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Vol. 19, Issue 2, 668-681, February 2008

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The Atg1 Kinase Complex Is Involved in the Regulation of Protein Recruitment to Initiate Sequestering Vesicle Formation for Nonspecific Autophagy in Saccharomyces cerevisiae

Life Sciences Institute and Departments of Molecular, Cellular, and Developmental Biology and Biological Chemistry, University of Michigan, Ann Arbor, MI 48109

Submitted August 24, 2007; Revised October 30, 2007; Accepted November 21, 2007
Monitoring Editor: Suresh Subramani

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Autophagy is the major degradative process for recycling cytoplasmic constituents and eliminating unnecessary organelles in eukaryotic cells. Most autophagy-related (Atg) proteins are recruited to the phagophore assembly site (PAS), a proposed site for vesicle formation during either nonspecific or specific types of autophagy. Therefore, appropriate recruitment of Atg proteins to this site is critical for their function in autophagy. Atg11 facilitates PAS recruitment for the cytoplasm-to-vacuole targeting pathway, which is a specific, autophagy-like process that occurs under vegetative conditions. In contrast, it is not known how Atg proteins are recruited to the PAS, nor which components are involved in PAS formation under nonspecific autophagy-inducing, starvation conditions. Here, we studied PAS assembly during nonspecific autophagy, using an atg11 mutant background to eliminate the PAS formation that occurs during vegetative growth. We found that protein complexes containing the Atg1 kinase have two roles for PAS formation during nonspecific autophagy. The Atg1 C terminus mediates an interaction with Atg13 and Atg17, facilitating a structural role of Atg1 that is needed to efficiently organize an initial step of PAS assembly, whereas Atg1 kinase activity affects the dynamics of protein movement at the PAS involved in Atg protein cycling.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Macroautophagy (hereafter autophagy) is a major degradative pathway that allows cells to respond to various types of environmental stress (Klionsky, 2005; Yorimitsu and Klionsky, 2007). Autophagy is an evolutionarily conserved pathway in all eukaryotic cells (Reggiori and Klionsky, 2002). After certain environmental cues such as nutrient deprivation or hormonal stimuli, cells dynamically sequester portions of the cytoplasm within double-membrane cytosolic vesicles, called autophagosomes (Klionsky and Ohsumi, 1999; Klionsky and Emr, 2000). The completed vesicles subsequently fuse with lysosomes/vacuoles, allowing the degradation of the sequestered components and the recycling of the resulting macromolecules. Autophagy has critical roles in cellular remodeling through degradation, thus acting at various stages of development (Klionsky, 2004; Levine and Klionsky, 2004), and it is involved in protection against a range of diseases such as pathogen infection, tumor formation and certain types of neurodegeneration (Shintani and Klionsky, 2004a; Rubinsztein et al., 2005).

Autophagy is usually considered a nonselective process for the bulk degradation of cytoplasmic components; however, there are many types of specific autophagy. For example, peroxisomes in methylotrophic yeasts are specifically degraded when cells are shifted from conditions where these organelles are required for metabolism to ones where they are no longer necessary (Dunn et al., 2005). In addition to its role in adapting to changing metabolic demands, selective autophagy is involved in removing damaged organelles such as mitochondria (Kissova et al., 2007; Zhang et al., 2007). Specific types of autophagy also occur in higher eukaryotes as seen, for example, with the targeting of pathogenic microbes (Birmingham and Brumell, 2006; Colombo et al., 2006; Talloczy et al., 2006; Vergne et al., 2006; Webster, 2006; Yoshimori, 2006). A particularly unique case of selective autophagy is the cytoplasm-to-vacuole targeting (Cvt) pathway, which is a biosynthetic autophagy-like pathway that occurs in growing conditions in yeast (Yorimitsu and Klionsky, 2005b; Farre et al., 2007). The precursor form of the vacuolar hydrolase aminopeptidase I (Ape1), and -mannosidase, are recognized and sequestered within double-membrane vesicles and transported into the vacuole through this alternative targeting route that overlaps extensively with bulk autophagy (Klionsky et al., 1992; Hutchins and Klionsky, 2001). There are clear morphological and mechanistic differences between the Cvt pathway and autophagy, even though both share most of the autophagy-related (Atg) proteins, the machinery that drives these processes. One of the key proteins required for both pathways, the serine-threonine kinase Atg1, plays a critical role in autophagy induction and is regulated by Tor-dependent signaling (Kamada et al., 2000). Recently, it was reported that the Atg1-interacting partners Atg13 and Atg17 participate in regulating the conversion between the Cvt pathway and autophagy through modulating the autophagic response (Kamada et al., 2000; Cheong et al., 2005; Kabeya et al., 2005).

The phagophore assembly site (also called the preautophagosomal structure; PAS) is thought to be an organizing center for formation of the sequestering vesicles, autophagosomes and Cvt vesicles, which form during bulk and specific autophagy, respectively. Although the PAS is presumed to play a prominent role in these processes, the nature of this structure is poorly understood. For example, through elegant circular reasoning the site where most Atg proteins reside is defined as the PAS, and the PAS is defined in part as the site where most Atg proteins reside. The crux of this logic is that the PAS is primarily detected through fluorescence microscopy as a perivacuolar punctum in yeast cells expressing fluorophore-tagged Atg proteins. Even though this site is poorly defined, the correct PAS targeting of Atg proteins seems to be required for their function (Suzuki et al., 2001; Kim et al., 2002; Nice et al., 2002). Although the role of the PAS is not fully understood, one model is that this site serves to facilitate the nucleation and/or expansion of the phagophore, the precursor to the autophagosome, through the recruitment of Atg proteins (Mizushima et al., 2001; Suzuki and Ohsumi, 2007). Accordingly, to understand the initial steps of sequestering vesicle formation, it is important to understand how Atg proteins are recruited to the PAS, which proteins interact at this site, and how they function.

