) C B F 3 associated with M T s plus-ends may represent a "pre-kinetochore" structure that participates in establishing M T attachment in the next cell cycle during the process of. "search and capture". To gain insight into the function of Ndc lO on the mitotic spindle, protein interacting partners of Ndc lO were identified with the goal of discovering proteins required for NdclO's spindle localization and subsequent spindle functions. Using a genome-wide two-hybrid screen, multiple interactions between Ndc lO and the sumoylation machinery of budding yeast were found, and subsequent analysis demonstrated that Ndc lO is a target for sumoylation in vivo, as are other kinetochore proteins ( B i r l , Cep3, and Ndc80). 58 Sumoylation plays a key role in many cellular processes including nuclear transport, signal transduction, transcriptional regulation, and maintenance of genome integrity (Hay, 2005; Johnson, 2004; Seeler and Dejean, 2003). Sumoylation is a process by which a Small Ubiquitin-like MOdi f i e r protein, S U M O (Smt3 in yeast), is conjugated to a target protein at a lysine residue (Figure 3-1). S U M O belongs to a family of ubiquitin-like proteins (Ubls) sharing -20% identity with ubiquitin, which was first discovered in mammals to be conjugated to R a n G A P l (Mahajan et al., 1997; Matunis et al., 1996). The conjugation of S U M O to target proteins makes use of an enzyme cascade that is similar to ubiquitination and includes an E l activating enzyme that binds to S U M O and activates S U M O for subsequent binding to an E 2 protein, or S U M O conjugating enzyme. A n E3 complex, S U M O ligase, may then facilitate the modification of a target protein by mediating the interaction between the target and E 2 - S U M O complex. However, one of the unique features of the sumoylation process is the ability of the S U M O E2 (Ubc9) to recognize substrate proteins without the aid of an E3 . Typically this occurs provided that the lysine to be modified is part of the S U M O conjugation motif ^ K x E where ¥ is any large hydrophobic residue and x is any residue (Rodriguez et al., 2001). It is worth noting that S U M O modification sites are known that do not conform to this consensus motif, nor are all matching consensus sites in a protein modified by S U M O . This suggests that although Ubc9 can recognize substrates, other factors are important for modification such as substrate presentation to Ubc9 or subcellular localization of the substrate. L ike most other ubiquitin-like proteins, S U M O must be post-translationally matured by proteolytic cleavage to reveal the di-glycine motif used to form the iso-59 Target Sumo Maturation of SUMO Deconj ligation 9  l i  ^ ^ P M k J ^ ^ ^ ^ Sumo ™ ^ Ulp J I I Activation by E l Target Sumo A o s l uba2 S u m o E3 mediated \ ligation J 1 ^  ^ Transesterfication to E2 E3 Figure 3-1. SUMO modification cycle. The process o f sumoylation starts by proteolytic cleavage o f Sumo to expose a di-glycine motif. Once matured, Sumo is activated and transferred to an E2 protein that then conjugates Sumo to the target protein via an iso-peptide bond with the help o f an E3 ligase. Sumo modified substrates can then be deconjugated by the action o f Ubiquitin-like proteases (Ulps) to regenerate Sumo and the unmodified form o f the target. peptide bond with its substrate. Maturation of S U M O is carried out by ubiquitin-like proteases (ULPs) that are also able to cleave S U M O from modified substrates making sumoylation a reversible process (L i and Hochstrasser, 1999; L i and Hochstrasser, 2000). The consequences of sumoylation are substrate specific, and can involve altering a substrate's interaction with other macromolecules (e.g. proteins and D N A ) , or blocking lysine residues on target proteins from being modified by other lysine-targeted modifications such as ubiquitin (Desterro et al., 1998; Hoege et al., 2002). In mammalian cells, defects in sumoylation cause abnormal nuclear architecture, chromosome mis-segregation, and embryonic lethality (Nacerddine et al., 2005). In the budding yeast Saccharomyces cerevisiae, sumoylation is essential for viability and is required for proper chromosome segregation with sumoylation-deficient cells arresting in G 2 / M with short spindles and replicated D N A (Li and Hochstrasser, 1999; L i and Hochstrasser, 2000; Seufert et al., 1995). SMT3, which was originally identified as a high copy suppressor of a mutation in the kinetochore protein M i f 2 (Meluh and Koshland, 1995), encodes the ubiquitin-like protein S U M O in yeast. These phenotypes highlight a role for S U M O modification in the cell cycle, and more specifically mitosis. For example, both D N A topoisomerase II and Pds5 have been shown to be sumoylated, with this modification being important for the role these two proteins play in sister chromatid cohesion (Bachant et al., 2002; Stead et al., 2003). Likewise, it was also found that D N A Topoisomerase II is a target for S U M O modification in Xenopus egg extracts, which is required for proper chromosome segregation. However, in budding yeast these proteins alone do not account for all the defects observed in S U M O deficient cells, thus the critical protein targets that lead to these phenotypes remain unknown. This is further 61 illustrated by the fact that in yeast strains defective for S U M O conjugation, A P C / C activity is downregulated and targets critical for cell cycle progression (e.g. Securin and mitotic cyclins) are not degraded, but the actual targets being modified by S U M O that are required for A P C / C activity are not known (Dieckhoff et al., 2004). Recent attempts in budding yeast to expand the number of S U M O substrates by proteomic means have identified greater than 400 potential sumoylation targets; thus, pinpointing the biologically relevant sumoylation events remains a challenge. (Denison et al., 2005; Hannich et al., 2005; Panse et al., 2004; Wohlschlegel et al., 2004; Wykoff and O'Shea, 2005; Zhou et al., 2004). Within this set of putative substrates are many proteins involved in chromosome segregation, including kinetochore proteins (Denison et al., 2005; Hannich et al., 2005; Hoege et al., 2002; Panse et al., 2004; Wohlschlegel et al., 2004; Wykoff and O'Shea, 2005; Zhou et al., 2004) However, the functional role(s) of sumoylation on these proteins are not understood. Work presented in this chapter demonstrate that sumoylation of Ndc lO, Ndc80, Cep3 and B i r l is differentially regulated in response to checkpoint activation suggesting that sumoylation has distinct roles in modulating the function of these kinetochore proteins. Importantly, lysine residues required for NdclO 's sumoylation are necessary for NdclO's proper localization to the mitotic spindle, suggesting that sumoylation plays a direct role in facilitating NdclO 's interaction with the spindle apparatus. Mis-localization of Ndc lO from the mitotic spindle is not associated with cytokinesis defects, suggesting that the spindle-bound form of NdclO does not have a role in cytokinesis as previously thought. Furthermore, loss of NdclO 's mitotic spindle association resulted in anaphase spindles of abnormal length, highlighting a role for NdclO in mitotic spindle dynamics. 62 M a t e r i a l s a n d M e t h o d s Yeast Strains, plasmids, and microbial techniques Yeast strains (S288C background) and plasmids used in this study are listed in Table 3-1 & 3-2. Mutant alleles were integrated into the yeast genome at the endogenous locus by co-transformation of a P C R product carrying the desired mutation(s) and a P C R product containing a nutritional marker (URA3). Integration of the desired mutation was confirmed by D N A sequencing. To arrest cell cultures, aF (1 mg/ml in methanol) or N Z (5 mg/ml in D M S O ) was added and cultures were incubated for 2hrs at 30°C. To assay benomyl sensitivity, benomyl was added at the indicated concentration to Y P D media, dimethyl sulfoxide was used in the control plate (0 pg/ml benomyl), and five fold serial dilutions were spotted on the plates and then grown at 30°C for 2 days. F low cytometry analysis to monitor D N A content was performed as previously described (Haase and Lew, 1997). Genome-wide Two-hybrid screen N D C 10 was cloned into p O B D 2 and p B D C using standard techniques (Cagney et al., 2000; Mi l l son et al., 2003). Fusion of the D N A binding domain to the C-terminus (pBDC), but not the N-terminus of NdclO (pOBD2), resulted in a functional protein as judged by the ability to rescue a ndclOA strain. Two independent genome-wide two-hybrid screens were performed using an activation domain array (Hazbun et al., 2003), as described previously (Uetz et al., 2000). The two-hybrid positives from these genome-wide screens were reconfirmed by repeating the two-hybrid assay. The identities of the activation domain fusions were confirmed by rescuing plasmids and sequencing. 63 T A B L E 3-1. Yeast St ra in L i s t Strain Genotype Source DBY1385 MATa his4 ura3-52 tub2-104 D. Botstein DBY2501 MATa ura3-52 ade2-101 tubl-1 D. Botstein PWY 194a MATa ade2-loc can]-100 his3-ll,15 leu2-3,l 12 trpl-lura3-l ade3A birlA::hphMX3 birl-9A-13MYC ::kanMX6::URA3 lys2A::HIS3 NDC10-YFP::kanMX6 (Widlund et al., 2006) PWY 204a MATa ade2-loc canl-100 his3-ll,15 leu2-3,112 trpl-lura3-l ade3A birlA::hphMX3 birlA1AP-13MYC ::kanMX6::URA3 lys2A::HIS3 (Widlund et al., 2006) PWY93-3ba MATa ade2-loc can!