Apg9p/Cvt7p Is an Integral Membrane Protein Required for Transport Vesicle Formation in the Cvt and Autophagy Pathways

Abstract

In nutrient-rich, vegetative conditions, the yeast Saccharomyces cerevisiae transports a resident protease, aminopeptidase I (API), to the vacuole by the cytoplasm to vacuole targeting (Cvt) pathway, thus contributing to the degradative capacity of this organelle. When cells subsequently encounter starvation conditions, the machinery that recruited precursor API (prAPI) also sequesters bulk cytosol for delivery, breakdown, and recycling in the vacuole by the autophagy pathway. Each of these overlapping alternative transport pathways is specifically mobilized depending on environmental cues. The basic mechanism of cargo packaging and delivery involves the formation of a double-membrane transport vesicle around prAPI and/or bulk cytosol. Upon completion, these Cvt and autophagic vesicles are targeted to the vacuole to allow delivery of their lumenal contents.Key questions remain regarding the origin and formation of the transport vesicle. In this study, we have cloned the APG9/CVT7 gene and characterized the gene product. Apg9p/Cvt7p is the first characterized integral membrane protein required for Cvt and autophagy transport. Biochemical and morphological analyses indicate that Apg9p/Cvt7p is localized to large perivacuolar punctate structures, but does not colocalize with typical endomembrane marker proteins. Finally, we have isolated a temperature conditional allele of APG9/CVT7 and demonstrate the direct role of Apg9p/Cvt7p in the formation of the Cvt and autophagic vesicles. From these results, we propose that Apg9p/Cvt7p may serve as a marker for a specialized compartment essential for these vesicle-mediated alternative targeting pathways.

PpATG9 Encodes a Novel Membrane Protein That Traffics to Vacuolar Membranes, Which Sequester Peroxisomes during Pexophagy inPichia pastoris

“…These vesicles distribute as a single peak on sucrose gradients, which can be resolved from the vacuole (Pho8), plasma membrane (Pma1), Golgi apparatus (Kex2), endoplasmic reticulum (Sec12), and endosomes (Pep12; Noda et al, 2000). In addition, our results suggest that those vesicles adjacent to the vacuole are not the prevacuolar compartment (Noda et al, 2000).…”

“…PpAtg9 is not essential for cell viability, but appears to be structurally conserved throughout a number of plant, fungi, insect, and mammalian species. PpAtg9 is also necessary for starvation-induced nonselective autophagy in P. pastoris, S. cerevisiae, and A. thaliana, and for the trafficking of Ape1 to the vacuole in S. cerevisiae (Noda et al, 2000;Hanaoka et al, 2002).…”

“…The results demonstrate that PpAtg9 does not colocalize with either Sec13 (Figure 3E) or Sec7 ( Figure 3F). In S. cerevisiae, the peripheral vesicles containing ScAtg9 do not colocalize with any defined organelle markers for the Golgi apparatus, endosomes, or the endoplasmic reticulum (Noda et al, 2000;Kim et al, 2002;Reggiori et al, 2004). Therefore, we will refer to these unique peripheral structures collectively as the Atg9 peripheral compartment (Atg9-PC).…”

Autophagy and Related processes in Trypanosomatids: Insights from Genomic and Bioinformatic Analyses

“…While we analyzed trypanosomatid proteins, domain composition of orthologous proteins across species is generally conserved so our conclusions are relevant to the structural biology of autophagy in general. Only one protein, ATG9, is an integral membrane protein, 55 of the kind that present special problems for crystallographers. One particularly attractive area would be the single UBL-conjugating system predicted for trypanosomatids, where the structure of the ubiquitin-like protein ATG8 is known for a reasonably close relative, 17 but where three enzymes ATG3, ATG4 and ATG7, are of unknown or near-unknown structure (Table 2).…”

Atg27 Is Required for Autophagy-dependent Cycling of Atg9

“…One of the most intriguing questions is the origin of the membrane for the double-membrane Cvt vesicles or autophagosomes. Recent data suggest that Atg9 may mark the membrane that is donated to the forming sequestering vesicles (Noda et al, 2000;Reggiori et al, 2004a). Atg9 localizes to mitochondria and cycles between this compartment and the pre-autophagosomal structure (PAS), the site of organization for Cvt vesicle and autophagosome formation (Tucker et al, 2003).…”