Recently, we reported that Cvt cargo complexes, composed of the cargo protein precursor Ape1 (prApe1), the receptor Atg19 and the adaptor Atg11, have a critical role in PAS organization under vegetative conditions (Shintani and Klionsky, 2004b). In contrast, PAS assembly in the absence of these proteins occurs normally during starvation, which suggests that Cvt cargo complexes are most likely involved in the formation of a Cvt-specific PAS, but they are not needed for PAS formation during nonspecific autophagy. These results raise questions about targeting of Atg proteins to the PAS during specific and nonspecific types of autophagy, and in particular about the mechanism of Atg protein recruitment under autophagy-inducing conditions. In this study, we examined the role of proteins that comprise the Atg1 kinase complex in PAS assembly. Our results suggest that Atg1, Atg13, and Atg17 have a critical role in PAS organization during nonspecific autophagy and that the interaction among these proteins is essential for PAS recruitment of Atg proteins.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Media
Yeast were grown in YPD medium (1% yeast extract, 2% peptone, and 2% glucose) or synthetic minimal medium (SMD; 0.67% yeast nitrogen base, 2% glucose, and auxotrophic amino acids and vitamins as required). Starvation medium was SD-N (0.17% yeast nitrogen base without ammonium sulfate or amino acids, containing 2% glucose).

Strains and Plasmids
The Saccharomyces cerevisiae strains used in this study are listed in Table 1. Tagging of proteins by the integration of modified genes at the corresponding chromosomal loci, and gene deletions were performed by a polymerase chain reaction (PCR)-based procedure (Longtine et al., 1998). For gene disruption, the entire coding region was replaced with the Escherichia coli kan^r, Schizosaccharomyces pombe HIS5, Kluyveromyces lactis URA3, Saccharomyces kluyveri HIS3, or S. cerevisiae TRP1, LEU2, or URA3 gene by using PCR primers containing 40 bases of identity to the regions flanking the open reading frame. Western blotting, PCR, or both verified putative gene knockout and tagged strains. The functionality of tagged constructs was confirmed by examining the processing of prApe1, green fluorescent protein (GFP)-Atg8, or both.

For yeast two-hybrid screening, we used an N-terminally truncated version of Atg1, lacking a kinase domain (Atg1N), that was introduced into the yeast two-hybrid bait vector pGBDU-C1 by using BamHI and SalI sites, as described previously (Yorimitsu and Klionsky, 2005a). The C-terminal deletion mutant (C20) of this version of Atg1 was generated by PCR and cloned into pGBDU-C1 by using the same restriction enzyme sites. The pGBDU-C1 plasmids expressing point mutants that substituted alanine for tyrosine and arginine at positions 878 and 885, respectively (Atg1^Y878A,R885A) and glutamate for arginine and lysine at positions 885 and 892, respectively (Atg1^R885E,K892E) were generated by PCR-based site-directed mutagenesis using the pGBD-Atg1N construct. Full-length Atg13 (pAD-Atg13), described previously (Scott et al., 2000), was used as prey. Each set of bait and prey plasmids were transformed into strain PJ69-4A, and interactions were assayed by streaking transformants on SD plates lacking adenine, and examining growth.

To generate mutant versions of native Atg1, we started with the wild-type ATG1 gene containing its endogenous promoter and terminator that was cloned into pRS315 [pATG1(315); Abeliovich et al., 2003], in which an NheI site was introduced after the stop codon. The Atg1C20, Atg1^Y878A,R885A and Atg1^R885E,K892E mutations were each cloned from the pGBDU-C1 vector containing these respective mutants, into pATG1(315) as SphI–NheI fragments. The mutations were verified by DNA sequencing. These constructs were used for the Pho860 alkaline phosphatase assay and the fluorescence and electron microscopy analyses.

For protein A (PA) affinity isolation, the Atg1 open reading frame with a BamHI site engineered just after the ATG start codon, was amplified by PCR and cloned as a SpeI–SalI fragment into the pCu416 vector (Labbe and Thiele, 1999). A PA tag on a BamHI fragment from plasmid pTS480 was used to create an N-terminal PA-tagged version of this construct, and the functionality was determined by monitoring prApe1 maturation in an atg1 strain expressing this protein. DNA fragments containing the Atg1C20 deletion or point mutations were excised as SphI–SalI fragments from the pGBDU-C1 vector, and they were used to replace the wild-type Atg1 sequence in the PA-tagged construct after digestion with the same set of enzymes.

Full-length ATG13, containing its endogenous promoter and terminator sequences, was cloned into pRS315 (Kamada et al., 2000). An AvrII site was introduced by site-directed mutagenesis, immediately after the start codon of ATG13. DNA containing three tandem copies of the hemagglutinin (HA) coding sequence was amplified from 3HA-Apg12 (a kind gift from Dr. Noboru Mizushima, Tokyo Medical and Dental University) by primers containing an XbaI site and ligated into the AvrII site to create an N-terminal fusion of 3xHA with Atg13. Western blotting and PCR were used for verifying this construct, and the functionality was assayed by examining prApe1 maturation. HA-tagged Atg11 has been described previously (Yorimitsu and Klionsky, 2005a).

Protein A Affinity Isolation
Cells were grown in 50 ml of SMD lacking the appropriate auxotrophic amino acids to OD₆₀₀ = 0.6. Rapamycin (0.2 µg/ml; Fermentek, Jerusalem, Israel) was added, and cells were cultured for an additional 2 h. PA-affinity isolation was performed as described previously (He et al., 2006), with only one exception: the cell extracts were incubated with immunoglobulin (Ig)G-Sepharose beads for 2 h at 4°C. The eluates were resolved by 6% SDS-polyacrylamide gel electrophoresis (PAGE), transferred to Immobilon polyvinylidene difluoride (IPVH00010; Millipore, Billerica, MA) and immunoblotted with monoclonal anti-HA antibody (Santa Cruz Biotechnology, CA).

