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Shortill, Shawn Peter

University of British Columbia

The endosome is a central sorting station for proteins and lipids. Retrograde protein sorting from the endosome is promoted by conserved SNX-BAR-containing coat complexes including retromer which enrich cargo at tubular microdomains and generate transport carriers. In humans, retromer cooperates with the VPS9-domain GEF VARP to direct an endosomal recycling pathway. Functions of the yeast VARP homolog Vrl1 have been overlooked due to an inactivating mutation in commonly studied strains. It is not clear how Vrl1 contributes to endosomal sorting and investigating this could provide insight to pathways controlled by VARP. Here, we show that Vrl1 is an endosomal protein with features of a SNX-BAR. We demonstrate that Vrl1 associates with Vin1, the paralog of retromer SNX-BAR Vps5, and that this is essential for its localization and function. Unique features in the Vin1 N-terminus allow Vrl1 to distinguish it from Vps5, thereby forming a complex that we have named VINE. Like other SNX-BAR coats, VINE localizes to endosomes in a PI3P-dependent manner and templates endosomal tubules, indicating a potential role in membrane trafficking. Vin1 and Vps5 specifically form VINE and retromer, respectively, through interactions at their shared unstructured N-terminal regions. We mapped determinants of VINE formation in the Vin1 N-terminus and identified a poly-basic region and a Leu-Phe motif. Structural prediction of the Vps5 N-terminal interaction with retromer subunit Vps29 suggest a bipartite mechanism which also involves a Leu-Phe motif. By establishing an ectopic expression system in S. cerevisiae, we demonstrate that the sole isoform of Vps5 in closely related yeast K. lactis forms both VINE and retromer. Structural predictions of the K. lactis Vps5 N-terminus suggest a regulated or competitive mechanism could control complex selection between VINE and retromer. In this work we identified and characterized mechanisms of assembly for a novel yeast VPS9 domain-containing SNX-BAR coat that we have named VINE. VINE combines features of a sorting complex and GEF, and may perform roles in endosomal membrane trafficking. The findings outlined in this dissertation advance our understanding of SNX-BAR assembly in yeast and humans and provide insight to the functional relationship between endosomal coats and VPS9-domain GEFs.

Text

2024-06-30

Attribution-NonCommercial-NoDerivatives 4.0 International

http://hdl.handle.net/2429/85185

Medical Genetics

University of British Columbia

UBCV

http://creativecommons.org/licenses/by-nc-nd/4.0/