Construction of a MKO strain to study autophagy
We were able to construct the MKO strain because all the ATG
genes directly required for autophagy and the Cvt pathway are nonessential, and the loxP
system allows the use and recycling of markers for MKOs (; Gueldener et al., 2002
). Among the 31 ATG
genes, 24 of them were deleted in strain YCY123 (). ATG15
, and ATG30
were not deleted because the first two function after autophagosomes fuse with the lysosomal/vacuolar membrane, and the rest are either not found in Saccharomyces cerevisiae
or are only required for peroxisome degradation in other methylotrophic yeasts (Cao and Klionsky, 2007
was not deleted because we finished generating the MKO strain and the experiments described in this manuscript before it was published (Kabeya et al., 2007
). We refer to strain YCY123 as the MKO strain. Additional deletions or the presence of ATG
genes are indicated in parentheses.
Figure 1. Generation and properties of the MKO strain. (A) Schematic representation of the MKO strategy. The loxP–Cre system allows disruption of up to five genes using different markers, and efficient removal of markers by Cre expression. Multiple rounds (more ...)
Yeast strains used in this study
The MKO strain does not have any obvious unexpected defect other than a slight growth delay (). In the MKO strain, we observed one abnormally enlarged vacuole, which is similar to the atg18Δ
phenotype (; Dove et al., 2004
). The normal vacuole morphology was restored when we introduced Atg18 back into the MKO strain (). We also tested whether the MKO strain was defective for other vacuolar targeting pathways. Carboxypeptidase Y (Prc1) transits through the ER, Golgi complex, and late endosome–multivesicular body (MVB) for delivery to the vacuole. Carboxypeptidase S (Cps1) is a cargo for the MVB pathway and transits through the early secretory pathway similar to Prc1, but is packaged into lumenal vesicles of the MVB before vacuolar delivery. Vacuolar alkaline phosphatase (Pho8) is transported through the ER to the Golgi, but it bypasses the endosome–MVB (Bowers and Stevens, 2005
). By pulse-chase analysis and immunoprecipitation, we followed Prc1, Cps1, and Pho8 processing in the MKO strain. As ATG6
is essential for the carboxypeptidase Y pathway, we transformed a plasmid expressing Atg6 into the MKO and the atg6Δ
strains. As shown in , although there was a kinetic delay in the MKO strain expressing Atg6, most of the precursor Prc1, Cps1 (not depicted), and Pho8 became mature after 45 min in chase medium. Taking the growth delay of the MKO strain into consideration, these data suggest that the MKO strain expressing Atg6 is not defective for the carboxypeptidase Y, MVB, and alkaline phosphatase vacuolar protein delivery pathways, which are nonautophagic.
Components necessary and sufficient for cargo packaging in the Cvt pathway
We next wanted to determine if the MKO strain faithfully reproduced different steps of autophagy when expressing the appropriate autophagy genes. Previous studies with standard deletion strains have only been able to demonstrate the requirement, but not the sufficiency, for various proteins involved in different steps of autophagy. In the next set of experiments, we reconstituted the cargo packaging step of the Cvt pathway. At the beginning of the cargo packaging process, prApe1 forms a large oligomer, and a bright single punctate structure representing the oligomer can be observed by fluorescence microscopy if the protein is tagged with a fluorophore (Kim et al., 1997
; Shintani et al., 2002
). The same pattern was also detected in the MKO (atg15Δ RFP-APE1
) strain expressing prApe1 tagged with RFP (, left). Additional knockout of ATG15
in the MKO strain slightly reduced its growth rate but did not affect Cvt vesicle/autophagosome formation (not depicted). In ape1Δ
cells, both the Atg19 receptor and the Atg11 adaptor are dispersed in the cytosol (Yorimitsu and Klionsky, 2005
). To determine whether the MKO strain reproduced these phenotypes, we constructed a version lacking APE1
and transformed it with a plasmid expressing either YFP-Atg19 or GFP-Atg11. In the absence of prApe1 (the cargo), YFP-Atg19 was cytosolic (, middle), but GFP-Atg11 formed a punctate structure (, right). Atg11 is able to interact with itself and several other Atg proteins, and the coiled-coil domains through which Atg11 self-interacts overlap with the interaction sites used by Atg1, Atg17, and Atg20 (Yorimitsu and Klionsky, 2005
). Therefore, it is possible that without competition from the other Atg proteins, Atg11 can form a large oligomer by self-interaction in the MKO strain. Analysis of the Atg11 protein by gradient fractionation suggests that it indeed forms a large complex in the MKO strain (unpublished data).