Enzyme Assays
The alkaline phosphatase assay using Pho860, and an in vitro phosphorylation assay using HA-tagged Atg1 were performed as described previously (Kamada et al., 1995; Noda et al., 1995). β-Galactosidase assays with AD-Atg13 and BD-Atg1 variants in strain PJ69-4A were performed as described previously (Rose et al., 1990).

Microscopy
For fluorescence microscopy, cells were grown to mid-log phase in YPD or SMD medium lacking the appropriate auxotrophic amino acids to maintain plasmids. For starvation, cells were shifted to SD-N medium for 2 h. The cells were viewed using a DeltaVision Spectris (Applied Precision, Issaquah, WA) microscope fitted with differential interference contrast (DIC) optics and an Olympus camera IX-HLSH100 with softWoRx software. For image quantification of fluorescence intensity, a stack of 12 z-sections at intervals of 0.5 µm was collected, which completely covered the whole cell along the z-axis. The stack of 12 z-sections was then projected into a two-dimensional image by softWoRx software. A circle was drawn around the PAS dot and the intensity within this area was measured. An area of the same size encompassing the adjacent cytosol was subtracted as background. For the time-lapse experiment, cells were immobilized on SD-N medium containing 2% agar on concavity slides (Fisher Scientific) and pictures were taken every minute.

Electron microscopy was performed as described previously (Kaiser and Schekman, 1990). To quantify size, the diameter of autophagic bodies with clear membrane boundaries was measured.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Atg17 Is Important for PAS Organization under Starvation Conditions
Atg17 and Atg11 were identified as interacting proteins of Atg1, which are required for either nonspecific autophagy or the specific Cvt pathway, respectively (Kamada et al., 2000; Kim et al., 2001b). Atg11 plays a role in cargo packaging and transport in the Cvt pathway, but it is not required for nonspecific autophagy (Shintani et al., 2002; Yorimitsu and Klionsky, 2005a). Conversely, Atg17 is not needed for the Cvt pathway, but it modulates the magnitude of the autophagic response; the atg17 mutant has fewer, and smaller, autophagosomes (Cheong et al., 2005; Kabeya et al., 2005). Except for this role, however, the physiological function of Atg17 in autophagy is not understood.

Recently, we reported that the Cvt-specific proteins Atg11 and Atg19 (the prApe1 receptor), along with the prApe1 cargo protein, recruit Atg proteins to the PAS to facilitate Cvt vesicle formation (Shintani and Klionsky, 2004). In contrast, these proteins are not needed for PAS formation during starvation conditions, which suggests that other proteins are involved in PAS formation during nonspecific autophagy. Accordingly, we hypothesized that one of the autophagy-specific Atg proteins has a similar role to Atg11 in PAS formation under starvation conditions. Atg17 is one promising candidate because of the unique autophagy-specific phenotype of the null mutant. In addition, some Atg proteins do not display a normal PAS localization in the absence of Atg17 under starvation conditions; for example, GFP-Atg13 does not localize at the PAS in the atg17 strain (our unpublished data; Suzuki et al., 2007). Accordingly, we decided to examine the role of Atg17 in PAS formation during nonspecific autophagy.

We first used GFP-tagged Atg1 and Atg8 as independent PAS markers and examined PAS formation under rich and starvation conditions. Cells expressing GFP-Atg8 or GFP-Atg1 were grown to mid-log phase and imaged by fluorescence microscopy. In wild-type cells, both Atg proteins usually displayed a prominent punctate signal and also a more faint cytosolic diffuse pattern (Figure 1). The punctate structure was located in a perivacuolar region and represents the phagophore assembly site (Suzuki et al., 2001; Kim et al., 2002). Approximately 30–40% of the cells contained a GFP-Atg8 or GFP-Atg1 punctate signal in vegetative (SMD medium) conditions. Under starvation conditions (SD-N medium), we could usually detect more than one punctum for GFP-Atg1, and many of the GFP-Atg8 cells displayed some level of vacuolar fluorescence, reflecting autophagic delivery of this chimera that remains associated with the completed autophagosome (Kim et al., 2001a). More than 50% of the cells still had clear punctate signals corresponding to the PAS in starvation conditions (Figure 1).

View larger version (41K): [in this window] [in a new window]   Figure 1. Atg17 is important for PAS organization under starvation conditions. Wild-type (SEY6210), atg11 (AHY001), atg17 (CWY239), and atg11 atg17 (HCY66) strains expressing GFP-Atg8 or GFP-Atg1 from centromeric plasmids were grown in SMD lacking auxotrophic amino acids and shifted to SD-N for 2 h before microscopy. The cells were examined by fluorescence microscopy as described in Materials and Methods.

Next, to test whether the defect in PAS recruitment of Atg1 and Atg8, and potentially other Atg proteins, in the atg11 atg17 strain affects autophagosome formation, we carried out a morphological examination by electron microscopy. One of the unique features of Cvt vesicles and autophagosomes is the presence of double membranes; after fusion of the autophagosome outer membrane with the vacuole, single-membrane autophagic bodies are released into the vacuole lumen (Takeshige et al., 1992). The autophagic bodies are subsequently degraded in a Pep4-dependent manner; pep4 mutants accumulate autophagic bodies, which can be observed by electron microscopy. Additionally, we deleted the VPS4 gene to eliminate the vesicles derived from the multivesicular body pathway (Reggiori et al., 2004). Cells in the pep4 vps4 background were grown in YPD, shifted to SD-N for 4 h to induce autophagy and observed by electron microscopy, as described in Materials and Methods.

