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UBC Theses and Dissertations

Insights into cargo adaptor function through the study of novel interactors Bean, Bjorn


In Eukaryotes, luminal and transmembrane proteins are moved to their functional locations by conserved membrane trafficking machinery. In this process, cargo adaptors bind motifs present on cargo, indirectly linking the proteins to coats, which deform membranes and form transport vesicles. Here, cargo adaptor recruitment and cargo recognition was studied by characterizing associated factors in the budding yeast Saccharomyces cerevisiae. Possible cargo adaptor-associated factors were identified in a proteomics study that grouped protein-protein interactions into 501 putative membrane associated complexes using a Markov clustering algorithm. Two clusters were selected for this work. The first contained the uncharacterized protein Ssp120 with the endoplasmic reticulum-to-Golgi trafficking complex Emp46/Emp47. Ssp120 stably interacted with the Emp46/Emp47 complex and depended on Emp47 for its punctate localization. The C-terminus of Ssp120 mediated the interaction. Homology with human MCFD2 suggests that Ssp120 may link a subset of cargo to Emp46/Emp47. The second cluster was comprised of retromer, an endosome-to-Golgi trafficking complex, and the Rab5-family guanine nucleotide exchange factor (GEF) Muk1. Both Muk1 and the other known Rab5-family GEF, Vps9, interacted with retromer and the presence of at least one was required for retromer recruitment to endosomes. Additionally, a new VPS9 domain-containing protein present was identified and shown to complement loss of MUK1 and VPS9. Retromer recruitment was shown to be dependent on putative GEF catalytic residues and the presence of their target Rabs. Furthermore, loss of GEFs resulted in mislocalization of the potential Rab5-family GTPase effector, Vps34, and its lipid product, phosphatidylinositol 3-phosphate (PI3P), to the vacuolar membrane. As retromer is recruited by PI3P, the data support a positive feedback model whereby retromer interacts with GEFs to indirectly modify the lipid composition of the membrane allowing further localized recruitment. This study validates the approach of studying novel interactors of cargo recognition complexes to better understand their function. It suggests that Ssp120 may recognize a subset of Emp46-Emp47 cargo, indicating that an associated factor can diversify the proteins recognized by a given cargo adaptor. Furthermore, the work on retromer suggests a novel mechanism for the reinforcement of cargo selective complex recruitment that may be conserved in humans.

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