A key early step in the membrane expansion and autophagic vesicle-formation processes may be defined by the recruitment of starvation-induced components to a membrane compartment involved in the formation of autophagic vesicles. The Aut7 protein is the first characterized transport vesicle component that is upregulated during starvation conditions (Kirisako et al. 1999
; Huang et al. 2000
). The membrane association of the hydrophilic Aut7 protein is an important requirement for transport vesicle formation and completion. In this study, we have characterized the steps and components required for Aut7p membrane binding. We have identified a function for Aut2p in mediating a proteolytic processing event at the COOH terminus of Aut7p ( C). The Aut7p processing reaction is rapid, occurring with a half time of <1 min by radioactive pulse-chase labeling analysis (data not shown). This modification is a requisite step for the subsequent membrane binding of Aut7p, as the Aut7 protein is found exclusively in a soluble fraction in an aut2
Δ strain defective for proteolytic processing ( D). Similarly, a GFPAut7p fusion protein that functions as an in vivo marker for the autophagy pathway displays a diffuse cytosolic staining pattern in the aut2
Δ mutant (). In contrast, in most of the autophagy mutants that possess the capacity to process Aut7p (e.g., apg1
Δ and apg9
Δ; and A, respectively), the GFPAut7p fusion protein localizes to prominent, punctate perivacuolar structures in addition to its cytosolic distribution. These punctate structures may represent a previously described intermediate compartment that appears during autophagosome formation (Kirisako et al. 1999
To further understand the role of Aut2p in the Aut7p cleavage reaction, we examined the Aut2 protein. Both subcellular fractionations ( B) and confocal microscopy of the Aut2pGFP fusion protein (data not shown) indicate that Aut2p is a cytosolic, soluble protein. These findings indicate that the Aut2p-mediated cleavage of Aut7p most likely occurs in the cytosol. In addition, the observation that Aut2p expression does not increase under starvation conditions (data not shown) suggests that Aut2p is not a stoichiometric partner of Aut7p, but, rather, interacts with Aut7p in a catalytic manner to facilitate Aut7p proteolysis. Recently, Kirisako et al. 2000
have demonstrated that Aut2p/Apg4p is a cysteine protease that cleaves the ultimate residue (arginine) from the COOH terminus of Aut7p. Moreover, the Aut7p C-terminal cleavage was determined to be required for its subsequent membrane association. Our subcellular fractionation results () as well as the localization of the GFPAut7p fusion protein in the aut2Δ background confirm these observations.
The recent two-hybrid analyses of the S. cerevisiae
genome have presented an array of interactions between Apg components (Ito et al. 2000
; Uetz et al. 2000
). These interactions included the association of Aut7p with Aut1p. In addition, Aut7p and Aut1p both interact with components of the Apg conjugation system that covalently links Apg12p to Apg5p through the action of Apg7p (Mizushima et al. 1998
). Specifically, Aut7p associates with Apg7p while Aut1p interacts with both Apg7p and Apg12p ( A). Based on these findings, we investigated the role of Aut1p and the Apg conjugation components in Aut7p function by examining Aut7p membrane binding in the aut1
Δ strain and in null mutants defective in the conjugation reaction. Both subcellular fractionations of the endogenous Aut7 protein and confocal microscopy analysis of the GFPAut7p fusion protein indicate that Aut1p and the Apg conjugation system are required for Aut7p membrane recruitment. Aut7p appears exclusively in the soluble, supernatant fractions in the aut1
Δ and Apg conjugation mutants, while a significant amount of Aut7p binds to a low-speed pellet fraction in the other apg
mutant strains ( A and 7 B, respectively). In agreement with the fractionation results, the GFPAut7p fusion protein is diffusely distributed in the cytosol in the aut1
Δ and in all of the Apg conjugation mutant strains. These findings establish a role for Aut1p and the Apg conjugation system in mediating Aut7p recruitment to an intermediate, punctate membrane compartment during autophagosome formation. Interestingly, the requirement of the conjugation component Apg16p, which facilitates the multimerization of the already-conjugated Apg12p–Apg5p species, suggests that it is not the conjugation reaction per se that plays a role in membrane binding of Aut7p, but rather some function of the multimeric Apg conjugation complex. Finally, as stated above, two-hybrid studies link Aut7p with Apg7p. It is interesting to note that the fluorescent staining pattern of GFPAut7p is similar to that of Apg7pGFP (Kim et al. 1999
). Both proteins display a cytosolic distribution along with punctate perivacuolar structures. It therefore appears likely that both proteins are recruited to the same, as yet unidentified, intermediate compartment during autophagosome formation.
Although Aut1p interacts with Apg7p and Apg12p of the conjugation system, defects in Aut1p do not prevent the final conjugation of Apg12p to Apg5p, indicating that Aut1p is not directly required for the Apg conjugation reactions ( B). Whether Aut1p and the conjugation system act in concert or independently through parallel pathways to recruit Aut7p to the membrane remains to be determined. However, neither Aut1p nor the Apg conjugation components are required for the Aut2p-mediated processing of Aut7p ( A). Taken together, these findings suggest that Aut1p and the conjugation system act at a post-processing step in Aut7p membrane recruitment.