The wild-type (pep4 vps4) vacuole was filled with a large number of autophagic bodies that accumulated after starvation (Figure 2). In contrast, the atg17 strain accumulated a reduced number and size of autophagic bodies in agreement with our previous results (Cheong et al., 2005). We then focused on two strains, atg11 and atg11 atg17, which showed vegetative-specific or complete (i.e., including starvation conditions) defects in PAS formation (Figure 1). Under starvation conditions, the atg11 vacuole accumulated a comparable number and size of autophagic bodies as seen in the wild type. In contrast, the atg11 atg17 strain essentially did not contain any detectable autophagic bodies, having at most <5% the number seen in the atg11 mutant (Figure 2). This result indicates that PAS formation in autophagy conditions is closely linked to normal autophagosome formation. Furthermore, Atg17 plays an important role in the PAS localization of Atg proteins, which presumably facilitates assembly of the autophagosome.

View larger version (28K): [in this window] [in a new window]   Figure 2. The atg11 atg17 double mutant is defective for autophagosome formation. (A) The wild-type (FRY143; pep4 vps4), atg17 (HCY31; pep4 vps4), atg11 (HCY37; pep4 vps4), and atg11 atg17 (HCY38; pep4 vps4) strains were grown to mid-log stage in YPD and transferred to SD-N medium for 4 h. Cells were fixed with permanganate and examined by electron microscopy as described in Materials and Methods. (B) Quantification of autophagic body accumulation. 50 sections for each strain were scored for autophagic body accumulation.

Similar to the results with the atg11 atg17 strain, the PAS localization of Atg8 or Atg1 was defective in the atg11 atg13 double mutant under starvation conditions, whereas PAS localization of these proteins was essentially normal in the atg13 single-deletion strain, presumably due to the presence of the Atg11-dependent PAS formed during vegetative growth (Figures 3 and 4, A and B). Similarly, the atg1 atg11 mutant showed a diffuse fluorescent signal of GFP-Atg8 in the cytosol (Figures 3A and 4A); we could not use the GFP-Atg1 marker in this strain, because the hybrid protein expressed from the plasmid complemented the chromosomal deletion of ATG1 (our unpublished data). Therefore, we examined Atg17-GFP as another PAS marker in the atg1 atg11 double mutant strain. In both vegetative and starvation conditions, the Atg17-GFP signal was diffuse in the cytosol (Figure 3C). Overall, these results suggest that Atg1 and its modulators, Atg13 and Atg17, have essential roles in PAS formation during nonspecific autophagy.

View larger version (43K): [in this window] [in a new window]   Figure 3. Atg13 and Atg1 are required for PAS recruitment of Atg proteins under starvation conditions. Localization of GFP-Atg8 (A) or GFP-Atg1 (B) in atg11 (AHY001), atg13 (CWY233), atg1 atg11 (TYY158), and atg11 atg13 (CWY235) strains, and GFP-Atg17 (C) in the atg1 atg11 (TYY158) strain was examined by fluorescence microscopy as described in Figure 1.

View larger version (21K): [in this window] [in a new window]   Figure 4. Atg1 and its modulators Atg13 and Atg17 are required for the PAS recruitment of various Atg proteins under starvation conditions. The localization of Atg proteins was examined by fluorescence microscopy as described in Figure 1. The number of cells that contained PAS puncta was quantified for GFP-Atg8 (A), GFP-Atg1 (B), Atg14-GFP (C), and Atg16-GFP (D), after shifting to SD-N for 2 h. Approximately 150–250 cells for each strain were analyzed for scoring the percentage of cells with fluorescent PAS puncta. ND, not determined.

Atg1 Kinase Activity Is Not Necessary for PAS Recruitment of Atg Proteins but Is Needed for Their Normal Localization
Atg13 and Atg17 modulate Atg1 kinase activity (Kamada et al., 2000), but the role of Atg1 kinase activity is not clear in either autophagy or the Cvt pathway (Nair and Klionsky, 2005). Conflicting reports suggest that higher kinase activity plays a more important role in autophagy (Kamada et al., 2000) or the Cvt pathway (Abeliovich et al., 2003; Nair and Klionsky, 2005). To examine the potential role of Atg1 kinase activity in PAS formation, we decided to use a mutant form of Atg1 with reduced kinase activity, which combined two previously characterized mutant alleles. The Atg1^K54A mutant has an alteration in the kinase active site, but still displays a low level of function based on the Pho860 assay (Kamada et al., 2000; Figure 5A). When combined with a mutation of methionine to alanine at position 102, the Atg1^K54A,M102A double mutant displays a level of autophagic activity similar to an atg1 strain (Figure 5A). To confirm that the loss of autophagic activity corresponded with a reduction in kinase activity we used an in vitro kinase assay. Cells expressing HA-tagged Atg1, Atg1^K54A, and Atg1^K54A,M102A were treated with 0.2 µg/ml rapamycin for 1.5 h. A protein extract from each mutant was subjected to immunoprecipitation with anti-HA antibody under native conditions, and then the immunocomplex was analyzed for in vitro kinase activity by using myelin basic protein as a substrate (Kamada et al., 1995). Compared with the wild-type Atg1 control, the Atg1^K54A and Atg1^K54A,M102A mutants showed nearly a complete loss of in vitro kinase activity (Figure 5B). The Pho860, but not the kinase assay, revealed a difference in the activity of the two Atg1 mutants; we suspect this reflects the nature of the assays, as the Pho860 assay is more quantitative.

View larger version (17K): [in this window] [in a new window]   Figure 5. Atg1 kinase activity is required for autophagy. (A) Atg1 mutants are defective for autophagy. The alkaline phosphatase activity from Pho860, a marker for nonspecific autophagy, was monitored to determine the level of autophagy activity. The wild-type (YTS159) and atg1 (UNY1) strains harboring plasmids expressing Atg1, Atg1K54A (Atg1K), Atg1K54A,M102A (Atg1KM), or empty vector (pRS415) were grown in SMD lacking auxotrophic amino acids and shifted to SD-N for 4 h. Samples were collected and protein extracts assayed for alkaline phosphatase activity as described in Materials and Methods. The results represent the mean and SD of three experiments. (B) Atg1 mutants are defective for kinase activity. The atg1 mutant (WHY001) cells expressing HA-Atg1, HA-Atg1K54A, or HA-Atg1K54A,M102A were treated with 0.2 µg/ml rapamycin for 2 h. The extract of each mutant was immunoprecipitated with anti-HA antibody, and then immunocomplexes were assayed for Atg1 kinase activity as described in Materials and Methods.

