Cellular homeostasis requires the precise regulation of protein synthesis and organelle biogenesis as well as the degradation of unnecessary proteins and organelles. The degradation profile of the cell is comprised of the selective degradation of proteins that occurs predominantly in the cytosol through the ubiquitin/proteasome pathway, and the degradation of bulk cytoplasm that takes place in the lysosome in mammals and plants and in the vacuole in fungi (1
). Autophagy is the major degradation route by which bulk cytoplasm is delivered to the lysosome/vacuole. This pathway is conserved from yeast to human. Although the autophagic machinery only functions at a basal level in nutrient-rich conditions, it plays an essential physiological role in starvation conditions, which are more likely to mimic the natural environment of organisms such as yeast. In yeast, when cells sense nitrogen starvation, cytosol and organelles are nonspecifically sequestered and delivered to the vacuole by the up-regulated autophagic machinery, where they can be recycled and used for critical processes, thus ensuring cell survival (3
Interestingly, in yeast, autophagy overlaps with the biosynthetic delivery of the vacuolar hydrolase aminopeptidase I (Ape1)1
by the cytoplasm to vacuole targeting (Cvt) pathway. Under vegetative growth conditions, precursor Ape1 (prApe1) is synthesized in the cytosol where it quickly forms a homododecamer (5
). These dodecamers subsequently assemble into a higher order complex termed the Cvt complex. The Cvt complex becomes enwrapped by a membrane resulting in a doublemembrane Cvt vesicle (150 nm diameter). The contents of the Cvt vesicles appear to exclude cytosol and are observed as an electron-dense structure by electron microscopy (6
). Autophagy is morphologically similar to the Cvt pathway, however, the cytosolic double membrane vesicles (autophagosomes) are much larger (300–900 nm diameter) than Cvt vesicles and they include bulk cytoplasm (7
). In either case, the completed cytosolic vesicles then dock and fuse with the vacuole membrane releasing single membrane Cvt or autophagic bodies into the vacuole lumen. During starvation conditions, prApe1 is specifically sequestered, along with bulk cytoplasm, inside the double membrane autophagosome for its delivery to the vacuole. Precursor Ape1 is proteolytically processed to mature Ape1 once it reaches the vacuole by either the Cvt pathway or autophagy. Therefore, prApe1 processing serves as a useful diagnostic marker for studying both the Cvt pathway in nutrient-rich conditions and the autophagy pathway during starvation (8
Genetic analyses support the morphological data that indicate an overlap between the Cvt pathway and autophagy. A genetic screen that was based on the accumulation of precursor Ape1 under nutrient-rich conditions was used to isolate mutants in the Cvt pathway (9
). These cvt
mutants exhibited an extensive genetic overlap with apg
mutants that were isolated as being defective in autophagy, indicating that the two pathways also share protein components (8
). Recently, it has been shown that the selective degradation of excess peroxisomes (pexophagy) occurs by a similar process using the same molecular machinery shared between the Cvt and autophagy pathways (11
). The analysis of the molecular components required for these pathways has provided significant insights into the process by which proteins are delivered post-translationally to the vacuole/lysosome.
While many of the details of cytoplasm to vacuole transport remain to be elucidated, some of the key questions have begun to be answered. For example, the Cvt pathway operates under nutrient-rich conditions while autophagy is induced by starvation. Degradation of peroxisomes occurs during adaptation to preferred carbon sources following growth in peroxisome proliferating conditions such as methanol or oleate. The identified mechanism of signal transduction includes a signaling pathway regulated by Tor, a phosphatidylinositol 3-kinase homologue. Tor represses autophagy under nutrient-rich conditions. During starvation or following treatment with rapamycin, Tor kinase activity is inhibited and autophagy is induced (12
). The Tor signaling cascade also affects the phosphorylation state of Apg13 and its affinity for the Apg1 kinase (13
). Recently, Cvt9 and Apg17 were characterized as additional proteins that associate with the Apg1 kinase (13
). Interestingly, Cvt9 is specific for the Cvt pathway and is also needed for pexophagy while Apg17 is specific for autophagy. These findings suggest that a potential protein complex including the Apg1 kinase and various regulatory proteins may determine which of these pathways operates within the cell.
One of the most distinctive features of the Apg/Cvt pathways is the formation of sequestering vesicles that engulf cytoplasm. A major difference between autophagy and the Cvt pathway is that autophagosomes are substantially larger than the Cvt vesicles that form under nutrient-rich conditions. Aut7 is the first identified component that localizes to both Cvt vesicles and autophagosomes. The level of Aut7 is up-regulated under starvation conditions, suggesting that it is a limiting component that is required for Cvt vesicle formation as well as autophagosome expansion (15
). The formation and completion of the Cvt vesicle/autophagosome also requires a novel protein conjugation system consisting of Apg5, Apg7, Apg10, Apg12, and Apg16 (17
). Recent findings suggest that Aut7 membrane association is dependent on the autophagy (Apg) conjugation system as well as a novel membrane lipidation process involving the Aut1 protein (21
). Finally, Apg9 is the only characterized integral membrane protein required for the vesicle formation/completion step for both the Cvt and autophagy pathways. It is localized at a perivacuolar punctate structure that appears to be distinct from typical plasma membrane and endomembrane marker proteins. Interestingly, the Apg9 protein does not appear to be localized to the Cvt vesicle/autophagosome suggesting that it might mark the site of the donor membrane for vesicle formation (23
Although some components have been cloned, many questions remain to be answered concerning the Cvt pathway, autophagy, and pexophagy. The identification of additional components that function in these pathways will help us to understand the molecular details of the import process including the origin of the sequestering membrane, the signal transduction cascade that senses the nutritional conditions, the mechanism of vesicle formation, and the subsequent delivery and breakdown of the subvacuolar vesicles. In the present study we have begun a characterization of the Apg2 protein that is required for both the Cvt and autophagy pathways and also for the related process of peroxisome degradation. The apg2Δ strain accumulated the precursor form of Ape1 in a protease-accessible, membrane-associated form, suggesting that Apg2 is required for the completion stage of Cvt vesicle and autophagosome formation. Subcellular fractionation and biochemical studies demonstrate that Apg2 is a peripheral membrane protein that localizes to the previously identified Apg9 compartment. Finally, microscopy experiments indicate that GFPApg2 localization is dependent on the Apg9 protein. This finding is supported by an in vitro pull-down assay that demonstrates a physical interaction between Apg2 and Apg9.