Kirisako et al. 2000
have recently investigated the role of Apg7p, the E1 ubiquitin-activating enzyme homologue of the Apg conjugation system, in the Aut7p membrane-binding step. While the function of Apg7p in forming an ATP hydrolysis-dependent thioester linkage to Apg12p has been well characterized (Mizushima et al. 1998
; Tanida et al. 1999
), Kirisako et al. 2000
also demonstrated that Apg7p is required for Aut7p membrane association after its Aut2p-mediated processing step. In their study, the initial removal of the COOH-terminal arginine residue of Aut7p by Aut2p resulted in a loosely membrane-bound conformation. Then, by a ubiquitination-like mechanism, Apg7p (and potentially other factors) mediate the conjugation of Aut7p to an as-yet unidentified “X” factor on the membrane, thus converting Aut7p from a loosely membrane-bound state to a tightly membrane-bound one. Furthermore, Kirisako et al. 2000
demonstrated that Aut7p in the tightly membrane-associated form was subsequently liberated from the X factor through an Aut2p-mediated cleavage reaction, thus completing the Aut7p membrane-association cycle. In this study, we demonstrate that the entire collection of Apg conjugation components, including Apg5p, Apg7p, Apg10p, Apg12p, and Apg16p, are all required for Aut7p membrane binding. The exact function of the other Apg conjugation components in mediating Aut7p membrane binding remains to be elucidated. It is possible that the Apg conjugation system involving Apg12p–Apg5p acts indirectly by affecting the membrane compartment to which Aut7p initially binds. Future studies will be aimed at determining the role of these proteins in Aut7p membrane recruitment. However, it appears that Apg7p may be involved in two parallel pathways that are both required for Aut7p membrane binding: one that involves Apg10p in the conjugation of Apg12p–Apg5p and a second pathway that uses a second conjugating enzyme to add the X factor directly to Aut7p.
Other recent studies have offered two views of Aut7p function. An initial study by Lang et al. 1998
proposed that Aut2p and Aut7p functioned as attachment proteins that link autophagosomes to the microtubule cytoskeleton for vectorial delivery to the vacuole. According to this model, defects in Aut2p and Aut7p would result in the cytoplasmic accumulation of prAPI-containing autophagosomes. An alternative view of Aut7p function was subsequently proposed (Kirisako et al. 1999
; Huang et al. 2000
). These latter studies demonstrated that precursor API in the aut7
Δ strain existed in a membrane-bound, protease-accessible state, suggesting that Aut7p was required at a step before vesicle completion. Our results indicate that prAPI in the aut1
Δ and aut2
Δ strains also exists in a membrane-associated but protease-accessible state, identical to the aut7
Δ prAPI phenotype (). Because Aut1p and Aut2p are required for Aut7p function, the common prAPI phenotype exhibited by the aut1
, and aut7
null mutants supports the view that Aut1p, Aut2p, and Aut7p all act at the same step(s) of vesicle formation and/or completion.
The findings from this study can be incorporated into the current model of prAPI import by the Cvt and autophagy pathways ( B). Immediately after synthesis, prAPI oligomerizes into a dodecamer, and then assembles into a higher-order Cvt complex. The Cvt complex then associates with a membrane of unknown origin. Interestingly, while deletions of AUT1
, and the Apg conjugation apparatus all prevent Aut7p membrane binding, prAPI in all cases retains its ability to bind to a pelletable membrane fraction (). While these autophagy components are not required for prAPI membrane binding, additional factors may play a role in this process. The Aut2p-mediated cleavage of Aut7p generates a binding-competent form of this protein. However, both Aut1p and the Apg conjugation components are essential in facilitating Aut7p membrane binding. Our previous results indicated that the initial binding of Aut7p results in a protease-accessible form of the protein (Huang et al. 2000
; B). Upon vesicle completion, Aut7p travels with prAPI to the vacuole. Subsequent fusion with the vacuole releases the prAPI and Aut7p-containing autophagic bodies into the vacuole lumen. The autophagic bodies are then degraded by resident hydrolases, thus allowing prAPI to be processed to the mature form. In this final step, both Aut7p and bulk cytoplasmic cargo are degraded by vacuolar proteases and recycled for essential biosynthetic processes required to survive starvation conditions.
The Cvt and autophagy pathways share a common set of molecular components. However, the autophagosomes that are formed under starvation conditions are much larger than their Cvt vesicle counterparts formed under vegetative conditions. These observations suggest that formation of the autophagosome requires an expansion of the enwrapping membrane, as well as the increased expression of structural components. Aut7p may represent one member in a family of such structural components localized to these autophagic transport vesicles. Accordingly, the level of Aut7p expression in starvation versus vegetative conditions appears to reflect the 10–20-fold increase in vesicle surface area between autophagosomes and Cvt vesicles (Huang et al. 2000
). In support of this hypothesis, a block in the upregulation of Aut7p results in the formation of abnormally small autophagosomes (Abeliovich et al. 2000
In this study, we have characterized three events that are required for Aut7p recruitment to the membrane, a critical step in the formation of autophagic vesicles. In addition, these data define a function of the Apg conjugation system in recruiting structural components to the forming autophagosome. Further studies will be aimed at determining the specific modification effected by the conjugation machinery, the function of Aut1p in membrane binding of Aut7p, and the source of the membrane compartment to which Aut7p is recruited. These studies will begin to provide detailed mechanistic information about the formation of the sequestering vesicle used in the Cvt and autophagy pathways.