View larger version (18K): [in this window] [in a new window]   Figure 6. Atg1 kinase activity is required for autophagosome formation. (A) Atg1 mutants are defective for autophagic body accumulation. The atg1 (HCY76; pep4 vps4) strains harboring plasmids expressing Atg1, Atg1K54A (Atg1K), Atg1K54A,M102A (Atg1KM), or the empty vector (pRS415) were grown in selective medium and shifted to SD-N for 4 h. Cells were fixed with permanganate and examined by electron microscopy as described in Materials and Methods. (B) Quantification of autophagic body accumulation. Fifty sections for each strain were scored for autophagic body accumulation in the vacuole.

View larger version (31K): [in this window] [in a new window]   Figure 7. Atg1 kinase activity is not essential for PAS localization of Atg8 and Atg17, but it is required for normal localization of these proteins under starvation conditions. (A) Cells from the atg1 atg11 strain expressing GFP-Atg8 (HCY116) and Atg17-3xGFP (HCY119) from the respective chromosomal loci were transformed with plasmids encoding Atg1, empty vector, or Atg1K54A,M102A. The cells were grown in SMD and shifted to SD-N for 2 h before fluorescence microscopy, which was performed as described in Materials and Methods. (B) For quantification of fluorescence intensity at the PAS, 30 individual cells for each strain in A were observed by fluorescence microscopy. The relative percentage of fluorescence intensity was calculated as described in Materials and Methods and normalized to the value for wild-type Atg1 cells, which were set at 100%. Error bars indicate the SD of three independent experiments. (C) Redistribution of Atg8 from the PAS is affected by Atg1 kinase activity under starvation conditions. The atg1 atg11 strain expressing GFP-Atg8 from the chromosomal locus (HCY116) was cotransformed with plasmids encoding ATG1ts and Atg1K54A,M102A or ATG1ts and empty vector. The cells were grown in SMD medium at 24°C and transferred to SD-N for 1 h before the temperature shift. For inactivation of Atg1ts, cells were shifted to 37°C for 2 h, and then shifted back to 24°C for 2 h. The cells were examined by fluorescence microscopy as described in Materials and Methods. (D) For quantification of fluorescence intensity at the PAS, 30 individual cells from each strain in C were observed by fluorescence microscopy. The relative percentage of fluorescence intensity was calculated and normalized based on the value for GFP-Atg8 in cells expressing Atg1ts and the empty vector, which was set at 100% at the permissive temperature. Error bars indicate the SD of three independent experiments. (E) Quantification of the number of cells that contained GFP-Atg8 puncta as a PAS marker from C. Approximately 150–200 cells for each strain were analyzed for scoring the percentage of cells containing fluorescent dots.

To demonstrate that the accumulation of Atg17 or Atg8 at the PAS in the presence of Atg1 mutants is not due to a chronic defect associated with the Atg1 kinase mutation itself, we took advantage of a temperature-sensitive allele of Atg1 (Atg1^ts) (Suzuki et al., 2001) to generate the equivalent of a conditional Atg1 kinase mutation. We cotransformed plasmids expressing Atg1^ts and Atg1^K54A,M102A into the atg1 atg11 strain that expressed either GFP-Atg8 or Atg17-GFP integrated at their respective chromosomal loci. As a control, we used an empty vector instead of the plasmid expressing Atg1^K54A,M102A. First, we analyzed GFP-Atg8 processing to monitor the rate of Atg1^ts inactivation under starvation conditions. GFP-Atg8 is delivered to the vacuole inside the autophagic body and after lysis in the vacuole lumen the Atg8 portion of the chimera is degraded, whereas GFP is relatively stable; the appearance of free GFP can then be used to monitor autophagy (Cheong et al., 2005). Cells expressing only Atg1^ts in the atg1 atg11 background were grown in SMD at 24°C and transferred to SD-N for 1 h before the temperature shift. The accumulation of free GFP resulting from autophagy-dependent cleavage of GFP-Atg8 was significantly reduced within 2 h, which suggested inactivation of Atg1 occurred within this time frame after the temperature shift to 37°C (our unpublished data). Next, we used this conditional Atg1^ts background to examine how the Atg1^K54A,M102A kinase mutant affected the dynamics of GFP-Atg8 association/dissociation with the PAS.

In vegetative conditions, GFP-Atg8 showed a diffuse cytosolic signal in the atg1 atg11 double mutant at either 24°C or 37°C due to the absence of Atg11 (our unpublished data). Under starvation conditions GFP-Atg8 displayed a clear signal at the PAS in addition to an obvious cytosolic diffuse pattern in strains coexpressing Atg1^ts and Atg1^K54A,M102A, or Atg1^ts and empty vector at 24°C (Figure 7C). This pattern was essentially the same as that of the wild-type strain, but distinct from the Atg1^K54A,M102A mutant with regard to the level of cytosolic staining (Figure 7A), suggesting that the Atg1^ts protein at the permissive temperature complemented the atg1 mutation and was dominant to the Atg1^K54A,M102A phenotype. After a shift to the nonpermissive temperature of 37°C, GFP-Atg8 was restricted to the PAS without cytosolic staining within 2 h, only in the presence of Atg1^K54A,M102A. In contrast, the strain expressing Atg1^ts alone, displayed an increase in the diffuse cytosolic signal and a decrease in the PAS puncta when Atg1 was inactivated. Therefore, the Atg1^K54A,M102A phenotype that resulted rapidly after the Atg1^ts mutant was inactivated was the accumulation of a strong GFP-Atg8 signal at the PAS. When the cells were shifted back to the permissive temperature, GFP-Atg8 in both strains regained the dual PAS and cytosolic localization originally seen, indicating that the accumulation at the PAS was not a terminal phenotype (Figure 7C).