-100 his3-ll,15 leu2-3,112 trpl-lura3-l ade3A BIR1-13MYC::kanMX6 (Widlund et al., 2006) YPH 1734 MATa ura3-52, lys2-801, ade2-101, his3A200, leu2Al, trplA63 NDC10-13MYC::kanMX6 P. Hieter YPH 1793 MATa ura3-52, lys2-801, ade2-101, his3A200, leu2Al, trplA63 SLI15-VFP: :kanMX6 SPC29-CFP: :hphMX3 This study YPH 1794 MATa ura3-52, lys2-801, ade2-101, his3A200, leu2Al, trplA63 CEP3-13MYC::His3MX6 This study YPH 1798 MATa ura3-52, lys2-801, ade2-101, his3A200, leu2Al, trplA63ASEl-GFP::kanMX6 This study YPH 1799 MATa ura3-52, lys2-801, ade2-101, his3A200, leu2Al, trplA63 NDC10-13MYC::kanMX6 nfil A:: His3MX6 This study YPH 1800 MATa ura3-52, lys2-801, ade2-101, his3A200, leu2Al, trplA63 NDC80-13MYC::His3MX6 This study YPH 1801 MATa ura3-52, lys2-801, ade2-101, his3A200, leu2Al, trplA63 NDC10-13MYC::kanMX6 ulp2A:His3MX6 This study YPH 1802 MATa ura3-52, lys2-801, ade2-101, his3A200, leu2Al, trplA63 NDC10-3MYC::His3MX6 This study YPH 1803 MATa ura3-52, lys2-801, ade2-101, his3A200, leu2Al, trplA63 NDC10-13MYC: :kanMX6 sizl A: :His3MX6 This study YPH 1804 MATa ura3-52, lys2-801, ade2-101, his3A200, leu2Al, trplA63 NDC10-13MYC::kanMX6pRS306-ULP2 (2<um,URA3) This study YPH 1805 MATa ura3-52, lys2-801, ade2-101, his3A200, leu2Al, trplA63 NDC10-13MYC::kanMX6 pRS306-ULPl (2jjm, URA3) This study YPH 1806 MATa ura3-52, lys2-801, ade2-101, his3A200, leu2Al, trplA63 ndclO K556R-J3MYC::kanMX6 This study YPH 1807 MATa ura3-52, lys2-801, ade2-101, his3A200, leu2Al, trplA63 ndclO K779R-13MYC::kanMX6 This study YPH 1808 MATa ura3-52, lys2-801, ade2-101, his3A200, leu2Al, trplA63 ndc80 K231R-13MYC:: kanMX6 This study YPH 1809 MATa ura3-52, lys2-801, ade2-101, his3A200, leu2Al, trplA63 CDC10-GFP::His3MX6 This study YPH 1810 MATa ura3-52, lys2-801, ade2-101, his3A200, leu2Al, trplA63 CDC10-GFP::His3MX6 ndclO K556.651,652, 779R::URA3 This study YPH 1811 MATa ura3-52, lys2-801, ade2-101, his3A200, leu2Al, trplA63 NDC10-13MYC::kanMX6sizlAr.TRPl nfilA::His3MX6 This study YPH 1812 MATa ura3-52, lys2-801, ade2-101, his3A200, leu2Al, trplA63 NDC10-13MYC::kanMX6 mms21-ll::TRPl This study YPH MATa ura3-52, lys2-801, ade2-101, his3A200, leu2Al, trplA63 SLI15- This study 64 1814 VFP::kanMX6 SPC29-CFP::hphMX3 ndclO K556,651,652,779R::URA3 YPH 1816 MATa ura3-51, lys2-801, ade2-J01, his3A200, leu2Al, trplA63 ASE1-GFP::kanMX6ndclO K556,651,651, 779R::URA3 This study YPH 1817 MATa/aura3-52/ura3-52, lysl-801/lysl-801, ade2-101/ade2-101, his3A200/his3A200, leulAl/leulAl, trplA63VtrplA63 ndclO K556,651,652,779R::URA3/ndclO K556,651,652,779R::URA3 CFIII CEN3.L TRP1 SUP 11 This study YPH 1818 MATa ura3-52, lys2-801, ade2-101, his3A200, leu2Al, trplA63 ndclO K651.652R- 13MYC::kanMX6 This study YPH 1819 MATa ura3-52, lysl-801, ade2-101, his3A200, leulAl, trplA63 CEP3-GFP::His3MX6 This study YPH* 1820 MATa ura3-51, lysl-801, adel-101, his3A100, leulAl, trplA63 ndclO K556,651,651,779R::URA3 CEP3-GFP::His3MX6 This study YPH 1821 MATa ura3-51, lysl-801, adel-101, his3A100, leulAl, trplA63 B1R1-. GFP::His3MX6 This study YPH 1822 MATa ura3-51, lysl-801, adel-101, his3A100, leulAl, trplA63 BIR1-GFP::His3MX6 ndclO K556,651,651, 779R::URA3 This study YPH 1823 MATa ura3-51, lysl-801, adel-101, his3A100, leulAl, trplA63 63 ndclO K556,651,651,779R::URA3 BIRl-13MYC::His3MX6 • This study YPH 1824 MATa ura3-51, lysl-801, adel-101, his3A100, leulAl, trplA63 CEP3-13MYC-:His3MX6 ndclO K556.651, 651,779R::URA3 This study YPH 1825 MATa ura3-51, lysl-801, adel-101, his3A100, leulAl, trplA63 B1R1-13MYC::His3MX6 This study YPH 1826 MATa ura3-51, lysl-801, adel-101, his3A100,leulAl, trplA63 BIR1-13MYC::His3MX6 ndclO-l This study YPH 1827 MATa ura3-51, lysl-801, adel-101, his3A100, leulAl, trplA63 NDC80-13MYC::His3MX6ndclOK556.651, 651,779R::URA3 This study YPH 1828 MATa ura3-51, lysl-801, adel-101, his3A200, leulAl, trplA63 ndclO K556,651,651,779R-3MYC:: His3MX6 This study YPH 1829 MATa ura3-51, lysl-801, adel-101, his3A100, leulAl, trplA63 NDC10-13MYC::kanMX6birlA::His3MX6pRS3I6-BIRl(pPW02CEN,URA3) This study YPH . 1830 MATa ura3-51, lysl-801, adel-101, his3A100, leulAl, trplA63 NDC10-13MYC::kanMX6 birlA::His3MX6 pRS316-birl-9A (pPW124 CENJJRA3J This study YPH 1831 MATa ura3-51, lysl-801, adel-101, his3A100, leulAl, trplA63 NDC10-13MYC::kanMX6 birlA::His3MX6pRS316-birl-AIAP(pPW08 CEN,URA3J This study YPH 1832 MATa ura3-51, lys2-801, ade2-101, his3A100,leulAl, trplA63 BIR1-13MYC::His3MX6cep3-l This study YPH 1833 MATa ura3-51, lysl-801, adel-101, his3A100, leulAl, trplA63 BIR1-13MYC::His3MX6ctf 13-30 • . ' This study YPH 1834 MATa ura3-51, lysl-801, adel-101, his3A200, leulAl, trplA63 NDC10-GFP::His3MX6 CFP-TUB1 ::URA3 This study YPH 1835 MATa ura3-52, lys2-801, ade2-101, his3A200, leulAl, trplA63 ndclO K556,651,651,779R -GFP::His3MX6 CFP-TUB1 ::URA3 This study YBM 1015 MATa ura3-51, lysl-801, adel-101, his3A100, leulAl, trplA63 NDC10-3MYC::HISMX3 pGAlL-3XHA -CDC5: :kanMX This study YBM 186 MATa ura3-51, lysl-801, adel-101, his3A100, leulAl, trplA63 NDC10-13MYC::kanMX6madlA::HlSMX3 This study YPH 499 MATa ura3-51, lysl-801, adel-101, his3A100, leulAl, trplA63 P. Hieter a = W303 background 65 TABLE 3-2. Plasmid list Plasmid Description Source or Reference BPH 995 pOBD2-NDC10 This study BPH 996 pBDC-NDCIO This study BPH 997 GST-NDC10inpGex4t-2 This study BPH 998 GST-ndclO K556R in PGex4t-2 This study BPH 999 GST-ndclO K651.652A in pGex4t-2 This study BPH 1000 GST.-ndc 10 K779R in pGex4t-2 This study pPW02 BIR1 in pRS316 (Widlund et al., 2006) pPW124 birl-9xA in pRS316 (Widlund et al., 2006) pPW08 birl-AIAP in pRS316 (Widlund etal., 2006)' In vitro and in. vivo sumoylation assays G S T - N d c l O was expressed in E. coli strain B L 2 1 from a pGex 4T-2 vector (Amersham Biosciences) and purified using glutathione beads. In vitro sumoylation reactions were carried out using bacterially expressed proteins (expression plasmids provided by Lawrence Mcintosh, University of British Columbia, Vancouver, B C , Canada) as previously described (Macauley et al., 2005), and western blotting was performed with an Ndc lO antibody (Measday et al., 2005a). To detect sumoylation in vivo, yeast cells (100-200 OD600) were lysed by bead beating (10 X 30sec) in 2.5 ml of lysis buffer (50 m M Tr i s -HCl [pH 8.0], 5 m M E D T A , 150 m M N a C l , 0.2% Triton X -100, Roche Complete protease inhibitor (1 tablet per 25mL), 10 m M N-ethylmaleimide [ N E M ] , 2 m M phenylmethylsulfonyl fluoride, and 20 pg each of leupeptin, aprotonin and pepstatin per ml) on ice. Lysates were cleared by centrifugation at 30,000g for 20 min, and soluble protein concentrations were determined by protein assay (Bio-Rad). Equal amounts of protein were incubated with a-Myc-conjugated beads for 3hrs at 4°C, and then washed four times with cold lysis buffer for 2 minutes. Immunoprecipitated protein was then eluted with lysis buffer containing 2% SDS at 42°C for 15 minutes. 2.5uL of the eluted protein was used for western blotting with a-Myc A b to confirm pull down of the tagged protein; 25ul was used for blotting with a - S U M O A b to detect sumoylated protein. To detect the Ndc lO B i r l interaction, 25ul of a B i r l IP was used for western blotting with a-NdclO Ab . a - S U M O polyclonal antibodies were generated in rabbits (Covance Research Products) as previously described (Johnson and Blobel , 1999). Initial experiments also made use of an a - S U M O polyclonal antibody provided by Erica Johnson (Thomas Jefferson University, Philadelphia, P A ) . 67 Light, fluorescence and immunofluorescence microscopy Strains used for microscopy were grown in either Y P D (for synchronous culture experiments) or in F P M (minimal medium supplemented with 2 X adenine and containing 6.5 g/L sodium citrate). Cells were imaged at room temperature using a Zeiss Axioplan 2 microscope with a P L A N A P O C H R O M A T 100X/1.4 D I C oi l immersion objective with Zeiss filter set #38, #47, and Chroma filters 488000. 3D Images (0.25 p m steps) were acquired with a C o o l S N A P H Q camera (Roper Scientific) and analyzed using Metamoprh software (Molecular Devices). Images are presented as maximum intensity two-dimensional projections. For fluorescence microscopy, wi ld type and mutant proteins were tagged at the endogenous locus with G F P or a G F P variant (Longtine et al., 1998). T u b l - C F P : : U R A 3 containing strains were generated by integrating plasmid pSB375 (a gift from Kerry Bloom, University of North Carolina, Chapel H i l l , N C ) digested with StuI at the U R A 3 locus. Spindle length measurements were performed on asynchronous cultures of Tub 1-CFP containing cells fixed in 70% ethanol and 200mM Tr i s -HCl [pH 8.0] judged to be in anaphase by the presence of part of the spindle in the daughter cell. To visualize haploid budding pattern, cells were incubated in P B S with 20 pg/ml Calcofluor white (Fluorescent Brightener 28; Sigma Aldrich) at 25°C for 5 min, washed in P B S , and visualized for bud scar staining. Chromosome instability assays Quantitative half-sector analysis was performed as described ((Hyland et al., 1999; Koshland and Hieter, 1987). To perform the diploid bimater assay, 10 single colonies were patched onto Y P D plates and then replica plated to both M A T a and M A T a lawns and mating products were selected. The median number of mating products from 68 the 10 patches was compared to W T in order calculate the increase in frequency over mating of a W T diploid strain. Results NdclO interacts with multiple components of the sumoylation machinery In two independent genome-wide two-hybrid screens using Ndc lO as bait, with the Gal4 DNA-bind ing domain fused to either the N - or C-terminus, ten proteins were identified as putative Ndc lO protein interactors (Table 3-3). Three of these interactions occurred with only one of the baits, suggesting that the presence of the Gal4 D N A -binding domain on the N - or C-terminus may be interfering with specific protein-protein interactions, as has been previously reported (Millson et al., 2003). Of these ten interacting proteins, B i r l and Ubc9 were previously identified in two-hybrid screens with NdclO (Jiang and Kol t in , 1996; Yoon and Carbon, 1999); B i r l has also recently been shown to play a role in the localization of Ndc lO to the mitotic spindle (Bouck and Bloom, 2005; Widlund et al., 2006). To investigate the role the remaining proteins may have in mediating the spindle localization of NdclO, proteins were tagged with C F P and microscopy was performed to assay for co-localization with N d c l O - V F P . Only B i r l - C F P co-localized with Ndc lO on the mitotic spindle as previously observed (Bouck and Bloom, 2005; Widlund et al., 2005), furthermore deletion of H E X 3 , NFI1 , S AP 1 or FIR1 resulted in no change in N d c l O - V F P localization (data not shown). Given that NdclO plays an essential role in chromosome segregation, deletion of H E X 3 , NFI1 , S A P 1 , or FIR1 were also checked for C I N , but no increase in C I N was observed (data not shown). It is worth noting here, that both F i r l and N i s i localize to the bud neck, placing them in the correct locale to participate in the recently described roles for N d c l O and the C B F 3 69 TABLE 3-3. NdclO two-hybrid interactions Prey p O B D 2 - N d c l 0 a b p B D C - N d c l O b c Biological process d BIR1 +++++ +++++ chromosome segregation, mitotic spindle elongation CTF13 - ++ chromosome segregation, kinetochore assembly FIR1 +++++ +++++ mRNA polyadenylylation HEX3 +++ +++ protein sumoylation NFI1 +++ +++ protein sumoylation NISI +++ +++ regulation of mitosis SAP1 ++++ - unknown SLI15 + + chromosome segregation SMT3 . + + protein sumoylation UBC9 - ++++ protein sumoylation a = encodes NdclO with the GAL DNA binding domain fused to the N-terminus b = two-hybrid positive colony growth from weak(+) to strong (+++++) c = encodes NdclO with the GAL DNA binding domain fused to the C-terminus d = obtained from the Saccharomyces Genome Database (http://www.yeastgenome.org/) Data in this table was generated by Tony Hazbun (Purdue University). 