Requirement of Rab5 GTPase during heat stress-induced endocytosis in yeast.

The plasma membrane (PM) is constantly exposed to various stresses from the extracellular environment, such as heat and oxidative stress. These stresses often cause denaturation of membrane proteins and destabilize PM integrity, which is essential for normal cell viability and function. For maintenance of PM integrity, most eukaryotic cells have the PM quality control (PMQC) system, which removes damaged membrane proteins by endocytosis. Removal of damaged proteins from the PM by ubiquitin-mediated endocytosis is a key mechanism for maintenance of the PM integrity, but the importance of the early endosome in the PMQC system is still not well understood. Here we show that key proteins in early/sorting endosome function, Vps21p (yeast Rab5), Vps15p (phosphatidylinositol-3 kinase subunit), and Vps3p/8p (CORVET complex subunits), are involved in maintaining PM integrity. We found that Vps21p-enriched endosomes change the localization in the vicinity of the PM in response to heat stress and then rapidly fuse and form the enlarged compartments to efficiently transport Can1p to the vacuole. Additionally, we show that the deubiquitinating enzyme Doa4p is also involved in the PM integrity and its deletion causes mislocalization of Vps21p to the vacuolar lumen. Interestingly, in cells lacking Doa4p or Vps21p the amounts of free ubiquitin are decreased, and overexpression of ubiquitin restored defective cargo internalization in vps9Δ cells, suggesting that defective PM integrity in vps9Δ cells is caused by lack of free ubiquitin.

Distinct role of TGN-resident clathrin adaptors for Vps21p activation in the TGN-endosome trafficking pathway.

Clathrin-mediated vesicle trafficking plays central roles in post-Golgi transport. In yeast (Saccharomyces cerevisiae), the AP-1 complex and GGA adaptors are predicted to generate distinct transport vesicles at the trans-Golgi network (TGN), and the epsin-related proteins Ent3p and Ent5p (collectively Ent3p/5p) act as accessories for these adaptors. Recently, we showed that vesicle transport from the TGN is crucial for yeast Rab5 (Vps21p)-mediated endosome formation, and that Ent3p/5p are crucial for this process, whereas AP-1 and GGA adaptors are dispensable. However, these observations were incompatible with previous studies showing that these adaptors are required for Ent3p/5p recruitment to the TGN, and thus the overall mechanism responsible for regulation of Vps21p activity remains ambiguous. Here, we investigated the functional relationships between clathrin adaptors in post-Golgi-mediated Vps21p activation. We show that AP-1 disruption in the ent3Δ5Δ mutant impaired transport of the Vps21p guanine nucleotide exchange factor Vps9p transport to the Vps21p compartment and severely reduced Vps21p activity. Additionally, GGA adaptors, the phosphatidylinositol-4-kinase Pik1p and Rab11 GTPases Ypt31p and Ypt32p were found to have partially overlapping functions for recruitment of AP-1 and Ent3p/5p to the TGN. These findings suggest a distinct role of clathrin adaptors for Vps21p activation in the TGN-endosome trafficking pathway.

Role of phosphatidylserine in the localization of cell surface membrane proteins in yeast.

Phosphatidylserine (PS) is a constituent of the cell membrane, being especially abundant in the cytoplasmic leaflet, and plays important roles in a number of cellular functions, including the formation of cell polarity and intracellular vesicle transport. Several studies in mammalian cells have suggested the role of PS in retrograde membrane traffic through endosomes, but in yeast, where PS is localized primarily at the plasma membrane (PM), the role in intracellular organelles remains unclear. Additionally, it is reported that polarized endocytic site formation is defective in PS-depleted yeast cells, but the role in the endocytic machinery has not been well understood. In this study, to clarify the role of PS in the endocytic pathway, we analyzed the effect of PS depletion on endocytic internalization and post-endocytic transport. We demonstrated that in cell lacking the PS synthase Cho1p (cho1Δ cell), binding and internalization of mating pheromone α-factor into the cell was severely impaired. Interestingly, the processes of endocytosis were mostly unaffected, but protein transport from the trans-Golgi network (TGN) to the PM was defective and localization of cell surface proteins was severely impaired in cho1Δ cells. We also showed that PS accumulated in intracellular compartments in cells lacking Rcy1p and Vps52p, both of which are implicated in endosome-to-PM transport via the TGN, and that the number of Snx4p-residing endosomes was increased in cho1Δ cells. These results suggest that PS plays a crucial role in the transport and localization of cell surface membrane proteins.Key words: phosphatidylserine, endocytosis, recycling, vesicle transport.

Functional complementation reveals that 9 of the 13 human V-ATPase subunits can functionally substitute for their yeast orthologs.

Vacuolar-type H+-ATPase (V-ATPase) is a highly conserved proton pump responsible for acidification of intracellular organelles and potential drug target. It is a multisubunit complex comprising a cytoplasmic V1 domain responsible for ATP hydrolysis and a membrane-embedded Vo domain that contributes to proton translocation across the membrane. Saccharomyces cerevisiae V-ATPase is composed of 14 subunits, deletion of any one of which results in well-defined growth defects. As the structure of V-ATPase and the function of each subunit have been well-characterized in yeast, this organism has been recognized as a preferred model for studies of V-ATPases. In this study, to assess the functional relatedness of the yeast and human V-ATPase subunits, we investigated whether human V-ATPase subunits can complement calcium- or pH-sensitive growth, acidification of the vacuolar lumen, assembly of the V-ATPase complex, and protein sorting in yeast mutants lacking the equivalent yeast genes. These assessments revealed that 9 of the 13 human V-ATPase subunits can partially or fully complement the function of the corresponding yeast subunits. Importantly, sequence similarity was not necessarily correlated with functional complementation. We also found that besides all Vo domain subunits, the V1 F subunit is required for proper assembly of the Vo domain at the endoplasmic reticulum. Furthermore, the human H subunit fully restored the level of vacuolar acidification, but only partially rescued calcium-sensitive growth, suggesting a specific role of the H subunit in V-ATPase activity. These findings provide important insights into functional homologies between yeast and human V-ATPases.

Rab5-independent activation and function of yeast Rab7-like protein, Ypt7p, in the AP-3 pathway.

The small GTPases, Rab5 and Rab7, are key regulators at multiple stages of the endocytic/endolysosomal pathway, including fusion and maturation of endosomes. In yeast, Vps21p (Rab5 homolog) recruits a GEF for Rab7 and activates the downstream Ypt7p (Rab7 homolog) on endosomal membrane. Although the model of this sequential activation from Vps21p to Ypt7p in the endocytic pathway has been established, activation mechanism of Ypt7p in the Vps21p-independent pathway has not been completely clarified. Here we show that Ypt7p is activated and mediates vacuolar fusion in cells lacking all yeast Rab5 genes, VPS21, YPT52, and YPT53. We also demonstrate that deletion of both VPS21 and YPT7 genes cause severe defect in the AP-3 pathway as well as the CPY pathway although the AP-3 pathway is mostly intact in each vps21Δ or ypt7Δ mutant. Interestingly, in vps21Δ ypt7Δ mutant cargos trafficked via the VPS or endocytic pathway accumulate beside nucleus whereas cargo trafficked via the AP-3 pathway disperse in the cytosol. These findings suggest that Ypt7p is activated and plays a Rab5-independent role in the AP-3-mediated pathway.