Quantification of the data indicated that the relative fluorescence intensity of GFP-Atg8 at the PAS was 2.5 times higher at the nonpermissive relative to the permissive temperature for the Atg1^K54A,M102A strain (Figure 7D). Similarly, Atg17-GFP also showed a stronger PAS intensity in the strain expressing Atg1^K54A,M102A at the nonpermissive temperature, increasing 1.5-fold (our unpublished results). In addition we counted the number of cells containing a GFP-Atg8 PAS dot in these conditions to examine whether Atg1 kinase activity affected the total number of PAS that were assembled (Figure 7E). The number of GFP-Atg8 puncta in the strain expressing Atg1^K54A,M102A was comparable with that of the wild-type Atg1 strain at both permissive and nonpermissive conditions. In contrast, the atg1 atg11 mutant expressing Atg1^ts alone showed a clear decrease in the number of PAS dots at the nonpermissive temperature, whereas the intensity of GFP-Atg8 at the few detectable PAS was unaffected (the latter presumably correspond to PAS sites that have not yet disassembled). These results support the idea that autophagy-specific PAS recruitment is a dynamic process depending on the Atg1 protein and that there may be two separable Atg1-dependent functions; recruitment of Atg proteins needed for assembly of the PAS (and ultimately for autophagosome formation), and subsequent dissociation from this site, steps that are independent of and dependent on Atg1 kinase activity, respectively. Although Atg1 kinase activity is not needed for PAS recruitment of the Atg proteins that we analyzed, in the absence of normal kinase activity the Atg proteins are not able to undergo normal dissociation from this site, which presumably reflects a defect in function that translates into a reduced ability to form autophagosomes.

During this quantitative analysis, we noticed that the GFP-Atg8 puncta displayed a variable intensity, increasing and decreasing over time. To examine this dynamic effect carefully, we performed time-lapse fluorescence microscopy as described in Materials and Methods. As shown in Figure 7A, GFP-Atg8 in the atg1 atg11 strain expressing Atg1 displayed normal recruitment at the PAS upon the shift to starvation conditions. The GFP-Atg8 fluorescence reached a peak at 4 min, followed by a loss of signal after 7 min (Figure 8). In contrast, the same strain harboring the plasmid expressing Atg1^K54A,M102A showed a strong punctate signal of GFP-Atg8 without any substantial change in the fluorescence pattern at the PAS over the 7-min time course (Figure 8). These results support the hypothesis that Atg1 kinase activity affects the dynamics of Atg8 retention at, or dissociation from, the PAS.

View larger version (13K): [in this window] [in a new window]   Figure 8. Atg1 kinase activity is important for Atg8 dynamics during starvation. Cells from the atg1 atg11 strain expressing GFP-Atg8 (HCY116) were transformed with plasmids encoding Atg1 and Atg1K54A,M102A (Atg1KM). The cells were grown in SMD medium and shifted to SD-N for 2 h before fluorescence microscopy. For time-lapse experiments, the cells were immobilized on SD-N medium containing 2% agar on concavity slides as described in Materials and Methods, and pictures were taken every minute. Images are shown over the 7-min time course.

View larger version (36K): [in this window] [in a new window]   Figure 9. The interaction between Atg1 and Atg13 is important for autophagy. (A) Mutations in the C terminus of Atg1 disrupt its interaction with Atg13 based on a yeast two-hybrid assay. PJ69-4A cells expressing Atg13 (AD-Atg13) and either Atg1 lacking the N-terminal kinase domain (BD-Atg1N), Atg1N deleted for the C-terminal 20 amino acids (BD-Atg1C20), Atg1N with substitutions of R885E and K892E (BD-Atg1R885E,K892E), Atg1N with substitutions of Y878A and R885A (BD-Atg1Y878A,R885A), or the BD-empty vector were grown for 5 d on plates lacking adenine. Growth or lack of growth on these plates was scored as + or –, respectively. The strength of the interaction for each set of proteins was quantified by measuring β-galactosidase activity from three independent experiments. (B and C) Atg1 C-terminal mutants are defective for interacting with Atg13 but not Atg11. The B, atg1 atg13 (UNY27) or C, atg1 atg11 (YTS157) cells were transformed with a plasmid expressing 3xHA-Atg13 under the control of its endogenous promoter or 3xHA-Atg11 under the control of the CUP1 promoter as indicated, and CUP1 promoter-driven protein A (PA)-tagged fusions of either wild-type Atg1 (WT), Atg1C20 (C20), Atg1R885E,K892E (RK), or Atg1Y878A,R885A (YR). As negative controls UNY27 and YTS157 cells were transformed with a plasmid expressing CUP1 promoter-driven wild-type PA-Atg1 and an empty pRS315 or pRS414 plasmid, respectively. Protein extracts were subjected to affinity isolation, eluted proteins were separated on a 6% SDS-PAGE gel and detected with anti-HA antibody as described in Materials and Methods. IP, immunoaffinity-purified isolate. (D) The Atg1 C-terminal mutants are defective in autophagy. The Pho860 assay was used to monitor autophagy activity for the atg1 (UNY3) strain expressing the indicated wild-type Atg1, empty vector (pRS315), or Atg1 mutant plasmids expressing Atg1 under the control of the endogenous promoter, as described in Materials and Methods. Alkaline phosphatase activity was monitored from protein extracts prepared from cells grown in SMD or after a 4 h shift to SD-N. The results represent the mean and SD of three independent experiments. (E) The Atg1C20 mutant is defective in forming autophagosomes (detected as autophagic bodies), whereas small autophagosomes are formed in cells expressing C-terminal point mutants. An atg1 pep4 vps4 strain (HCY76) was transformed individually with empty vector, or plasmids expressing wild-type Atg1 or the indicated Atg1 mutant. Cells were grown to mid-log phase in SMD medium, shifted to SD-N medium to induce starvation, fixed with potassium permanganate, and examined by electron microscopy as described in Materials and Methods. No autophagic bodies were detected in cells transformed with the empty vector. (F) Quantification of the diameters of the autophagic bodies was carried out from 50 sections for each strain. Essentially no autophagic bodies were detected in the strain expressing Atg1C20.