70 complex in cytokinesis(Bouck and Bloom, 2005; Gi l l i s et al., 2005). Within this set of two-hybrid interacting proteins, four components of the sumoylation machinery were also identified (Ubc9, Smt3, Hex3 and N f i l ) . SMT3, which was originally identified as a high copy suppressor of a mutation in the kinetochore protein M i f 2 (Meluh and Koshland, 1995), encodes the ubiquitin-like protein S U M O . N d c l O is sumoylated in vitro and in vivo Given SMT3's genetic interaction with the kinetochore, and the number of two-hybrid interactions between Ndc lO and the sumoylation machinery, it is likely that these interactions occur because Ndc lO is a target for sumoylation. To address this possibility, bacterially expressed G S T - N d c l O was tested as a substrate for sumoylation in an in vitro sumoylation reaction. Western blot analysis of the reaction products revealed two SUMO-modif ied forms of Ndc lO that were generated in an ATP-dependent manner, indicating that Ndc lO is sumoylated in vitro (Figure 3-2a). To determine whether Ndc lO is modified by S U M O in vivo, Ndc lO was immunoprecipitated (IP) from a yeast cell lysate and detected SUMO-modif ied proteins by western blot analysis using an antibody that recognizes the yeast S U M O protein. Two signals were apparent by western blotting that corresponded to the correct molecular weight to be SUMO-modif ied forms of NdclO (Figure 3-2b). Both of these signals changed electrophoretic mobility upon switching the tag on NdclO from thirteen to three copies of M Y C , demonstrating that both of these signals represent in vivo SUMO-modif ied forms of Ndc lO and not a co-precipitated protein (Figure 3-2b). The presence of two modified forms of Ndc lO both in vitro and in vivo suggests that Ndc lO may be sumoylated on at least two lysine residues. Other post-translational modifications and/or poly-sumoylation on a single lysine residue, however, 71 A. ATP B. 13Myc3Myc 2 5 0 — 150. i in i tint a-Ndc10 150 m^0 a-SUMO 150. a-Myc IP: NddO c. 250. 150 S i ^ , - ^ a-SUMO a-Myc IP: NddO Figure 3-2. Sumoylat ion of the kinetochore protein Ndc lO . (A) Ndc lO is sumoylated in vitro. ATP-dependent sumoylation reactions were performed with recombinant proteins purified from E. coli without (-) or with (+) A T P . The asterisk and arrows denote unmodified and S U M O modified forms of NdclO, respectively. (B) Ndc lO is sumoylated in vivo. (C) The E 3 proteins S i z l and N f i l function in Ndc lO sumoylation. 72 cannot be ruled out as reasons for the appearance of multiple modified forms of Ndc lO from these data alone. The fraction of S U M O conjugated Ndc lO is estimated to be - 1 % or less, which is consistent with the level of modification of most known S U M O substrates. The addition of S U M O is often facilitated by a protein ligase (E3) (Hay, 2005; Johnson, 2004) and the observed two-hybrid interaction between the E3 protein N f i l and Ndc lO suggests that N f i l may act as an E3 for Ndc lO sumoylation. Mutation of known E3 proteins (nfilA , sizlA, and mms21-ll) individually had no effect on Ndc lO S U M O modification levels (Figure 3-2c); however, in the absence of both NFI1 and SIZ1, Ndc lO sumoylation was reduced (Figure 3-2c), indicating a functional redundancy between N f i l and S i z l in targeting NdclO for sumoylation. It is worth noting that Ndc lO has also been implicated as a substrate for ubiquitination (Kopski and Huffaker, 1997; Yoon and Carbon, 1995), raising the possibility of alternative regulation through both sumoylation and ubiquitination; however, in preliminary experiments a ubiquitinated form of NdclO has not been identified. During the course of this work, Ndc lO was also identified as a sumoylated protein in a proteomic analysis of sumoylated proteins in yeast (WohlschlegeJ et al., 2004). Dynamics of N d c l O sumoylation To-begin investigating the possible function(s) of Ndc lO sumoylation, the modification state of Ndc lO at discrete arrest points within the mitotic cell cycle was assayed. The mating pheromone alpha factor (aF) and the microtubule-destabilizing drug nocodazole (NZ) were used to arrest cells in G I and G 2 / M , respectively. The terminal 73 arrest of each population after treatment was verified by flow cytometry (data not shown). IP/westerns showed that sumoylation of NdclO was decreased to almost undetectable levels in those cells treated with N Z , while it was maintained in aF-treated cells (Figure 3-3a). Loss of sumoylation is not a general consequence of N Z treatment since other sumoylated proteins were not affected (see Ndc80, Figure 3-5a) or exhibit increased levels of sumoylation upon N Z treatment (Johnson and Blobel , 1999). Moreover, the loss of sumoylation is not a result of the G 2 / M arrest since Ndc lO does not become desumoylated in cells that are arrested in G 2 / M with temperature-sensitive alleles in the anaphase promoting complex/cyclosome (Figure 3-3a). To further analyze NdclO sumoylation during the cell cycle, the modification state of Ndc lO was monitored in a synchronized population of cells as they progressed through mitosis following release from aF arrest (Figure 3-3b). Timing of the progression of cells through the cell cycle was monitored by flow cytometry (Figure 3-3c). The modification state of NdclO remained relatively constant throughout the synchronized cell cycle, and in replicate experiments, the fluctuation observed between individual timepoints was not consistent, suggesting that Ndc lO sumoylation does not change dramatically over the cell cycle. These results suggest that the loss of NdclO sumoylation in N Z arrested cultures may be a direct consequence of N Z addition relating to checkpoint activation and/or loss of microtubules. Specific cysteine proteases are responsible for cleaving S U M O from modified substrates and for maturation of the S U M O protein itself (Johnson, 2004). Of the two S U M O proteases in yeast, Ulp2 localizes to the nucleus, placing it in the proper location to mediate the removal of S U M O from NdclO during N Z exposure. In ulp2A cells with 74 A. B. 2 5 0 . 150 . Log A F NZ cdc26A IP:Ndc10 2 5 0 . IP:Ndc10 c. a-SUMO "'MV 0 150 . 135 120 0 15 30 45 60 75 90 105 120 135 Time (min) 1 0 5 D. ULP2 + + + A A A 90 ^ Nocodazole - + + - + + eo 2 5 ° " b a-SUMO 45 -430 15 0 min 150 ^\^y~*^iamt*mOt-MyC 2hrs 3hrs 2hrs 3hrs IP:Ndc10 Figure 3-3. Dynamics of NdclO sumoylation. (A) NdclO sumoylation is reduced in N Z treated cells. IP/western blots were performed on protein extracts from logarithmic, G I (aF), G 2 / M (NZ) arrested yeast cell cultures or cdc26A culture arrested in G 2 / M by shift to the non-permissive temperature of 37°C for 3hrs. (B) Ndc lO sumoylation is constant over an unperturbed cell cycle. A n a F synchronized population was released into rich media at 30°C and samples for analysis by IP/western were taken every 15 minutes. (C) D N A content was analyzed at each 15 minute interval by flow cytometry to determine cell cycle progression. (D) Loss of sumoylation in N Z requires Ulp2. Cells wild-type (+) or mutant (A) for the S U M O protease Ulp2 were assayed for sumoylation after treatment of cells with D M S O (-) or D M S O + N Z (+). 7 5 N Z , it was found that the sumoylated forms of NdclO were still present (Figure 3-3d); therefore, Ulp2 is required for the loss of NdclO sumoylation in response to N Z . Loss of NdclO Sumoylation is linked to checkpoint activity To distinguish between the possibility that Ndc lO sumoylation is lost during N Z treatment due to checkpoint activation or the loss of microtubules, Ndc lO sumoylation was assayed in two conditions where microtubules are absent, but the spindle checkpoint remains inactivated. The first condition tested for a loss of Ndc lO sumoylation in G I arrested cells (non-mitotic), and the second tested Ndc lO sumoylation in anaphase arrested cells (checkpoint is silenced through A P C / C mediated proteolysis (Palframan et al., 2006)), which are arrested by controlling the expression of C D C 5 through the G A L promoter. In both cases, after cells were arrested and treated with N Z there was no observable change in the level of NdclO S U M O modification (Figure 3-4a).. This demonstrates that in response to nocodazole, Ndc lO ' s S U M O modifications are not lost due to an absence of microtubules, but is likely removed in a checkpoint dependent fashion. To verify this, Ndc lO sumoylation was assayed in a yeast strain that has had the checkpoint abolished through deletion of M A D 2 . A s expected, in the mad2A strain, the addition of nocodazole did not result in a loss of sumoylation supporting the conclusion that loss of Ndc lO sumoylation is checkpoint dependent (Figure 3-4b). Kinetochore proteins Cep3 and Ndc80 are sumoylated To understand i f sumoylation is a common modification on kinetochore proteins, a panel of fourteen proteins comprised of inner (Cep3, Mif2) , central (Chl4, C n n l , Ctf3, C t f l9 , Iml3, Ndc80, Nuf2, Spc24) and outer kinetochore components (Daml , Spc34) were tested for modification by S U M O . The spindle checkpoint protein B u b l and the 76 A. 250 150 B. o o a-SUMO —• -150. • . a-Myc • IP:Ndc10 IP:Ndc10 C. <? .