Figure 2. Reconstitution of the cargo recognition and packaging step of the Cvt pathway. (A) Localization of the cargo prApe1 in the MKO (atg15Δ RFP-APE1) strain, and localization of the receptor Atg19 and the adaptor Atg11 in the MKO (ape1Δ atg15Δ (more ...)
In wild-type cells, Atg19 binds the propeptide of, and colocalizes with, prApe1. Atg11 interacts with Atg19 and targets the complex to the PAS (Scott et al., 2001
; Shintani et al., 2002
; Yorimitsu and Klionsky, 2005
). Thus, there is a temporal order of protein interaction, whereby Atg11 cannot bind the prApe1 complex in the absence of Atg19. To test whether this temporal organization is faithfully retained in the MKO strain, we transformed the MKO (atg15Δ RFP-APE1
) strain with plasmids expressing YFP-Atg19, CFP-Atg11, or both. In agreement with the previous model, RFP-Ape1 and YFP-Atg19 colocalized with each other in the absence of other Atg proteins (, top). In contrast, without Atg19, RFP-Ape1 did not colocalize with CFP-Atg11 (, middle). The three proteins colocalized in a single punctum when all of them were coexpressed (, bottom). These data suggest that prApe1, Atg19, and Atg11 are necessary and sufficient for cargo packaging in the Cvt pathway. We note, however, that only ~50% of the puncta representing the cargo complex (comprised of prApe1, Atg19, and Atg11) were at the PAS (i.e., perivacuolar), which suggests that additional Atg proteins are required to facilitate the targeting of the cargo complex to this site.
Assembly of the initial starvation-specific PAS
The analysis of cargo packaging verified that the MKO strain faithfully replicated the temporal order of Atg protein interactions that have been deduced from previous studies. Next, we decided to use the MKO strain to address a question that would otherwise be extremely difficult to approach. In particular, we wanted to determine the order of assembly of Atg proteins at the PAS. In theory, it might be possible to order the proteins through an extensive analysis of multiple deletion strains, generating a series of epistatic relationships. However, a definitive study is best performed in the complete absence of the other (i.e., those not being examined) Atg proteins. Recent studies suggest that Atg11 and Atg17 are the two initial factors that establish the assembly sequence for Atg proteins at the PAS (Shintani et al., 2002
; Suzuki et al., 2007
). In particular, Atg11 is critical for PAS assembly during vegetative growth, whereas Atg17 is proposed to act as a scaffold for recruiting other Atg proteins during starvation-specific PAS formation.
To test whether Atg17 itself is sufficient to assemble the initial PAS, we observed the localization of Atg17 tagged with GFP in the MKO strain by fluorescence microscopy. In wild-type cells, Atg17-GFP displayed a clear PAS punctum as well as a diffuse cytosolic signal in both vegetative and starvation conditions; in some cells, we could detect more than one punctum, particularly under starvation conditions (). In contrast, Atg17-GFP was largely cytosolic in the MKO (ATG17-GFP) strain (, vector), which suggests that Atg17 is not sufficient for the initial PAS assembly even during starvation. Therefore, we decided to determine what additional factors were needed to allow correct localization of Atg17 during autophagy.
Figure 3. Reconstitution of the initial step of starvation-specific PAS assembly. (A) Localization of Atg17-GFP in the wild-type (WT) and MKO (ATG17-GFP) strains. The WT (ATG17-GFP) strain; the MKO (ATG17-GFP) strain transformed with vector (pRS415), a plasmid (more ...)