Cells that contain protein-A tagged wild-type Atg1 or mutant Atg1 derivatives, and Atg13 tagged with the HA epitope, were treated with rapamycin to induce autophagy and examined by affinity isolation as described in Materials and Methods. The wild-type and mutant protein A-tagged Atg1 were isolated with IgG-Sepharose and the presence of HA-Atg13 was analyzed by western blot. HA-Atg13 was coisolated with wild-type Atg1 (Figure 9B). By comparison, the amount of HA-Atg13 coisolated with the Atg1 mutants was substantially reduced; the point mutants showed essentially an equivalent loss of interaction with Atg13 as seen with the C20 truncation. As a control, we examined a strain expressing PA-Atg1 without HA-Atg13 and were not able to detect any coisolating bands. These results indicate that the mutations at the C terminus of Atg1 caused a substantial reduction in the ability of this protein to interact with Atg13. We also examined the ability of the different Atg1 proteins to interact with Atg11. Under starvation conditions, wild-type Atg1 and all of the Atg1 C-terminal mutants interacted strongly with Atg11 (Figure 9C). Thus, the extreme C terminus of Atg1 is important for forming a complex with Atg13, but apparently not with Atg11. Recently, it was reported that the interaction between Atg17 and Atg1 is mediated through Atg13 (Cheong et al., 2005; Kabeya et al., 2005); thus, it is possible that these Atg1 mutants might also show altered interactions with Atg17.

The function of Atg1 is required for both autophagy and the Cvt pathway. In contrast, the function of Atg13 seems to be more significant for autophagy rather than the Cvt pathway; an atg13 mutant exhibits a partial maturation of prApe1, but it is completely blocked in autophagy (Abeliovich et al., 2003; Kamada et al., 2000). To determine the physiological function of disrupting the Atg1–Atg13 interaction, we tested whether the Atg1 mutants were capable of prApe1 maturation via the Cvt pathway and/or autophagic activity. Under vegetative conditions, prApe1 maturation was completely blocked in the Atg1C20 mutant, indicating a defect in the Cvt pathway (our unpublished data). Conversely, an atg1 strain harboring each of the point mutants, Atg1^Y878A,R885A and Atg1^R885E,K892E, exhibited wild-type prApe1 maturation patterns (our unpublished data). Thus, in contrast to an atg13 mutant, which only partially affects the Cvt pathway, the Atg1C20 mutant shows a complete block in the Cvt pathway. Because the Atg1C20 mutant shows a greater Cvt defect compared with an atg13 mutant, we conclude that the last 20 amino acids are not merely important for interacting with Atg13 but also are required for other Atg1-specific functions.

We quantitatively monitored bulk autophagy by measuring the activity of a nonspecific autophagy marker, Pho860, which encodes an altered form of alkaline phosphatase; Pho860 remains in the cytosol and can only be delivered to the vacuole via autophagy (Noda et al., 1995; Klionsky et al., 2007). In the vacuole, Pho860 is processed to its mature, active form. Thus, measuring Pho860 activity under starvation conditions provides a quantitative analysis of autophagy. The alkaline phosphatase activity was measured in atg1 cells transformed with a plasmid expressing wild-type Atg1, an empty vector, Atg1C20, Atg1^Y878A,R885A, or Atg1^R885E,K892E, in vegetative and starvation conditions (Figure 9D). The atg1 cells transformed with an empty vector were defective in bulk autophagy, and showed only a basal level of Pho860 activity after 4 h starvation. In contrast, the Pho860 activity in atg1 cells expressing wild-type Atg1 increased after starvation, indicating that the wild-type Atg1 protein complemented the autophagy defect in atg1 cells. The Atg1C20 mutant was completely blocked for bulk autophagy, displaying a level of activity after starvation that was essentially the same as cells transformed with an empty vector. The atg1 cells expressing the Atg1^R885E,K892E and Atg1^Y878A,R885A mutations exhibited 35 and 30%, respectively, of the Pho860 activity seen with wild-type cells, indicating a substantial defect in bulk autophagy. Together, these results show that the C terminus of Atg1 is essential for both the Cvt and autophagy pathways. Furthermore, the reduced autophagic capacity in strains harboring each set of point mutants indicates that the interaction between Atg1 and Atg13 that is mediated through the Atg1 C terminus is important for efficient nonspecific autophagy.