* ^  & 250. 150-250-150-a-SUMO a-Myc IP:Ndc10 Figure 3-4. Loss of S U M O from Ndc lO in nocodazole treated cells is checkpoint dependent. (A) IP/western blots were performed on protein extracts from logarithmic or G I arrested cultures with or without the addition of nocodazole (NZ). (B) Cells carrying G A L - 3 x H A - C D C 5 were arrested in anaphase by growth in dextrose containing media to shutoff Cdc5 expression and tested for Ndc lO sumoylation in the absence or presence of N Z . (C) Cells wild-type (WT) or mutant (mad2A) for the checkpoint gene M A D 2 were assayed for sumoylation after treatment with or without N Z . 77 kinetochore-associated microtubule-binding protein B ik lwere also tested. Of all proteins tested, Cep3 and Ndc80 were found to be sumoylated by IP/western, with the detection of a doublet signal for Cep3, and a ladder of sumoylated forms of Ndc80 (Figure 3-5a). Like NdclO, the fraction of S U M O conjugated Cep3 is 1% or less, while Ndc80 is modified at slightly higher levels (-1-5%). Ndc80 and Cep3 were also found to be sumoylated throughout the cell cycle, and in replicate experiments the fluctuations observed between individual timepoints was not consistent, suggesting that Ndc80 and Cep3 sumoylation, as observed for NdclO, is not altered dramatically over the cell cycle (Figure 3-5b). Ndc80 was recently identified as a sumoylated protein in a proteomic analysis of sumoylated proteins in yeast by mass spectrometry, along with the kinetochore proteins B i r l , SH15, and Mcm21(Bachant et al., 2002; Denison et al., 2005; Hannich et al., 2005; Hoege et al., 2002; Panse et al., 2004; Stead et al., 2003; Wohlschlegel et al., 2004; Wykoff and O'Shea, 2005; Zhou et ah, 2004). Analysis revealed that Cep3, a member of the C B F 3 complex with Ndc lO that also localizes to the mitotic spindle (Bouck and Bloom, 2005), showed decreased sumoylation in response to N Z , an effect similar to that seen for Ndc lO (Figure 3-5a). For Ndc80, which does not localize to the mitotic spindle, sumoylation does not change in NZ-treated cells, suggesting that desumoylation during the N Z induced spindle checkpoint arrest (Figure 3-5a) may be associated only with those proteins interacting with the mitotic spindle. Furthermore, the differing response in N Z is indicative of a distinct role for Ndc80 sumoylation relative to that of Ndc lO and Cep3. Identification of lysine residues required for NdclO, Ndc80, and Cep3 sumoylation Sumoylation often occurs on lysine residues found in the consensus motif Y K x E 78 A. Log NZ , o o _ L ° 9 N Z mm:z a -SUMO JL 7 5 « v a«| " " 1 5 0 IP: Cep3 Ndc80 B. IP:Ndc80 250 — 1 5 0 — • " • I P O < U ~ | S L i ! « -SUMO TEA tan I < ECJ h t J ted M t ' 100- — a-Myc 0 15 30 45 60 75 90 105 120 135 Time (min) i o o — IP: Cep3 :*M<, avssUeJtafll a-SUMO 7 5 . 0 15 30 45 60 75 90 105 120 135 Time (min) Figure 3-5. Sumoylation of the kinetochore proteins Cep3 and Ndc80. (A) Cep3 and Ndc80 are sumoylated in vivo. (B) Ndc80 and Cep3 sumoylation is constant over an unperturbed cell cycle. A aF synchronized population was released into rich media at 30°C and samples for analysis by IP/western were taken every 15 minutes. D N A content was analyzed at each 15 minute interval by flow cytometry to determine cell cycle progression (data not shown; see Figure 3-3c for reference). 79 where *F is any large hydrophobic residue and x is any residue (Johnson, 2004). To identify the possible site(s) of sumoylation in NdclO, Ndc80, and Cep3, select lysine residues in amino acid sequences that resemble the consensus sequence for sumoylation were mutated. In total, 11, 14, and 6 potential sumoylation sites were mutated in NdclO, Ndc80, and Cep3, respectively (Table. 3-4). Strains expressing each K—»R mutant were assayed for growth at 37°C, chromosome instability (CIN), and changes in sumoylation by IP/western. None of the 31 K—>R individual mutations in Ndc lO, Cep3 or Ndc80 caused a Ts phenotype or C I N . However, lysine residues were identified that when mutated caused an overall decrease in the S U M O modification state of Ndc lO (K651, 652R and K779R) or abolished one specific SUMO-modif ied form of Ndc lO (K556R) (Figure 3-6a). Given Ndc lO ' s localization to the mitotic spindle, these three Ndc lO K—>R mutants were also assayed for proper sub-cellular localization. In all three cases, the localization of Ndc lO was indistinguishable from that of W T (data not shown). Of the 14 K—>R mutations in Ndc80, one lysine mutation (K231R) abolished the majority of Ndc80 sumoylation (Figure 3-6b), while none of the 6 K—>R mutations in Cep3 affected its sumoylation state (data not shown). The complete loss of sumoylation in Ndc80 through a single mutation suggests that K231 may be required for S U M O modification of other lysine residues, which leads to the ladder of modified species, or that this site is poly-sumoylated. To distinguish between these possibilities, Ndc80 sumoylation was checked in a strain that carries a form of S U M O with mutations at positions K l l , 15, and 19 that eliminate the formation of poly-sumoylated chains (Bylebyl et al., 2003). In Ndc80 IP/Westerns using this strain, the ladder of modified Ndc80 proteins recognized by the S U M O antibody remained present, indicating that the 80 T A B L E 3-4. Potent ia l s u m o y l a t i o n sites targeted b y site d i rec ted mutagenesis NdclO Ndc80 Cep3 K102R: E K R E K155R: L K Q P K23R: V K C D K240R: L K L G K231R: I K L D K265R: F K N F K243R: G K R D K292R: L K L E K383R: Y K V D K260,261R: E K K D K305R: L K L G K449R: A K S E K410R: FKSP K354R: L K S D K503R: S K L D K421R: A K K D K377R: G K L E K551R: L K N D K556R: Q K Q E K382R: M K S E K606R: E K L E K388R: L K E E K651.652R: I K K E K448R: R K L E K695R: F K K D K554R: L K H D K779R: L K R P K566R: E K L E K598R: E K M E K627R: L K L E K632R: L K V D A. A J? £' # 250. 150. La LA I i w l l i i l l i l l i P:Ndc10 a-SUMO a-Myc •250 •150 -100 IP:Ndc80 Figure 3-6. Identification of lysine residues in NdclO and Ndc80 that affect sumoylation. (A) Residues K556, K651-652, and K779 affect sumoylation of NdclO in vivo. (B) Residue K231 is required for sumoylation of Ndc80 in vivo. Mutations were introduced at the endogenous locus for each mutant strain and protein extracts were generated for assaying sumoylation by IP/western. 82 Ndc80 K231 site is not poly-sumoylated (data not shown). NdclO mitotic spindle localization is lost in the ndclO 4xK^R mutant Both in vitro and in vivo NdclO sumoylation data showed two modified forms of NdclO, indicating that NdclO may be modified by SUMO on more than one site (Figure 3-2). Moreover, lysine mutations in single SUMO consensus sites within NdclO did not completely block sumoylation, suggesting that there is more than one sumoylation site in NdclO (Figure 3-6a). If each of these lysine residues represents a potential sumoylation site, then defects caused by loss of one site, may be masked due to sufficient levels of modification at one of the other two sites. To circumvent this problem, all three potential sumoylation site mutations were combined to create NdclO-4xK—>R, which carries four lysine to arginine mutations at residues 556, 651, 652 and 779. By IP/western, NdclO-4xK—>R lacked any detectable levels of sumoylation, indicating that these three sites are required for the majority of NdclO sumoylation (Figure 3-7a). Combining the mutations at residues 556, 651, 652 and 779 also caused a dramatic reduction in the amount of NdclO localized to the anaphase mitotic spindle both along the length of the spindle and at the spindle mid-zone (Figure 3-7b), which was not due to a change in overall protein levels of NdclO-4xK—>R (Figure 3-7c). Using a cdc5-10 allele to arrest ndclO-4xK-+R strains in anaphase with elongated spindles, the localization defects associated with these mutations were quantified and it was found that 57% of cells had undetectable levels of NdclO-4xK—>R on the spindle, while 43% had faint staining that was observable, but well below WT levels (n=200). This result is in contrast to WT cells, which showed robust spindle staining in 88% of cells, and faint staining in only 12% of the population (n=200). NdclO may be on the spindle as part of the CBF3 complex since Cep3 was 83 Figure 3-7. Loss of N d c l O spindle localization. (A) Combining mutations that individually affect Ndc lO sumoylation results in loss of sumoylation in vivo. NDC10 was mutated at the endogenous locus to encode K556R, 651,652,779R (4xK—»R). (B) NdclO 4xK—>R mis-localizes from the mitotic spindle. Mutant and wi ld type protein was tagged with G F P and imaged in live cells. To visualize the mitotic spindle, Tub 1-CFP was also introduced into each strain. (C) NdclO 4xK—>R is expressed at normal levels. Whole cell protein extracts were analyzed by Western blot using cc-GFP antibodies (Roche). (D) Cep3 mis-localizes from the mitotic spindle in ndclO 4xK—+R strains. Cep3 was tagged with G F P in a wild-type or ndclO 4xK—*R strain and imaged in live cells. Scale bar represents 5 p.m. 84 r e c e n t l y s h o w n t o l o c a l i z e t o t h e m i t o t i c s p i n d l e ( B o u c k a n d B l o o m , 2 0 0 5 ) . C e p 3 w a s a l s o m i s - l o c a l i z e d f r o m m i t o t i c s p i n d l e s i n ndclO-4xK-^R m u t a n t s , s u p p o r t i n g t h e i d e a t h a t N d c l O a n d C e p 3 a r e p r e s e n t o n t h e s p i n d l e a s a c o m p l e x ( F i g u r e 3 - 7 d ) . T h e c h r o m o s o m a l p a s s e n g e r p r o t e i n S l i l 5 a n d t h e m i c r o t u b u l e - a s s o c i a t e d p r o t e i n A s e l s t i l l l o c a l i z e d n o r m a l l y i n t h e ndc!0-4xK—*R m u t a n t ( d a t a n o t s h o w n ) , a s d i d B i r l ( F i g u r e 3 -1 1 a ) , s u g g e s t i n g t h a t t h e m i s - l o c a l i z a t i o n o f N d c l O a n d C e p 3 i s n o t d u e t o a g r o s s d e f e c t i n s p i n d l e s t r u c t u r e . ndclO-4xK-^R s t r a i n s a r e a l s o n o t T s , a n d d i d n o t s h o w c h a n g e s i n N d c l O ' s a b i l i t y t o d i m e r i