Atg17 is part of a putative multiprotein complex, including Atg1 kinase. In addition to Atg17, most of the other proteins in the putative complex have been characterized as being relatively specific for either autophagy or the Cvt pathway (; Kamada et al., 2000
; Scott et al., 2000
; Kim et al., 2001b
; Nice et al., 2002
), leading to the hypothesis that this complex plays a role in regulating the conversion between these two pathways; however, the assignment of a particular mutant as “specific” has been somewhat arbitrary. For example, vac8
Δ cells are completely blocked for import of prApe1 under vegetative but not starvation conditions (Scott et al., 2000
). Thus, the Vac8 protein has been classified as specific for the Cvt pathway; however, the vac8
Δ mutant displays a substantial defect in autophagy based on the Pho8Δ60 assay (Scott et al., 2000
; ). Similarly, in the present report, we find based on electron microscopy data and analysis of GFP-Atg8 processing that the vac8
Δ mutant displays a significant autophagic defect (Figures and ). One conclusion from these observations is that it is insufficient to rely on one particular assay to reliably characterize an atg
mutant as being specific to autophagy versus the Cvt pathway.
The initial characterization of the atg17
Δ mutant suggested that it was specific for autophagy; however, this conclusion was based largely on the fact that prApe1 is matured in the atg17
Δ mutant under vegetative conditions, whereas the strain is defective for autophagy as determined by the inability to activate Pho8Δ60 (Kamada et al., 2000
); maturation of prApe1 was not determined under starvation conditions. Partly for the reasons described above, we decided to undertake a more detailed analysis of the role of Atg17. Although we found that atg17
Δ cells are normal for processing of prApe1 in vegetative conditions, the same was true for cells incubated in SD-N to induce autophagy (). Nonetheless, in agreement with the data of Kamada et al
), we found essentially a complete block in uptake of Pho8Δ60 (). These two sets of data are somewhat contradictory with regard to the requirement of Atg17 for autophagy; however, prApe1 is delivered to the vacuole by a specific autophagic mechanism that uses a receptor (Scott et al., 2001
; Shintani et al., 2002
), whereas Pho8Δ60 is a nonspecific marker for bulk autophagy. To resolve this discrepancy, we undertook three additional assays to monitor autophagy. We found that the atg17
Δ mutant displayed an intermediate starvation-sensitivity phenotype relative to the atg1
Δ strain (our unpublished data), showed limited vacuolar import of the autophagosome marker GFP-Atg8 (), and was essentially defective for pexophagy ().
Our conclusion from these various assays was that the atg17
Δ mutant was severely, but not fully, defective for autophagy. To fully understand the nature of this phenotype, we carried out an analysis of atg17
Δ cells by using electron microscopy (). We used a pep4
Δ background to allow accumulation of autophagic bodies within the vacuole lumen and to eliminate the presence of Mvb-derived vesicles. We found that the atg17
Δ mutant accumulated autophagic bodies that were smaller, and fewer in number compared with those seen in the wild-type strain. Thus, Atg17 is not absolutely required for autophagy but seems to play an important role in determining the magnitude of the autophagic response. The electron microscopy data provide an explanation for the results with prApe1, Pho8Δ60 and peroxisome degradation. The reduction in autophagosome capacity apparently causes a severe block in uptake of Pho8Δ60. In contrast, prApe1 is still imported with essentially wild-type kinetics due to its use of a specific receptor. Even though pexophagy is a specific process, the smaller autophagosomes are apparently unable to sequester an organelle of this size (); peroxisomes from oleate-grown cells are typically in the range of 0.2–0.4 μm in diameter (Thieringer et al., 1991
). Because Pho8Δ60 is a soluble cytosolic protein, it seems surprising that a low level is not taken up inside the aberrant autophagosomes that are produced in the atg17
Δ strain. Pho8Δ60 uptake, however, is not efficient even in wild-type cells, and the level of uptake by atg17
Δ cells may be below the level of detection. This possibility is supported by the observation that GFP is liberated from GFP-Atg8 in the atg17
Δ mutant, albeit at a lower level relative to the wild-type strain, similar to the vac8
Δ mutant (). Atg8 is a component of the autophagosome; hence, any autophagosome that fuses with the vacuole will deliver some Atg8 into the lumen. Thus, cleavage of GFP from GFP-Atg8 is a very sensitive marker for autophagy.
Δ double mutant resulted in a complete block in uptake of prApe1 () or processing of GFP-Atg8 (). Similarly, we observed by electron microscopy that the atg17
Δ double mutant was completely unable to form autophagic bodies and presumably autophagosomes. One explanation for these data is that Vac8 and Atg17 have a similar function; in the absence of either individual protein there is sufficient activity to carry out autophagy. Indeed, both the vac8
Δ and atg17
Δ mutants are only partially defective in autophagosome formation (). Although the vac8
Δ strain was previously reported to produce normal autophagosomes (Scott et al., 2000
), in our present analysis the vac8
Δ mutant generated smaller and fewer autophagic bodies relative to the wild-type strain (), in agreement with the autophagic defect detected by the Pho8Δ60 and GFP-Atg8 assays (Figures and ). We do not consider the explanation of similar functions for Vac8 and Atg17 likely, however, because the vac8
Δ mutant is completely defective for import of prApe1 under vegetative conditions, whereas atg17
Δ cells are not blocked in prApe1 import. Finally, Vac8 interacts with several proteins and plays a role in a range of processes, including organelle inheritance (Wang et al., 1998
) and piecemeal microautophagy of the nucleus (Roberts et al., 2003
), suggesting that it has different functions than Atg17. Because the vac8
Δ mutant displayed a severe, although not complete, defect in autophagy based on uptake of Pho8Δ60 (), we propose that the two mutations have a synthetic defect. A synthetic defect also would fit with the fact that these two proteins are part of a common complex. Atg13 modulates Atg1 kinase activity and Atg17 may have a similar role, via Atg13. The function of Vac8 in the Cvt and autophagy pathways is not known, but its interaction with Atg13 may allow it to also modulate Atg1 kinase activity.
