Apg5p Functions in the Sequestration Step in the Cytoplasm-to-Vacuole Targeting and Macroautophagy Pathways

Abstract

The cytoplasm-to-vacuole targeting (Cvt) pathway and macroautophagy are dynamic events involving the rearrangement of membrane to form a sequestering vesicle in the cytosol, which subsequently delivers its cargo to the vacuole. This process requires the concerted action of various proteins, including Apg5p. Recently, it was shown that another protein required for the import of aminopeptidase I (API) and autophagy, Apg12p, is covalently attached to Apg5p through the action of an E1-like enzyme, Apg7p. We have undertaken an analysis of Apg5p function to gain a better understanding of the role of this novel nonubiquitin conjugation reaction in these import pathways. We have generated the first temperature-sensitive mutant in the Cvt pathway, designated apg5 ts. Biochemical analysis of API import in theapg5 ts strain confirmed that Apg5p is directly required for the import of API via the Cvt pathway. By analyzing the stage of API import that is blocked in theapg5 ts mutant, we have determined that Apg5p is involved in the sequestration step and is required for vesicle formation and/or completion.