z e ( R u s s e l l e t a l . , 1 9 9 9 ) , s u g g e s t i n g t h a t t h e s e m u t a t i o n s h a v e n o t a l t e r e d t h e s t r u c t u r e o f t h e p r o t e i n ( d a t a n o t s h o w n ) . B e y o n d N d c l O ' s c a n o n i c a l f u n c t i o n a t t h e k i n e t o c h o r e i n c h r o m o s o m e s e g r e g a t i o n , r e c e n t r e p o r t s h a v e d e s c r i b e d r o l e s f o r t h e C B F 3 c o m p l e x a n d N d c l O i n c y t o k i n e s i s ( B o u c k a n d B l o o m , 2 0 0 5 ; G i l l i s e t a l . , 2 0 0 5 ) . F o r t h i s r e a s o n , ndc!0-4xK—>R m u t a n t s w e r e t e s t e d f o r m u l t i - b u d d i n g , p r o p e r s e p t i n r i n g m a t u r a t i o n , a n d f o r d e f e c t s i n t h e a x i a l p a t t e r n o f h a p l o i d b u d d i n g . I n t h e s e a s s a y s , n o d i f f e r e n c e s w e r e o b s e r v e d b e t w e e n W T a n d ndclO-4xK-+R ( F i g u r e 3 - 8 ) . K779 is the key sumoylation site T o f u r t h e r i n v e s t i g a t e t h e l y s i n e r e s i d u e s b e i n g m o d i f i e d b y S U M O , t h e p a i r w i s e c o m b i n a t i o n s o f l y s i n e t o a r g i n i n e m u t a t i o n s w e r e m a d e i n N d c l O (ndclO-2xK-+R), w h i c h i n c l u d e d K 5 5 6 R w i t h K 6 5 1 / 6 5 2 R , K 5 5 6 R w i t h K 7 7 9 R , a n d K 6 5 1 / 6 5 2 R w i t h K 7 7 9 R . I n e a c h c a s e , o n l y t h o s e d o u b l e m u t a n t s c o n t a i n i n g m u t a t i o n s i n s i t e K 7 7 9 R h a r b o r e d a C T F a n d N d c l O m i s - l o c a l i z a t i o n p h e n o t y p e ( F i g 9 ) . T h i s s u g g e s t s t h a t t h e k e y s i t e f o r m o d i f i c a t i o n i s K 7 7 9 , w h i c h a c t s i n c o n c e r t w i t h e i t h e r o f t h e o t h e r t w o s i t e s t o b e f u n c t i o n a l w i t h r e g a r d s t o C T F a n d s p i n d l e l o c a l i z a t i o n . H o w e v e r , t h e o b s e r v a t i o n t h a t 8 5 A . WT 4xK-»R B. WT 4xK->R Figure 3-8. ndclO 4xK-+R strains do not have defects related to cytokinesis. (A) Septin ring formation is normal in ndclO 4xK-+R strains. CdclO-GFP was used as a marker for the septin ring in wild type and ndclO 4xK-> R strains, and unlike ndclO-1 (Bouck and Bloom, 2005), septin rings mature and split normally. (B) Haploid axial bud pattern is unaffected in ndclO 4xK-+R mutants. Wild type and ndclO 4xK-* R strains were grown at 30°C and fixed. Bud scars were stained with calcofluor white. Scale bar represents 5um. 86 A . Figure 3-9. K779 is the key residue affecting chromosome segregation and spindle localization. (A) ndclO strains lose an artificial chromosome fragment at elevated rates when K779 is mutated in combination with one of the other modification sites. Formation of a colored sector indicates loss of the chromosome fragment. (B) Combining mutations with K779 also cause NdclO to mis-localizes from the mitotic spindle. Mutant and wild type protein was tagged with GFP and imaged in live cells. Scale bar represents 5pm. 87 the single K779R mutant does not have a C T F or localization defect suggests that when both K556 and K651/652 are present they can compensate for the loss of the K779 site with regards to these two functions. ndclO-4xK-^R mutants display chromosome instability In addition to the spindle localization defects, ndclO-4xK-^R strains showed increased C I N in a color sector assay (Figure 3-10a). When quantified by half-sector analysis (Hyland et aL, 1999; Koshland and Ffieter, 1987), ndclO-4xK^R strains had rates of chromosome loss and chromosome nondisjunction 160X and 6 0 X greater, respectively, than W T . In comparison, loss of checkpoint function in bublA or bubSA strains causes a 50X increase in the rate of chromosome loss (Warren et al., 2002). These strains also lose endogenous chromosomes at an increased rate as determined by a diploid bi-mating assay (Spencer et al., 1990). In this assay, ndclO-4xK—*R homozygous diploid strains formed mating colonies at a rate 10X that of W T diploids, presumably due to the loss of chromosome III (2N-1) (Figure 3-10b). Although ndcl0-4xK—*R strains showed increased rates of C I N , there is no observable delay in cell cycle progression of a aF-synchronized cell culture (Figure 3-10c). ndclO-4xK—*R strains are also G 2 / M checkpoint proficient in both their ability to arrest and recovery from N Z exposure (data not shown). These results suggest that the defect causing chromosome missegregation is not eliciting a checkpoint response and frequent repair, but is more likely to be either a rare event that always leads to failure, or an event that does not trigger a G 2 / M checkpoint response at all . ndcl0-4xK^>R mutants have mitotic spindle defects A common phenotype associated with mutations affecting kinetochore and/or 88 Spindle length (um) Figure 3-10. ndclO 4xK-+ R strains have increased rates of chromosome mis-segregation and mitotic spindle defects. (A) ndclO 4xK-+ R strains lose an artificial chromosome fragment at elevated rates. Formation o f a colored sector indicates loss o f the chromosome fragment. (B) ndclO 4xK-+ R strains lose endogenous chromosomes. Independent isolates o f wi ld type and ndclO 4xK-+ R diploid strains were mated with haploid tester strains and mating products were selected. (C) Ce l l cycle progression is not delayed in ndclO 4xK-> R strains. W i l d type and ndclO 4xK-> R strains were arrested in G I with a F , and then released. D N A content was analyzed by flow cytometry to determine cell cycle progression. (D) ndclO 4xK-> R strains are benomyl sensitive. Serial dilutions o f each strain were spotted plates containing 0, 10, 15, or 20ug/mL of benomyl and incubated at 30°C for 2 days. Benomyl hypersensitive tubl-1 and resistant strain tub2-104 are included as controls. (E) ndclO 4xK-+ R strains have mitotic spindles o f abnormal length. Anaphase mitotic spindle lengths were measured in asynchronous cultures o f wi ld type and ndclO 4xK-> R strains containing T u b l - C F P . 8 9 spindle function is increased sensitivity to the microtubule-destabilizing drug benomyl. ndclO-4xK-^R strains displayed benomyl sensitivity (Figure 3-10d), which taken together with the mitotic spindle mis-localization phenotype raise the possibility that the loss of NdclO sumoylation may be causing specific defects in spindle function. To investigate this possibility, the lengths of mitotic spindles in anaphase stage cells of an asynchronous culture were measured. In the ndclO-4xK—*R mutant, anaphase spindles were of abnormal length, with spindles averaging 7.30 p m ± 0.08 (n=483) compared to 6.62 pm ± 0.06 (n=443) for W T cells. In comparing the distribution of spindle lengths graphically, it was noted that the ndclO-4xK-+R mutants had abnormally long anaphase spindles, ranging up to 10-12pm, lengths that are never seen in W T cells (Figure 3-10e). The observed spindle defect and C I N , which is associated with no observable G 2 / M delay, suggests that the defect(s) caused by loss of NdclO sumoylation is related to events in anaphase after the G 2 / M checkpoint has been' silenced (e.g. spindle elongation). Birl and NdclO-4xK—>R physically interact In anaphase, Ndc lO and B i r l co-localize on the mitotic spindle in a B i r l -dependent manner, suggesting that NdclO and B i r l are part of a complex on the mitotic spindle (Bouck and Bloom, 2005; Widlund et al., 2006). In the ndclO-4xK—>R strain, Ndc lO no longer localized to the mitotic spindle (Figure 3-7b), whereas B i r l remained spindle-bound (Figure 3-1 la). Therefore, the spindle.length defect and mis-localization of NdclO may be due to a disruption of the N d c l O - B i r l protein interaction. However, co-IP's demonstrated that the NdclO-4xK—>R interaction with B i r l was comparable to that seen in W T cells (Figure 3-1 lb) . The interaction between Ndc lO and B i r l was not disrupted in cells arrested with aF or N Z , demonstrating that this interaction is not 90 Figure 3-11. Birl is sumoylated in an NdclO dependent manner independent of CBF3 function. (A) Bi r l -GFP remains spindle localized in ndclO 4xK-^*R anaphase cells. B1R1 was tagged with GFP in a WT and ndclO 4xK—+R strain and imaged in live cells. For panels B-E & G, Birlwas immunoprecipitated with a-Myc conjugated beads and used for Western blot analysis with a-Myc , a-NdclO, or a-SUMO antibodies as indicated from wild type or ndclO 4xK—*R strains. (B) NdclO and B i r l interact in vivo independently of NdclO sumoylation. (C) The interaction between NdclO and B i r l does not require microtubules (NZ) nor is it specific to mitotic cells (aF). (D) B i r l is sumoylated in vivo, but reduced in response to NZ treatment. (E) B i r l sumoylation is reduced in an ndclO 4xK—*R strain. (F) Cep3 and Ndc80 sumoylation is unchanged in the ndclO 4xK-^R mutant strain. (G) B i r l sumoylation is not affected by mutations in CBF3 components other than NdclO. Protein extracts from WT, ndel0-1, cep3-l, and ctf 13-30 were made after 3hrs at 37°C. Scale bar represents 5pm. 91 specific to mitosis and is not microtubuie-dependent (Figure 3-1 lc). These results suggest that the observed interaction between NdclO and Birl occurs independently of the mitotic spindle, and that the spindle length defect in the ndclO-4xK-^R strain is not due to the failure of NdclO-4xK—>R to interact with Birl. Birl is sumoylated in an NdclO-dependent manner Birl is a phosphorylated protein that was also recently identified as a sumoylated protein in proteomic studies (Widlund et al., 2006; Wohlschlegel et al., 2004; Zhou et al., 2004). By IP/western, it was confirmed that Birl is sumoylated, with an estimated 1% of Birl being SUMO conjugated in an asynchronous culture, and as seen for NdclO, treatment of cells with NZ resulted in loss of Birl sumoylation (Figure 3-1 Id). Both the phosphorylation and sumoylation state of Birl were checked in the ndclO-4xK—>R mutant strain, and westerns show that the overall appearance of Birl phosphorylation (as indicated by the diffuse western signal representing phosphorylated forms of Birl) is unchanged in the mutant as compared to WT (Figure 3-1 le, aMYC blot), but that sumoylation of Birl is