Apg5p functions in the sequestration step in the cytoplasm-to-vacuole targeting and macroautophagy pathways

The cytoplasm-to-vacuole targeting (Cvt) pathway and macroautophagy are dyn amic events involving the rearrangement of membrane to form a sequestering vesicle in the cytosol, which subsequently delivers its cargo to the vacuol e. This process requires the concerted action of various proteins, includin g Apg5p. Recently, it was shown that another protein required for the, impo rt 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 o f 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 the apg5(ts) str ain 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 the a pg5(ts) mutant, we have determined that Apg5p is involved in the sequestrat ion step and is required for vesicle formation and/or completion.