To gain further information about the interaction among the components of the Atg1 kinase complex, we mapped interacting domains in Atg13 and Atg17 (). The domains in Atg13 that interact with Atg1 and Vac8 have been mapped previously (Kamada et al., 2000
; Scott et al., 2000
). We found that the Atg17-interacting domain overlapped partially with that of Atg1 but was separate from that of Vac8. A finer degree of analysis would probably separate the Atg1- and Atg17-binding domains; however, we chose to focus on Atg17. Two of the five predicted coiled-coil domains of Atg17 were needed for interaction with Atg1 and Atg13 (Figures , , and ). It is possible that the interaction between Atg17 and Atg1 is mediated through Atg13; however, we obtained conflicting data on this point. Two-hybrid assays indicated a direct interaction between Atg17 and Atg1 (), whereas affinity isolation suggested that these two proteins did not interact in the absence of Atg13 (). These differences may reflect the greater stringency of the affinity isolation, or a higher sensitivity of the two-hybrid assay. Although the two-hybrid assay also is better suited to detect transient interactions, it is limited by the use of hybrid proteins that must interact in a nonphysiological context. We extended the analysis of protein interactions by carrying out affinity isolation with the mutated Atg17 proteins and were able to confirm that the first and third coiled-coils of Atg17 are necessary for binding Atg1 and Atg13 (). During the course of these studies, Ohsumi and colleagues published an analysis of Atg17 (Kabeya et al., 2005
). They found that a mutation of cysteine at position 24, corresponding to a site within the first coiledcoil domain, eliminated the interaction between Atg17, Atg13, and Atg1, further supporting our results; however, they were unable to determine whether the interaction between Atg1 and Atg17 was direct or mediated through Atg13.
When the level of autophagy was examined with Atg17 mutants lacking predicted coiled-coil domains, the Atg17ΔCC2 mutant showed low Pho8Δ60 activity after starvation, similar to the result seen with the Atg17ΔCC1 or ΔCC3 mutants (). Although Atg17ΔCC2 is not involved in the interaction with Atg1 and Atg13, we have not determined whether the Atg17CC2 domain is involved in interactions with other proteins that might be important for autophagy. Similarly, the Atg17ΔCC5 mutant may be defective for Pho8Δ60 activity due to the loss of other binding sites that do not involve Atg1 or Atg13. Alternatively, this large deletion may affect the folding of the protein resulting in a loss of function, although the mutant protein was stable.
When we analyzed the localization of GFP-Atg17 mutants, we found that GFP-Atg17ΔCC1 displayed a diffuse cytosolic signal and did not show a punctate dot, a localization pattern different from that seen for Atg17-GFP in the atg1Δ and atg13Δ mutants (). As part of our analysis, we generated a set of smaller deletions within the coiled-coil regions in a preliminary study to more finely map the Atg17 interaction sites. We found that smaller deletions within the first coiled-coil region resulted in mutant GFP-Atg17 proteins that displayed single, bright punctate dots and that were also defective in interacting with Atg13 (our unpublished data). These data suggest that a specific region(s) within the first coiled-coil domain of Atg17 may be involved in its localization to the PAS, and that this region(s) might be separate from those that interact with Atg1 and Atg13.
The role of Atg1 kinase activity is not clear, and there have been conflicting reports suggesting that higher kinase activity plays a more important role in the autophagy (Kamada et al., 2000
) or Cvt (Abeliovich et al., 2003
) pathways. In the present analysis, we found that the localization of Atg17 was dependent on Atg1 kinase activity particularly in rich conditions (; our unpublished data). Atg17 localization was more severely affected in starvation conditions in the atg1
Δ strain than in the presence of the kinase dead Atg1K54A
mutant (our unpublished data). This finding is in agreement with previous studies that suggest that Atg1 may have a structural role in autophagy (Abeliovich et al., 2003
). We suggest that higher kinase activity is more important for the Cvt pathway but that Atg1 kinase activity is needed for both the Cvt and autophagy pathways (Abeliovich et al., 2003
; Reggiori et al., 2004a
) in agreement with the recent study by Kabeya et al
Finally, it may be informative to note that ATG17
only has homologues in yeast, whereas ATG13
do not have homologues in higher eukaryotes outside of plants. Autophagosomes in higher eukaryotes seem to be of a uniform size before and after starvation (Dunn, 1990
). Thus, regulation of sequestering vesicle size seems to be peculiar to yeast. We propose that the direct interaction between Atg17 and Atg13, and the direct or indirect interaction with Atg1 is critical for regulating autophagosome size. Although the Atg1 complex is proposed to be involved in controlling the conversion between the autophagy and Cvt pathways, this regulation may not take place at the stage of autophagic induction. We have shown that Atg1 and Atg13 participate at a later stage, retrieval of Atg9 from the PAS (Reggiori et al., 2004a
). An Atg1-dependent defect in Atg9 retrieval that is mediated through Atg17 could provide one explanation for the smaller and fewer autophagosomes seen in the atg17
Δ mutant. That is, the absence of Atg17 may partially interfere with Atg9 cycling (), resulting in a reduced supply of membrane to the sequestering vesicles. In contrast, atg1
Δ or atg13
Δ strains are completely defective in this step and are unable to form vesicles of any size. Additional studies concerning the functions of the components of the Atg1 complex should provide further insight into the mechanism of vesicle size regulation.