Recently, it was found that Atg1 and Atg13 are required along with Atg17 in assembly of the starvation-specific PAS (Cheong et al., 2008
). Accordingly, we hypothesized that Atg1, Atg13, and Atg17 are all required for the initial PAS assembly during nonspecific autophagy. To examine this, we transformed plasmids expressing Atg1, Atg13, or both into the MKO (ATG17-GFP
) strain and observed localization of Atg17-GFP by fluorescence microscopy. Atg17-GFP showed clear punctate structures under starvation conditions only when Atg1 and Atg13 were expressed together (, Atg1 + Atg13). In contrast, expression of either Atg1 or Atg13 alone or in combination with Atg11 did not facilitate Atg17-GFP puncta formation (). The same localization pattern was detected with GFP-Atg1; this protein was cytosolic when expressed by itself, and formed punctate structures in starvation conditions only when Atg13 and Atg17 were both present (unpublished data). Furthermore, ~75% of Atg17-GFP or GFP-Atg1 puncta were perivacuolar (detected with the lipophilic dye FM 4-64), which indicates that Atg1, Atg13, and Atg17 are sufficient for the initial PAS assembly during starvation (). All together, these data suggest that Atg17 is not sufficient for starvation-specific PAS formation; rather, Atg1, Atg13, and Atg17 all appear to have an initial role in PAS assembly during starvation.
Finally, we quantified the percentage of cells containing Atg17-GFP puncta in the MKO strain expressing different Atg proteins. Approximately 40% of the cells coexpressing Atg1 and Atg13 showed Atg17-GFP puncta under starvation conditions, whereas <5% of the cells displayed any punctate structure when Atg17-GFP was expressed by itself (). Additional expression of other components in the Atg17 complex further increased the percentage of cells displaying Atg17-GFP puncta. For example, Atg29 by itself did not significantly affect Atg17-GFP puncta formation, but expression of Atg29 in the presence of Atg1 and Atg13 increased the percentage of cells with puncta to ~53% during starvation. Atg11, when expressed together with Atg1 and Atg13, resulted in an even greater increase to ~80% (). Collectively, these results indicate that starvation-specific PAS formation initially requires a minimal set of Atg proteins consisting of Atg1, Atg13, and Atg17; other components in the Atg1 complex, such as Atg29 and Atg11, further enhance assembly.
The Atg8 conjugation system affects formation of the Atg12–Atg5 conjugate
Having established that the MKO strain reproduces the temporal order of action of components involved in cargo packaging in growing conditions, and determined the initial factors for PAS assembly during starvation, we decided to extend our analysis of the utility of this system. In particular, we chose to examine the Atg12–Atg5 conjugation system because this part of the autophagy process has been reconstituted in vitro. Therefore, components have been identified that are both necessary and sufficient for the reaction. The question we posed was whether the information gained from these previous experiments accurately reflected the complete in vivo situation. Previous data show that Atg5, Atg7, Atg10, and Atg12 are essential for the conjugation (Mizushima et al., 1998
), and Atg16 is required for the efficiency of this reaction (Mizushima et al., 1999
), but Atg3, Atg4, and Atg8 from the Atg8–PE system are not involved in Atg12–Atg5 conjugation (Kuma et al., 2002
). Accordingly, we expressed various combinations of these proteins in the MKO strain. We used 3× HA-tagged Atg12 to allow detection of the Atg12–Atg5 conjugate. When expressed in an atg12Δ
strain, we detected both free HA-Atg12 and HA-Atg12 conjugated to Atg5 (, lane 2). As expected, when only Atg5 and HA-Atg12 were expressed in the MKO strain, we did not detect the conjugated species (, lane 4). In contrast, if Atg7 and Atg10 were also present, we detected a faint band corresponding to the HA-Atg12–Atg5 conjugate; however, the majority of the HA-Atg12 was in the free form (, lane 5). When we added Atg16, we found that a substantially greater amount of conjugate was formed (, lane 6).
Figure 4. Reconstitution of the Atg12–Atg5 conjugation system. MKO, MKO (ATG3), and atg12Δ cells transformed with various plasmids were grown in selective SMD medium, collected at mid-log phase, and subjected to Western blot analysis using an anti-HA (more ...)
Next, we examined possible contributions from the Atg8 conjugation system. When we expressed Atg4 and Atg8 together with Atg5, Atg7, Atg10, Atg12, and Atg16, the Atg12–Atg5 conjugation efficiency was further enhanced (, lane 7). Atg8–PE was not generated in this strain because of the absence of Atg3 (not depicted). Thus, it seems that even without the formation of Atg8–PE, the presence of some proteins in the Atg8–PE system can facilitate the conjugation of Atg12 to Atg5. Furthermore, the level of the Atg12–Atg5 conjugate was even greater when Atg3 was added (, lane 8). Thus, our data suggest that Atg5, Atg7, Atg10, and Atg12 are minimum requirements for Atg12–Atg5 conjugate formation, and Atg16 facilitates the conjugation, in agreement with published data. We further found, however, that components in the Atg8–PE system also enhance the conjugation reaction. The same conclusions were reached in either vegetative () or starvation (not depicted) conditions.
