Biology subjects

Shortill, S. P.

Publications and source records attributed to Shortill, S. P..

3 recordsLinked to original sources

bioRxiv2024

N-terminal signals in the SNX-BAR paralogs Vps5 and Vin1 guide coat complex formation

Endosomal coat complexes assemble by incorporating membrane-binding subunits such as those of the sorting nexin (SNX) family. The S. cerevisiae SNX-BAR paralogs Vin1 and Vps5 are respective subunits of the endosomal VINE and retromer complexes that arose from a fungal whole genome duplication. Interactions mediated by the Vin1 and Vps5 BAR domains are required for protein complex assembly and membrane association. However, a degree of promiscuity is predicted for yeast BAR-BAR pairings, suggesting that another mechanism guides the formation of specific endosomal coat complexes. Previous work by our group and others has implicated the unstructured N-terminal domains of Vin1 and Vps5 in complex assembly. Here, we map N-terminal signals in both SNX-BAR paralogs that contribute to the formation and function of two distinct endosomal coats in vivo. Whereas Vin1 leverages a polybasic region and adjacent hydrophobic motif to bind Vrl1 and form VINE, the N-terminus of Vps5 interacts with the retromer subunit Vps29 at two separate sites. We show that one of these Vps5 motifs binds to a conserved hydrophobic pocket in Vps29 that is shared with other accessory proteins and targeted by a bacterial virulence factor in humans. Lastly, we examined the sole isoform of Vps5 from the milk yeast K. lactis and found that ancestral yeasts may have used a nested N-terminal signal to form both VINE and retromer. Our results suggest that the specific assembly of Vps5-family SNX-BAR coats depends on inputs from unique N-terminal sequence features in addition to BAR domain coupling, expanding our understanding of endosomal coat assembly mechanisms.

cell biology↗

bioRxiv2021

The VINE complex is a VPS9-domain GEF-containing SNX-BAR coat involved in endosomal sorting

Membrane trafficking pathways perform important roles in establishing and maintaining the endolysosomal network. 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 metazoans, retromer cooperates with VARP, a conserved VPS9-domain GEF, to direct an endosomal recycling pathway. The function of the yeast VARP homolog Vrl1 has been overlooked due an inactivating mutation in commonly studied strains. Here, we demonstrate that Vrl1 has features of a SNX-BAR coat protein and forms an obligate complex with Vin1, the paralog of the retromer SNX-BAR protein Vps5. Unique features in the Vin1 N-terminus allow Vrl1 to distinguish it from Vps5, thereby forming what we have named the VINE complex. VINE occupies endosomal tubules and promotes the delivery of a conserved mannose 6-phosphate receptor-like protein to the vacuolar membrane. In addition to sorting functions, membrane recruitment by Vin1 is essential for Vrl1 GEF activity, suggesting that VINE is a multifunctional coat complex that regulates trafficking and signaling events at the endosome.

cell biology↗

bioRxiv2021

Systematic analysis of membrane contact sites in Saccharomyces cerevisiae uncovers modulators of cellular lipid distribution

Actively maintained close appositions, or contact sites, between organelle membranes, enable the efficient transfer of biomolecules between the various cellular compartments. Several such sites have been described together with their tethering machinery. Despite these advances we are still far from a comprehensive understanding of the function and regulation of most contact sites. To systematically characterize the proteome of contact sites and support the discovery of new tethers and functional molecules, we established a high throughput screening approach in Saccharomyces cerevisiae based on co-localization imaging. We imaged split fluorescence reporters for six different contact sites, two of which have never been studied before, on the background of 1165 strains expressing a mCherry-tagged yeast protein that have a cellular punctate distribution (a hallmark of contact sites). By scoring both co-localization events and effects on reporter size and abundance, we discovered over 100 new potential contact site residents and effectors in yeast. Focusing on several of the newly identified residents, we identified one set of hits as previously unrecognized homologs to Vps13 and Atg2. These proteins share their lipid transport domain, thus expanding this family of lipid transporters. Analysis of another candidate, Ypr097w, which we now call Lec1 (Lipid-droplet Ergosterol Cortex 1), revealed that this previously uncharacterized protein dynamically shifts between lipid droplets and the cell cortex, and plays a role in regulation of ergosterol distribution in the cell.

cell biology↗