Competitive organelle-specific adaptors recruit Vps13 to membrane contact sites

Abstract

The regulated expansion of membrane contact sites, which mediate the nonvesicular exchange of lipids between organelles, requires the recruitment of additional contact site proteins. Yeast Vps13 dynamically localizes to membrane contacts that connect the ER, mitochondria, endosomes, and vacuoles and is recruited to the prospore membrane in meiosis, but its targeting mechanism is unclear. In this study, we identify the sorting nexin Ypt35 as a novel adaptor that recruits Vps13 to endosomal and vacuolar membranes. We characterize an interaction motif in the Ypt35 N terminus and identify related motifs in the prospore membrane adaptor Spo71 and the mitochondrial membrane protein Mcp1. We find that Mcp1 is a mitochondrial adaptor for Vps13, and the Vps13-Mcp1 interaction, but not Ypt35, is required when ER-mitochondria contacts are lost. All three adaptors compete for binding to a conserved six-repeat region of Vps13 implicated in human disease. Our results support a competition-based model for regulating Vps13 localization at cellular membranes.

A VPS13D spastic ataxia mutation disrupts the conserved adaptor binding site in yeast Vps13

“…We found all adaptors respond similarly to alteration of the VAB domain repeats: mutation of the 6 th repeat abolished the binding of all three adaptors tested, while mutation of the 1 st repeat caused a partial defect. Moreover, we found that repeats 5 and 6 alone bound strongly to all three adaptors, suggesting adaptors recognize a single site within these repeats, consistent with our previous finding that the adaptors compete to recruit Vps13 to different membranes (24).…”

“…We have previously shown that soluble chimeric proteins carrying a PxP motif compete with adaptors to block Vps13 recruitment to membranes (24). Therefore, if a new adaptor is required for Vps13's CPY sorting function, overexpression of soluble PxP motif proteins should cause cells expressing wild type Vps13 to secrete CPY.…”

“…To determine the contribution of each repeat to adaptor binding, we generated a series of Vps13 point mutants with alanine substitutions in the highly conserved asparagine of each repeat (24) (Fig 1A). Herein these mutants are referred to as Vps13 N1 , Vps13 N2 , and so on, indicating the repeat with the substitution.…”

“…Vps13 has also been localized to other organelles, including peroxisomes (22), which could require yet other adaptors. These unidentified adaptors may be identified by bioinformatics approaches that identify predicted PxP motifs (24).…”

Nuclear envelope-vacuole contacts mitigate nuclear pore complex assembly stress

“…Several outer NE membrane proteins including Nvj1 (Pan et al, 2000) and Mdm1 (Henne et al, 2015) are present at NVJs and tether the NE to the vacuole through specific interactions with corresponding vacuolar proteins like Vac8 (Pan et al, 2000;Jeong et al, 2017) or phospholipid species such as phosphatidylinositol 3-phosphate (Henne et al, 2015). NVJs expand when cells are grown using carbon sources other than glucose (Hariri et al, 2018;Bean et al, 2018) or to promote piecemeal autophagy of the nucleus (PMN) in response to nutrient starvation , which involves autophagy-dependent degradation of specific nuclear proteins in the vacuole (Krick et al, 2008). Proteins that promote lipid droplet (LD) formation are also enriched at NVJs (Kohlwein et al, 2001), which serve as non-exclusive sites for LD synthesis.…”