Apg7p/Cvt2p is required for the cytoplasm-to-vacuole targeting, macroautophagy, and peroxisome degradation pathways

Proper functioning of organelles necessitates efficient protein targeting t o the appropriate subcellular locations. For example, degradation in the fu ngal vacuole relies on an array of targeting mechanisms for both resident h ydrolases and their substrates. The particular processes that are used vary depending on the available nutrients. Under starvation conditions, macroau tophagy is the primary method by which bulk cytosol is sequestered into aut ophagic vesicles (autophagosomes) destined for this organelle. Molecular ge netic, morphological, and biochemical evidence indicates that macroautophag y shares much of the same cellular machinery as a biosynthetic pathway for the delivery of the vacuolar hydrolase, aminopeptidase I, via the cytoplasm -to-vacuole targeting (Cvt) pathway. The machinery required in both pathway s includes a novel protein modification system involving the conjugation of two autophagy proteins, Apg12p and Apg5p. The conjugation reaction was dem onstrated to be dependent on Apg7p, which shares homology with the Fl famil y of ubiquitin-activating enzymes. In this study, we demonstrate that Apg7p functions at the sequestration step in the formation of Cvt vesicles and a utophagosomes. The subcellular localization of Apg7p fused to green fluores cent protein (GFP) indicates that a subpopulation of Apg7pGFP becomes membr ane associated in an Apg12p-dependent manner. Subcellular fractionation exp eriments also indicate that a portion of the Apg7p pool is pelletable under starvation conditions. Finally, we demonstrate that the Pichia pastoris ho mologue Gsa7p that is required for peroxisome degradation is functionally s imilar to Apg7p, indicating that this novel conjugation system may represen t a general nonclassical targeting mechanism that is conserved across speci es.