Atg8–PE formation and Atg4 cleavage
We extended our analysis with the MKO strain to the reconstitution of Atg8–PE conjugation. Although there have been extensive studies on the in vitro reconstitution of Atg8–PE, in vivo reconstitution has only been described in Escherichia coli
where autophagy does not occur (Ichimura et al., 2004
). The C-terminal arginine of nascent Atg8 is normally removed by Atg4, which can also release Atg8 from PE. When the C-terminal arginine is removed, as in Atg8ΔR, the initial processing step is bypassed. Previous experiments using purified Atg8ΔR, Atg3, Atg7, and PE-containing liposomes suggest that Atg8–PE formation is at its peak when the PE content reaches 70% of the total membrane, but with a concentration close to that of yeast organelle membranes, the efficiency is very low (Ichimura et al., 2004
). However, the efficiency of conjugation is boosted by the presence of the Atg12–Atg5 conjugate, and further addition of Atg16 only slightly increases Atg8–PE formation (Hanada et al., 2007
). Our data support this current view of Atg8 lipidation. Moreover, they provided additional insights into both the conjugation and deconjugation reactions.
We first tested if Atg8, Atg4, Atg7, and Atg3 are sufficient for Atg8–PE formation. When we transformed a plasmid expressing these proteins into the MKO (ATG3) strain, Atg8–PE was essentially not detected in both growing and starvation conditions (, lane 3); even when we expressed all the components for the Atg12–Atg5 conjugation system, we were still not able to detect a signal for Atg8–PE (, lane 4). These data do not agree with the in vitro reconstitution data. One of the differences between these previous studies and our experiments is that the previous analyses used the modified Atg8ΔR to bypass the need for the initial Atg4 cleavage, and omitted Atg4 from the subsequent reactions. However, the absence of Atg4 also blocks the deconjugation of Atg8–PE and thus favors the accumulation of the conjugated species. Considering the role of Atg4 in determining the balance between Atg8 and Atg8–PE, we introduced a plasmid expressing Atg8ΔR, Atg7, and Atg10, into the MKO (ATG3) strain; in this case, a significant amount of Atg8–PE was detected (, lane 5). When we additionally expressed Atg5, HA-Atg12, and Atg16, almost all of the Atg8 was in its conjugated form (, lane 6). Thus, when Atg4 is present, as is the case in vivo, Atg8–PE levels are extremely low in the MKO strain, even in the presence of the Atg12–Atg5 conjugation system. These results suggest that the in vitro system does not faithfully reflect the contribution of other factors that are required to control the activity of Atg4.
Figure 5. Reconstitution of the Atg8–PE conjugation system. (A) The role of Atg4 and the Atg12–Atg5 conjugation system in Atg8–PE formation. MKO (ATG3) cells transformed with different combinations of plasmids were grown in selective SMD (more ...)
Finally, we attempted to further define the reason for the low level of Atg8–PE in this system. In wild-type cells, a population of Atg8 is constantly degraded inside the vacuole, so the total amount of Atg8 is lower than that in the MKO strain. Therefore, we used a pep4Δ strain, which blocks Atg8 degradation, as a control (, lane 11). When deconjugation occurs, an Atg8–PE band was not detected (, lane 3), whereas expression of Atg8ΔR in an atg4Δ atg8Δ strain defective in deconjugation causes all of the protein to accumulate in the lipidated form (, lane 10). When we expressed Atg8ΔR, Atg7, and Atg10 along with Atg5, Atg12, or Atg16 separately, Atg8–PE conjugation was not increased (, lane 4–7). However, the presence of the Atg12–Atg5 conjugate facilitated Atg8–PE formation (, lane 8; and not depicted). In this in vivo system, Atg16 also facilitated the formation and/or enhanced the stability of Atg8–PE (, lane 9), which could be indirect through its action on the Atg12–Atg5 conjugate (, lane 6).