reduced in the ndclO-4xK-^R mutant to undetectable levels (Fig 7e, aSUMO blot). Cep3 and Ndc80 were also tested for changes in sumoylation state in the ndclO-4xK-^R mutant (Figure 3-1 If), but no significant differences were found compared to WT; thus, the loss of sumoylation in the ndclO-4xK-+R mutant appears to be specific to the modification of Birl. These data suggest that sumoylation of NdclO is required for Birl sumoylation. B i r l sumoylation is independent of CBF3 function NdclO's effect on Birl sumoylation may be dependent on the activity of the CBF3 complex or may occur via a CBF3-independent mechanism. To distinguish these 92 two possibilities, B i r l sumoylation in strains carrying Ts mutations in the C B F 3 component Cep3 (cep3-l) or C t f l3 (ctfl3-30) were tested. In both cases, when cells were arrested at the non-permissive temperature, B i r l sumoylation was detected; however, loss of sumoylation was observed when the ndclO-1 Ts allele was used to abrogate Ndc lO ' s function. This result indicates that NdclO 's effect on B i r l sumoylation is not due to altered C B F 3 activity (Figure 3-1 lg) . Discussion These data demonstrate that the kinetochore proteins Ndc lO, Cep3, Ndc80, and B i r l are substrates for sumoylation. In the case of NdclO, the effect of lysine mutations on NdclO 's localization demonstrates that sumoylation demarcates a specific subset of NdclO that associate with the mitotic spindle. This demarcation most likely occurs through an alteration of NdclO 's binding interactions with microtubules or microtubule-associated proteins, leading to the establishment or maintenance of spindle localization. This data also assigns a function to the fraction of Ndc lO protein on the mitotic spindle in controlling mitotic spindle dynamics. Ndc lO contribution to spindle stability was previously demonstrated using the Ts allele ndclO-1 (Bouck and Bloom, 2005). At the non-permissive temperature the ndclO-1 protein becomes unstable resulting in protein degradation and loss of both kinetochore function and spindle checkpoint function (Fraschini et al., 2001; Gardner et al., 2001; Goh and Kilmartin, 1993). Interestingly, the phenotype observed using the ndclO-1 strain was an enrichment of cells with 2-4|im spindles that cycled through states of elongation, catastrophe, and rescue. This was attributed to Ndc lO having a role in promoting spindle stability during anaphase spindle elongation. However, this work demonstrated that when 93 NdclO mis-localizes specifically from the mitotic spindle the result was abnormally long spindles, which suggests NdclO has a role in destabilizing the mitotic spindle. These contradictory results, can be explained by the loss of kinetochore function in the ndclO-1 allele, since the kinetochore is known to have a stabilizing effect on microtubule dynamics (Mcintosh et al., 2002). Moreover, Ndc lO ' s localization to the mitotic spindle has been linked to a role in regulating cell separation during cytokinesis, using the ndclO-1 allele, strains at the non-permissive temperature were shown to form multi-cell clusters, and aberrant septin ring formations (Bouck and Bloom, 2005). In contrast, the ndclO 4xK—yR strain shows no such defects, suggesting that the pleiotropic phenotypes observed in the ndclO-1 strain are likely due complete loss of Ndc lO protein in the cell and not just spindle localization. This interpretation of the data is support by recent work on B i r l , which when mutated causes mis-localization of Ndc lO from the mitotic spindle, but no defects observed in cytokinesis (Widlund et al., 2006). Overall, this supports a role for Ndc lO on the mitotic spindle in controlling spindle dynamics and suggests that there is no role for spindle bound NdclO in cytokinesis. The recent discovery of a checkpoint termed, NoCut, which halts cytokinesis in the presence of spindle damage supports this conclusion. The authors of this study found that NoCut was activated in response to spindle damage and when abolished, allows ndclO-1 cells to complete cytokinesis (Norden et al., 2006). This suggests that the cytokinesis defects in ndclO-1 cells are not due to a requirement of Ndc lO in cytokinesis, but due to the spindle damage that is present in this mutant causing activation of the NoCut checkpoint. A number of other kinetochore proteins also localize to the spindle during anaphase including B i r l , Cep3, Cin8, D a m l , D u o l , I p l l , Sl i 15, S l k l 9 , and Stu2, most of 94 which have also been shown to play a role in controlling spindle dynamics (Biggins et al., 1999; Bouck and Bloom, 2005; Hofmann et al., 1998; Hoyt et al., 1992; Jones et al., 1999; K o s c o e t al., 2001; Widlund et al., 2006; Zeng et al., 1999). The localization of I p l l , SH15, and Ndc lO to the spindle requires the function of the phosphatase C d c l 4 that is activated in anaphase to regulate mitotic exit (Bouck and Bloom, 2005; Pereira and Schiebel, 2003; Stegmeier and Amon, 2004). Moreover, Ndc lO , B i r l , Cep3, and SH15 are S U M O substrates (this work, (Wohlschlegel et al., 2004)) indicating that these spindle associated kinetochore proteins may be regulated via common mechanisms to control mitotic spindle dynamics during anaphase. Regulation of spindle dynamics likely includes delivery of these kinetochore proteins to the spindle mid-zone, which may be used as a signal to co-ordinate the collapse of the mitotic spindle and to initiate other late stage mitotic events once chromosome segregation has occurred. Given ndclO-4xK-^-R's long spindle phenotype, it is possible that the signal to commence spindle disassembly is delayed due to the absence of Ndc lO on the mitotic spindle, providing time for additional spindle elongation. It is currently unknown i f the altered spindle dynamics are also responsible for the increase in C I N observed in ndclO-4xK-^R mutants, but the lack of an observable cell cycle delay is suggestive of events that are invisible to the mitotic checkpoint machinery or occur during stages of the cell cycle when the checkpoint has already been satisfied (e.g. anaphase). Intriguingly, Ndc lO also localizes to M T ' s in telophase and into G i of the next cell cycle raising the possibility that M T associated NdclO may be required in non-mitotic stages of the cell cycle (Bouck and Bloom, 2005), which may include S phase when C E N D N A is replicated and kinetochore microtubule attachments are being established. 95 NdclO's localization to the mitotic spindle is dependent on sumoylation and B i r l (this study; (Bouck and Bloom, 2005; Widlund et al., 2006)). However, this work demonstrates that the interaction between Ndc lO and B i r l occurs in non-mitotic cells and is independent of microtubules, Ndc lO spindle localization, and sumoylation of NdclO or B i r l . How then does B i r l facilitate NdclO 's spindle association i f not by physically interacting with Ndc lO on the mitotic spindle? Based on localization data, the other structure at which these two proteins co-localize is the kinetochore (Widlund et al., 2006). A t the kinetochore, B i r l could direct Ndc lO to microtubules in a manner dependent on the function of the B i r l - S l i l 5 - I p l l kinase complex. In support of this possibility, Ndc lO has a weak two-hybrid interaction with Sl i 15 (Table 3-3). Moreover, Ndc lO has been shown to be an I p l l substrate for phosphorylation in vitro, and ipll mutants, like ndclO-4xK-*R mutants, contain spindles of abnormal length (Biggins et al., 1999; Buvelot et al., 2003). In experiments that use the ipll-321 temperature sensitive mutant, Ndc lO shows only slightly lower levels of mitotic spindle localization (Bouck and Bloom, 2005). It is known, however, that at the restrictive temperature, ipll-321 retains a low level of kinase activity, and a phosphorylation-dependent mechanism is therefore still possible (Pinsky et al., 2006). In contrast to NdclO, mutations that abolish B i r l sumoylation (Figure 3-12a) do not result in a loss of B i r l mitotic spindle localization (Widlund et al., 2006), implying that B i r l ' s association with the mitotic spindle does not rely on sumoylation. A clue to the function of B i r l ' s sumoylation may come from recent work on the mammalian homolog of B i r l , Survivin. In mammals, modification of B i r l /Surv iv in with ubiquitin appears to regulate dynamic protein-protein interactions important for chromosome 96 segregation at the centromere (Vong et al., 2005). Intriguingly, these modifications map to the I A P repeat region of mammalian Bi r l /Surv iv in (Vong et al., 2005), deletion of which in yeast B i r l results in a loss of sumoylation (Figure 3-12a). Thus, the I A P repeat domain may be used to regulate both yeast and mammalian Bi r l /Surv iv in ' s function by lysine-directed modifications. Once Ndc lO and B i r l are on the mitotic spindle, it appears that these two proteins function to regulate spindle dynamics in an opposing manner given that the spindle length defect observed in ndclO-4XK—>R mutants is in contrast to that seen in B i r l mutants where spindles fail to fully elongate and are shorter than W T on average (this study; (Widlund et al., 2006)). The interplay between these two proteins is further illustrated by the fact that in the ndclO-4xK—+R mutant, B i r l sumoylation is undetectable, even though the NdclO mutant protein and B i r l still interact normally. This dependence is not reciprocal in that Ndc lO sumoylation is not affected by mutations that block B i r l ' s sumoylation or that disrupt the interaction between B i r l and Ndc lO (Figure 3-12b). The reliance on Ndc lO being competent for sumoylation suggests that Ndc lO requires prior modification with S U M O before sumoylation of B i r l can occur, and is indicative of a cascade of S U M O modification events where Ndc lO functions in trans to facilitate modification of B i r l by S U M O . Although Ndc lO is not conserved in higher eukaryotes, B i r l and Ndc80 are well conserved. A n important question is whether sumoylation of kinetochore proteins including B i r l and Ndc80 function in an analogous manner in these evolutionarily diverse organisms. While neither B i r l nor Ndc80 homologues have been shown to be sumoylated in higher eukaryotes, there is evidence linking sumoylation to kinetochore 97 B. a-SUMO . a-Myc • 250 -150 IP: NddO Figure 3-12. B i r l phosphorylation and the IAP domain are required for B i r l sumoylation. (A) B i r l sumoylation is undetectable in the birl-9A and birl-AIAP mutants. B i r l - A I A P (lacks the inhibitor of apoptosis repeat) interacts with N d c l O and the mitotic spindle while B i r l - 9 X A (lacking nine potential phosphorylation sites) does not interact with NdclO but remains spindle bound (Widlund et al., 2006). (B) Ndc lO sumoylation is not dependent on B i r l sumoylation or a physical interaction with B i r l . 