Aspartyl Aminopeptidase Is Imported from the Cytoplasm to the Vacuole by Selective Autophagy in Saccharomyces cerevisiae

Masaki Yuga,Katsuya Gomi,Daniel J Klionsky,Takahiro Shintani

doi:10.1074/jbc.m110.173906

Masaki Yuga, Katsuya Gomi + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m110.173906

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Abstract

Macroautophagy is a catabolic process by which cytosolic components are sequestered by double membrane vesicles called autophagosomes and sorted to the lysosomes/vacuoles to be degraded. Saccharomyces cerevisiae has adapted this mechanism for constitutive transport of the specific vacuolar hydrolases aminopeptidase I (Ape1) and α-mannosidase (Ams1); this process is called the cytoplasm to vacuole targeting (Cvt) pathway. The precursor form of Ape1 self-assembles into an aggregate-like structure in the cytosol that is then recognized by Atg19 in a propeptide-dependent manner. The interaction between Atg19 and autophagosome-forming machineries allows selective packaging of the Ape1-Atg19 complex by the autophagosome-like Cvt vesicle. Ams1 also forms oligomers and utilizes the Ape1 transport system by interacting with Atg19. Although the mechanism of selective transport of the Cvt cargoes has been well studied, it is unclear whether proteins other than Ape1 and Ams1 are transported via the Cvt pathway. We describe here that aspartyl aminopeptidase (Yhr113w/Ape4) is the third Cvt cargo, which is similar in primary structure and subunit organization to Ape1. Ape4 has no propeptide, and it does not self-assemble into aggregates. However, it binds to Atg19 in a site distinct from the Ape1- and Ams1-binding sites, allowing it to "piggyback" on the Ape1 transport system. In growing conditions, a small portion of Ape4 localizes in the vacuole, but its vacuolar transport is accelerated by nutrient starvation, and it stably resides in the vacuole lumen. We propose that the cytosolic Ape4 is redistributed to the vacuole when yeast cells need more active vacuolar degradation.

Highlights

In the yeast Saccharomyces cerevisiae, 34 autophagy-related (ATG)2 genes have been identified as important genes for selective and non-selective autophagy
The vacuole preparation from the pep4⌬ prb1⌬ cells resulted in 39-fold purification of the vacuole fraction as estimated by immunoblot of the precursor 2 (p2) form of the marker protein Prc1 that travels to the vacuole via the vacuolar protein sorting pathway (Fig. 1B, lane 1 versus lane 2), whereas there was relatively little contamination from cytosol (Pgk1, 0.40-fold purification)
The cargo proteins maintain their folding and oligomeric states during membrane translocation with this transport system because they do not need to pass through a transport channel that is used for conventional translocation mechanisms

Summary

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MATa his3-⌬200 leu112 lys801 trp1-⌬901 ura suc2-⌬9 GAL SEY6210; atg1⌬::his5ϩ SEY6210; atg11⌬::LEU2 K.l. Because Atg and Atg further associate with Atg and Atg, PAS components required for phagophore expansion, the Cvt complex acts as a scaffold for the formation of a double membrane-bound vesicle so that the vesicle excludes cytosolic components (13, 18 –22) This mechanism could ensure efficient packaging with a limited membrane supply in nutrient-rich conditions. The second Cvt cargo protein, is synthesized in the cytosol as a homo-oligomeric enzyme but does not form a higher order structure by itself in contrast to prApe1 [24] Instead, it is concentrated on the Ape complex through its interaction with Atg, and its import to the vacuole is largely dependent on prApe in nutrient-rich conditions [13].

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