Membrane recruitment of Aut7p in the autophagy and cytoplasm to vacuole targeting pathways requires Aut1p, Aut2p, and the autophagy conjugation complex

Autophagy is a degradative pathway by which cells sequester nonessential, b ulk cytosol into double-membrane vesicles (autophagosomes) and deliver them to the vacuole for recycling. Using this strategy, eukaryotic cells surviv e periods of nutritional starvation. Under nutrient-rich conditions, autoph agy machinery is required for the delivery of a resident vacuolar hydrolase , aminopeptidase I, by the cytoplasm to vacuole targeting (Cvt) pathway. In both pathways, the vesicle formation process requires the function of the starvation-induced Aut7 protein, which is recruited from the cytosol to the forming Cvt vesicles and autophagosomes. The membrane binding of Aut7p rep resents an early step in vesicle formation. In this study, we identify seve ral requirements for Aut7p membrane association. After synthesis in the cyt osol, Aut7p is proteolytically cleaved in an Aut2p-dependent manner. While this novel processing event is essential for Aut7p membrane binding, Aut7p must undergo additionalphysical interactions with Aut1p and the autophagy ( Apg) conjugation complex before recruitment to the membrane. Lack of these interactions results in a cytosolic distribution of Aut7p rather than local ization to forming Cvt vesicles and autophagosomes. This study assigns a fu nctional role for the Apg conjugation system as a mediator of Aut7p membran e recruitment. Further, we demonstrate that Aut1p, which physically interac ts with components of the Apg conjugation complex and Aut7p, constitutes an additional factor required for Aut7p membrane recruitment. These findings define a series of steps that results in the modification of Aut7p and its subsequent binding to the sequestering transport vesicles of the autophagy and cytoplasm to vacuole targeting pathways.