98 and mitotic spindle functions. For example, S U M O - 2 modified proteins are enriched at the inner centromere of chromatids in Xenopus egg extracts, and alteration of S U M O modification of chromosomal substrates by S U M O - 2 causes a block in the segregation of sister chromatids in anaphase (Azuma et al., 2005; Azuma et al., 2003). Moreover, RanGapl , the first identified sumoylated substrate, is targeted to mitotic spindles and kinetochores, and like NdclO, mutations that abolish sumoylation lead to R a n G A P l mis-localization from the spindle (Joseph et al., 2004; Joseph et al., 2002; Mahajan et al., 1997; Matunis et al., 1996; Saitoh et al., 1997). Complexed with R a n G A P l is the S U M O E3 ligase RanBP2, which when depleted in mitotic cells results in mis-localization of R a n G A P l , the spindle checkpoint proteins M a d l and Mad2, and the kinetochore proteins C E N P - E and C E N P - F (Joseph et al., 2004). The phenotypic consequences of these effects include the accumulation of mitotic cells with multipolar spindles and unaligned chromosomes. The localization of RanBP2 to kinetochores places an E3 protein near the kinetochore, highlighting the possibility that there may be many proteins of the kinetochore targeted for sumoylation. The number of sumoylated substrates in yeast and higher eukaryotes continues to grow rapidly, but the biological functions of the majority of these modifications remains elusive. The data presented here provides evidence for the regulation of chromosomal passenger proteins and kinetochore proteins by S U M O modification including the localization of Ndc lO to the mitotic spindle during anaphase. Findings in higher eukaryotes provide evidence that the mechanism for localizing proteins to microtubules via sumoylation may be conserved (e.g. R a n G A P l ) . Overall, regulation of the. kinetochore is still not well defined, and sumoylation of kinetochore proteins could 99 represent a novel mode of regulation that may be related to cell cycle checkpoint function. Recent publications provide evidence for sumoylation at mammalian kinetochores (Joseph et al. 2004, Chung et al. 2004) suggesting evolutionary conservation of this modification and possible mechanisms of action. 100 Chapter 4: Future Directions F u t u r e d irect ions i n the field of k ine tochore re search In the past two decades the progress made in the field of chromosome segregation and kinetochore function has been astounding. This is highlighted by the discovery of more than 65 proteins that function at the budding yeast kinetochore alone (McAinsh et al., 2003). Despite these advances, for the majority of these proteins little more is known than the fact that these proteins localize at the kinetochore and are required for chromosome segregation. This presents a challenge to the kinetochore field in the coming years, which wi l l be to shift from simple identification to thorough characterization of the biochemical activities and mechanisms of action of the large number of proteins comprising the kinetochore. These studies wi l l be aimed at addressing a number of major questions still left to answer including: 1.) How does a kinetochore physically attach to a microtubule? 2.) How does the attachment of kinetochores to microtubules allow for the generation of a motive force to move sister chromatids during anaphase? 3.) How is the mechanical signal of attachment and tension converted into a biochemical signal that is processed by the spindle assembly checkpoint? 4.) What are the underlying processes that regulate the formation and function of a kinetochore? To answer these questions the field wi l l likely have to move in the direction of developing an in vitro system of kinetochore assembly and function, coupled with solving the structure of many of these proteins and characterizing the structural characteristics of the complexes they reside in. Beyond understanding how a kinetochore is built and regulated as an isolated biochemical system, the kinetochore field wi l l also be challenged with understanding how other cellular processes impact on the function of this supermolecular complex. For example, a critical role for chaperones (proteins that prevent non-specific aggregation of 102 nascent polypeptides and promote their correct folding) was recently demonstrated for the activation of the C B F 3 complex components S k p l and C t f l 3 , which is required for proper CBF3-C£7V complex formation (Stemmann et al., 2002). Tubulin chaperones also show genetic interactions with many of the non-essential kinetochore components (Tong et al., 2004), and several genes involved in tubulin folding in yeast were first identified in a screen for mutants that displayed a C I N phenotype (Hoyt et al., 1997; Hoyt et al., 1990; Stearns et al., 1990). As discussed in Chapter 2, the kinetochore also interacts with components of the nuclear envelope. Examples include spindle assembly checkpoint proteins that bind to the nuclear pore, where they are sequestered until activation of checkpoint function (Campbell et al., 2001; Iouk et al., 2002), and nuclear pore components that reloclalize to the kinetochore and SPB in mitosis (Salina et al., 2003). Furthermore, mutants in a component of the M t w l complex, N n f l , was originally described to have phenotypes similar to nuclear pore and nucleocytoplasmic transport mutants suggesting there are shared subunits and/or functions between these complexes (Shan et al., 1997). These examples highlight the highly complex and integrated pathways that function to control chromosome segregation. While a good start has been made in identifying the building blocks of a kinetochore, a mechanistic and biochemical understanding of this process wi l l require much more work. In relation to the work presented in this thesis, future research directions are discussed below. Spc24, Spc34 and A p q l 2 in chromosome segregation The work presented in Chapter 2 provides the groundwork for a variety of future research opportunities. First, the analysis of Ts alleles of both Spc24 and Spc34 that 103 differ in their terminal arrest phenotypes suggests that these two proteins are multifunctional and provide hints as to the location of specific functional domains in each protein. For instance, spc24-9, which carries a mutation in the C-terminus of Spc24, was SS or S L with the chl4, ctf3, ctfl9, iml3 and mcm21 central kinetochore mutants. spc24-8 and spc24-10, which carry mutations in the N-terminal region of Spc24 that contains two coiled-coil domains, did not display genetic interactions with central kinetochore mutants. spc24-9 mutants also have a much higher rate of chromosome loss than spc24-8 and spc24-10 mutants. Thus, it is likely that the C-terminal mutation in spc24-9 affects a different Spc24 function or protein-protein interaction than the spc24-8 and spc24-10 mutants. These data are consistent with a recently published structural analysis of the Ndc80 complex which demonstrates that the C-terminus of Spc24 is a globular domain that likely interacts with the kinetochore (Wei et al., 2005). In the case of Spc34, although spc34-6 and spc34-7 have a common amino acid mutation (S18P) they do not display similar genetic interactions. This may be due to the K 1 9 8 E mutation present in spc34-6, which is directly adjacent to an I p l l phosphorylation site (T199) (Cheeseman et al., 2002a). It w i l l be of interest to investigate this site further for possible phosphorylation events given that the D a m l complex is a critical target of the I p l l kinase in regulating kinetochore microtubule attachments, of which Spc34 is a component (Cheeseman et al., 2002a). Therefore, future work focused on determining the alterations caused by each individual mutation on protein-protein interactions and post-translational modifications of Spc24 and Spc34 could be very informative, especially in light of the recent structural information published for both the Ndc80 complex and D a m l complex (Miranda et al., 2005; Wei et al., 2005; Westermann et al., 2005). 