Cvt19 is a receptor for the cytoplasm-to-vacuole targeting pathway.

Cvt19 is specifically required for the transport of resident vacuolar hydrolases that utilize the cytoplasm-to-vacuole targeting (Cvt) pathway. Autophagy (Apg) and pexophagy, processes that use the majority of the same protein components as the Cvt pathway, do not require Cvt19. Cvt19GFP is localized to punctate structures on or near the vacuole surface. Cvt19 is a peripheral membrane protein that binds to the precursor form of the Cvt cargo protein aminopeptidase I (prAPI) and travels to the vacuole with prAPI. These results suggest that Cvt19 is a receptor protein for prAPI that allows for the selective transport of this protein by both the Cvt and Apg pathways.

Vacuolar localization of oligomeric alpha-mannosidase requires the cytoplasm to vacuole targeting and autophagy pathway components in Saccharomyces cerevisiae.

One challenge facing eukaryotic cells is the post-translational import of proteins into organelles. This problem is exacerbated when the proteins assemble into large complexes. Aminopeptidase I (API) is a resident hydrolase of the vacuole/lysosome in the yeast Saccharomyces cerevisiae. The precursor form of API assembles into a dodecamer in the cytosol and maintains this oligomeric form during the import process. Vacuolar delivery of the precursor form of API requires a vesicular mechanism termed the cytoplasm to vacuole targeting (Cvt) pathway. Many components of the Cvt pathway are also used in the degradative autophagy pathway. alpha-Mannosidase (Ams1) is another resident hydrolase that enters the vacuole independent of the secretory pathway; however, its mechanism of vacuolar delivery has not been established. We show vacuolar localization of Ams1 is blocked in mutants that are defective in the Cvt and autophagy pathways. We have found that Ams1 forms an oligomer in the cytoplasm. The oligomeric form of Ams1 is also detected in subvacuolar vesicles in strains that are blocked in vesicle breakdown, indicating that it retains its oligomeric form during the import process. These results identify Ams1 as a second biosynthetic cargo protein of the Cvt and autophagy pathways.

Peroxisome degradation in Saccharomyces cerevisiae is dependent on machinery of macroautophagy and the Cvt pathway.

Organelle biogenesis and turnover are necessary to maintain biochemical processes that are appropriate to the needs of the eukaryotic cell. Specific degradation of organelles in response to changing environmental cues is one aspect of achieving proper metabolic function. For example, the yeast Saccharomyces cerevisiae adjusts the level of peroxisomes in response to differing nutritional sources. When cells are grown on oleic acid as the sole carbon source, peroxisome biogenesis is induced. Conversely, a subsequent shift to glucose-rich or nitrogen-limiting conditions results in peroxisome degradation. The degradation process, pexophagy, requires the activity of vacuolar hydrolases. In addition, peroxisome degradation is specific. Analyses of cellular marker proteins indicate that peroxisome degradation under these conditions occurs more rapidly and to a greater extent than mitochondrial, Golgi, or cytosolic protein delivery to the vacuole by the non-selective autophagy pathway. To elucidate the molecular mechanism of selective peroxisome degradation, we examined pexophagy in mutants that are defective in autophagy (apg) and the selective targeting of aminopeptidase I to the vacuole by the cytoplasm to vacuole targeting (Cvt) pathway. Inhibition of peroxisome degradation in cvt and apg mutants indicates that these pathways overlap and that peroxisomes are delivered to the vacuole by a mechanism that utilizes protein components of the Cvt/autophagy pathways.

V1-situated stalk subunits of the yeast vacuolar proton-translocating ATPase.

The proton-translocating ATPase of the yeast vacuole is an enzyme complex consisting of a large peripheral membrane sector (V1) and an integral membrane sector (V0), each composed of multiple subunits. The V1 sector contains subunits that hydrolyze ATP, whereas the V0 sector contains subunits that translocate protons across the membrane. Additional subunits in both sectors couple these activities. Here we have continued our examination of intermediate subunits primarily associated with the V1 but also implicated in interactions with the V0. Interactions between Vma7p (F) and Vma8p (D) and between Vma4p (E) and Vma10p (G) are described. Although Vma7p and Vma10p have been observed to interact with the V0 sector, our results indicate that these subunits behave primarily as canonical V1 sector subunits. We categorize these four subunits as "stalk" subunits to distinguish them from the known catalytic (A and B) and proton-translocating (c, c', and Vma16p) subunits and to highlight their intermediate nature. Furthermore, we show that the in vivo stability of Vma4p is dependent upon interaction with Vma10p. This may be important in the regulation of assembly, since these two subunits add to the V1 during later stages of V1 assembly. This is the first demonstration of interdependence between ATPase subunits for structural stability.