104 A second opportunity would involve the characterization of A p q l 2 ' s role in cell cycle control and C I N , since it is unclear how the deletion of A P Q 1 2 affects these processes. Deciphering the role of a protein in a given process is often aided by determining its physical interaction partners, but in the case of A p q l 2 , very little is known. Therefore, a first step in the characterization of A p q l 2 could involve a mass spectrometry or two-hybrid based screen for binding partners. Because A p q l 2 is a nuclear envelope protein and contains a transmembrane domain, it may be beneficial to perform these screens with the N - and C-terminal domains as well as full length protein. Expressing different domains of A p q l 2 to attempt rescue of particular defects in a null mutant could also be used to map function onto different domains of the protein. This is important because of the possibility that the observed phenotypes in the apql2 deletion are a secondary consequence of wide scale perturbation of m R N A export. It may also be interesting to use the benomyl resistance phenotype of apql2A strains as a basis for a suppressor screen to define other processes that work in concert with A p q l 2 . Finally, the S L screens using Spc24 and Spc34 identified many genetic interactions that were not investigated, but offer interesting possibilities for further investigation. This includes the S L interactions observed between two negative regulators of the c A M P pathway, ira2 and pde2, in the spc24-9 screen. PDE2 was also recently identified as a high copy suppressor of D a m l kinetochore complex mutants (L i et al., 2005), and five negative regulators of the c A M P pathway, including ira2 and pde2, were also identified as benomyl sensitive mutants in genome-wide screens (Pan et al., 2004). Therefore, upregulation of the c A M P pathway by mutation of its negative regulators 105 appears to have a deleterious effect on kinetochore function, and may hint at a novel mode of regulation that involves c A M P signaling. Sumoylation: Future bouts with chromosome segregation The original identification of S U M O in budding yeast was through a high copy suppressor screen of a mutation in the kinetochore protein M i f 2 (Meluh and Koshland, 1995). Since then; S U M O and the process of sumoylation has been linked to chromosome segregation in numerous ways. This includes the observation that sumoylation is required for passage through mitosis, and more specifically that the S U M O machinery is required for checkpoint function, sister chromatid cohesion and chromosome stability (Bachant et al., 2002; L i and Hochstrasser, 1999; L i and Hochstrasser, 2000; Seufert et al., 1995; Stead et al., 2003). The work presented in Chapter 3 adds to these findings by demonstrating that the kinetochore proteins NdclO, Ndc80, Cep3, and B i r l are modified by S U M O . However, with the possible exception of NdclO, the purpose of these modifications remains unclear and wi l l require further investigation to wrestle the meaning of these modifications into the spot light. In the case of Cep3, mutation of lysine residues found in S U M O consensus sites failed to identify a modification site, which leaves another 35 lysine residues that could be targeted by this modification. Without the structure of Cep3 to prioritize surface lysines, possible approaches to tackling this problem are performing mutagenesis on all remaining lysine residues or using mass spectrometry to identify S U M O modifications on Cep3 purified from yeast cell lysate or modified in vitro. Identification of the modification site wi l l be important not only in characterizing the role of Cep3 106 sumoylation, but in combination with the ndclO-4xK-^*R mutant may shed light on the role sumoylation plays in regulating C B F 3 function. Mutations in Ndc80 and B i r l that effectively abolish sumoylation were both identified in this work (ndc80 K231R and b i r l - A I A P ) , but no phenotypes related to chromosome segregation were observed. It is almost certain that these modifications are important for the function of these proteins, and it is likely that the relevant phenotypes are not being tested. One approach that could be used to place sumoylation of these two substrates into a biological context is to perform genome-wide synthetic lethal screening with these alleles. The usefulness of identifying synthetic lethal genetic interactions is that they can be used to identify genes whose products buffer one another or impinge on the same essential pathway. S L screens also provide a list of genetic interactions that can be used as a fingerprint to identify genes and gene products functioning in a similar manner. It is known that the number of common interactions observed between two query genes correlates with known protein-protein interactions between the corresponding gene products. In other words, each allele that is screened gives a pattern of interactions that would be similar to other genes that function in the same process. For example, the CTF8 and CTF18 gene products physically interact in the same complex, and although these two genes are not connected in genetic networks, they share a large number of common interactions (Tong et al., 2004). This is further illustrated by the fact that 28 of 333 gene pairs with more than 16 common interactions encode physically interacting proteins, which is an 11-fold enrichment over those proteins that do not share a similar frequency of common interactions (Tong et al., 2004). The predictive nature of this approach is limited by the size of the genetic network, but genes involved in chromosome biology are 107 over-represented in the S L data produced to date, so it is likely that both the individual S L interactions and overall pattern of interactions' would provide clues to the function of Ndc80 and B i r l sumoylation. S U M O modification of NdclO can be linked to both Ndc lO and Cep3 spindle localization, regulation of mitotic spindle dynamics and chromosome segregation. Exactly how the S U M O modification mediates these events is unknown, but since neither NdclO nor Cep3 have microtubule binding or motor protein properties it seems likely that S U M O is responsible for establishing the interaction between Ndc lO and the protein(s) required to bridge NdclO and Cep3 to the spindle. This would predict that in the S U M O bound state, Ndc lO or S U M O itself is bound by other factors to properly localize. Given the capacity to generate in vitro sumoylated Ndc lO, one strategy to identify such factors could be to generate an affinity column with immobilized N d c l O - S U M O , on to which, whole cell yeast lysate from various conditions (e.g. anaphase) could be loaded. After extensive washing, proteins bound to the column could be eluted and identified by mass spectrometry. Alternatively, two-hybrid screening systems have been designed to detect interactions that are dependent on phosphorylation (Guo et al., 2004), which could be similarly designed to detect S U M O dependent interactions. This has the added benefit of detecting these interactions in vivo. The identification of a spindle localizing factor that recognizes sumoylated substrates could be very exciting in light of the fact that NdclO, Cep3, and human RanGapl all localize to the mitotic spindle, and that the localization of NdclO and RanGapl is S U M O dependent (Matunis et a l , 1996). Finally, data in Chapter 3 suggests that NdclO 's spindle localization may rely on the interaction of Ndc lO with the I p l l / B i r l / S l i l 5 complex. This is reflected in the 108 observation that Ndc lO has a two-hybrid interaction with B i r l and S l i l 5 , is a substrate for Ip l l phosphorylation in vitro, and is mis-localized from the spindle when B i r l is mutated. Furthermore, the data suggests that this interaction occurs prior to spindle loading since the B i r l interaction can still be observed in the ndclO-4xK-^R mutant. To investigate this further, it w i l l be necessary to investigate the effect of SH15 and I p l l mutants on the localization of NdclO. Interestingly, this has already been done in i p l l -321 mutants and there appears to be only a slight decrease in the levels of mitotic spindle localization (Bouck and Bloom, 2005). It is known, however, that at the restrictive temperature, ipll-321 retains a low level of kinase activity, and a phosphorylation-dependent mechanism is therefore still possible (Pinsky et al., 2006). In fact, evidence of an interaction between phosphorylation and sumoylation has been shown for many substrates, and has been termed a phospho-SUMO switch. It has been shown for these substrates that these modifications can inhibit or promote one another (Yang and Gregoire, 2006). In preliminary experiments using ndclO-4xK-^R mutant, it can be observed that reduction of Ipj l kinase activity in an ipl l -321 allele results in hyper-sumoylation of NdclO, but not Cep3 or Ndc80, suggesting that this effect is specific to NdclO and-does not reflect whole cell changes in modification levels (Figure 4 - l a and data not shown). This argues for the presence of a phospho-SUMO switch in NdclO. Inspection of the Ndc lO sequence reveals a number of potential phosphorylation sites near two of the lysine residues in NdclO that are critical for sumoylation (Figure 4-lb) . Mutational analysis of these sites wi l l be critical in understanding the function of such a switch, since phenotypes arising in the ipl l -321 allele are not specific to loss of NdclO phosphorylation and subsequent hyper-sumoylation given the number of known I p l l 109 A. <s £ | £ | 2 5 0 — | m a-SUMO > - y BUS 150— L_} a-Myc ^BBJBBr ^^^^^^ short exp. long exp. IP:Ndc10 B. K556 LTFASSHNPDTHPTQKQESEGPLQMSQLDTT K779 IEQKLGSHTGKFSTLKRPQLYMTEEHNVGFD Figure 4-1. NdclO is hypersumoylated when Ipll kinase activity is abolished. (A) IP/western blots were performed on protein extracts from W T or ipl l -321 strains grown at 37°C for 3hrs. A short and long exposure are shown of the same westerns. Note that in the ipl l-321 lane a sumoylated form o f Ndc lO can be seen in the M Y C blots, which is normally unobservable in W T (arrow). (B) Inspection of the N d c l O sequence reveals a number o f potential phosphorylation sites (blue) near lysine residues K556 and K779 (red), which are critical for sumoylation in Ndc lO. 110 targets (Cheeseman et al., 2002a). One intriguing possibility is that phosphorylation of NdclO may be used to regulate NdclO sumoylation in response to checkpoint activation, since NdclO sumoylation is decreased in N Z challenged cells. If this is the case, inactivating this switch by mutation should provide some insight into the function of NdclO in spindle checkpoint function (Fraschini et al., 2001). In conclusion, sumoylation of various kinetochore proteins provides evidence for a novel means of regulating chromosome segregation. It remains to be seen i f these S U M O modifications also occur on the human homologs of these proteins, but given the severe defects in chromosome segregation and nuclear architecture that is seen in S U M O knockout mice, it is certain that sumoylation plays a central role in chromosome segregation in mammalian systems (Nacerddine et al., 2005). Conclusion The work presented in this thesis set out to further characterize the function(s) and regulation of a few essential kinetochore proteins in budding yeast to better understand their roles in chromosome segregation. 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