We decided to complete our analysis of the Atg1 mutants by examining their effect on autophagosome formation. Deletion of Atg17 results in cells with fewer and smaller autophagosomes (Cheong et al., 2005). Therefore, we examined the morphology of autophagosomes produced in cells expressing either the Atg1C20, Atg1^Y878A,R885A or Atg1^R885E,K892E mutants by electron microscopy. We transformed atg1 pep4 vps4 mutant cells with plasmids expressing wild-type or mutant Atg1. The transformants were grown in selective media, shifted to SD-N for 4 h to induce autophagy, fixed with potassium permanganate, and embedded in Spurr's resin as described in Materials and Methods. Similar to the results shown in Figure 6, after 4-h starvation, cells expressing wild-type Atg1 showed an accumulation of 10–14 autophagic bodies within the vacuole (Figure 9E). Consistent with the basal level of pho860 activity seen in cells expressing Atg1C20, most cells harboring this mutant version of Atg1 exhibited an empty vacuole, whereas a few cells exhibited vacuoles with a very small number of autophagic bodies (Figure 9E). Also in agreement with the reduced bulk autophagy seen in cells expressing the Atg1 point mutants, electron microscopy analysis revealed that the Atg1^Y878A,R885A or Atg1^R885E,K892E mutants produced smaller autophagic bodies (i.e., having a reduced diameter), with presumably reduced autophagic capacity compared with those in the wild-type strain (Figure 9, E and F). However, the total number of autophagic bodies produced in cells expressing these Atg1 point mutants (12.87 ± 4.83 for Atg1^Y878A,R885A and 11.23 ± 4.04 for Atg1^R885E,K892E) was not significantly different from the wild type (12.28 ± 3.63). Together, these data suggest that the interaction between Atg1 and Atg13 regulates the size of the autophagosomes (and the resulting autophagic bodies), and thereby the optimal magnitude of the autophagic response.

The Interaction of Atg1 with Atg13-Atg17 Is Needed for Starvation-induced PAS Organization
Finally, we used the various Atg1 C-terminal mutants to examine the effect of the loss of interaction between Atg1 and Atg13-Atg17 on PAS formation. GFP-Atg8 and Atg17-GFP were monitored in the atg1 atg11 strain expressing Atg1, Atg1C20, Atg1^Y878A,R885A, or Atg1^R885E,K892E. Under starvation conditions, wild-type Atg1 complemented the PAS localization defect of GFP-Atg8 and Atg17-GFP in this strain, and once again we detected puncta at the PAS and weak cytosolic signals (Figure 10, A and B). GFP-Atg8 also displayed some level of vacuolar fluorescence under these conditions. In contrast, the Atg1C20 or Atg1^Y878A,R885A mutants showed only a diffuse cytosolic fluorescent signal for GFP-Atg8 and Atg17-GFP, with no evidence for PAS recruitment (Figure 10, A and B). The Atg1^R885E,K892E mutant in this strain occasionally showed weak puncta for both chimeras but the predominant pattern was a diffuse cytosolic signal. To quantify the results for PAS recruitment of GFP-Atg8 and Atg17-GFP, we counted the number of cells containing a PAS dot in the strains that harbored the various Atg1 mutants (Figure 10C). The atg1 atg11 strain expressing Atg1C20, Atg1^Y878AR885A or Atg1^R885E,K892E showed a significant reduction in the number of GFP-Atg8 and Atg17-GFP dots at the PAS compared with the same strains expressing wild-type Atg1. These results support the hypothesis that the interaction among Atg1, Atg13, and Atg17 plays an important role in PAS assembly/organization for nonspecific autophagy, a function that is distinct from Atg1 kinase activity.

View larger version (38K): [in this window] [in a new window]   Figure 10. The interaction of Atg1 with Atg13 is required for PAS formation under starvation conditions. Cells from the atg1 atg11 strain expressing GFP-Atg8 (HCY116) (A) or Atg17-GFP (HCY119) (B) from the chromosomal locus were transformed with plasmids encoding wild-type Atg1, or the Atg1C20 (C20), Atg1R885E,K892E (RK), or Atg1 Y878A,R885A (YR) mutants. The cells were grown in SMD and shifted to SD-N for 2 h before fluorescence microscopy, which was carried out as described in Materials and Methods. (C) Quantification of the number of cells that contained GFP-Atg8 or Atg17-GFP puncta as a PAS marker from A and B. Approximately 150–200 cells for each strain were analyzed for scoring the percentage of cells with fluorescent dots.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Atg17 is characterized as a protein that is only needed for nonspecific autophagy, and it thus seemed like a reasonable candidate for playing an equivalent role to Atg11 under starvation conditions. We examined the role of Atg17 in strains deleted for ATG11 to eliminate the PAS that would otherwise form under vegetative conditions. The atg11 atg17 double mutant displayed a strong block in PAS formation in starvation conditions based on the localization of GFP-Atg8 and GFP-Atg1 (Figure 1), supporting this hypothesis. Similarly, the double mutant showed a much greater defect in autophagosome/autophagic body formation compared with the atg17 mutant (Figure 2). Thus, Atg11 is sufficient in the absence of Atg17 for dictating PAS assembly in vegetative conditions, whereas Atg17 is necessary for allowing assembly in starvation conditions; the absence of both proteins prevents PAS recruitment of GFP-Atg8, GFP-Atg1, and other Atg marker proteins including Atg14 and Atg16 under either condition.

During the course of our analysis, a report was published suggesting that Atg11 and Atg17 are the two initial factors that establish the assembly sequence for Atg proteins at the PAS (Suzuki et al., 2007). Atg17 is proposed to act as a scaffold for recruiting other Atg proteins, similar to the function we proposed for Atg11 (Yorimitsu and Klionsky, 2005a; He et al., 2006). However, in our present analysis, we found that PAS assembly under starvation conditions requires more than Atg17; cells deleted for ATG11 and either ATG1 or ATG13 displayed essentially the same phenotype as the atg11 atg17 double mutant (Figure 3). The analysis by Suzuki et al., 2007) relied primarily on an examination of strains disrupted for individual ATG genes in the presence of rapamycin, whereas the present analysis includes double deletion strains, and autophagy was induced by starvation. Both studies note the role of Atg17 as a scaffold that functions during autophagy-inducing conditions in a similar manner to that of Atg11 during vegetative growth. However, single deletions of ATG1, ATG13, or ATG17 did not result in defective PAS localization phenotypes, especially for GFP-Atg1 or GFP-Atg8 (Figure 4). Thus, the role of Atg1 and Atg13 was not examined in the previous analysis, which only noted the effect of the atg11 atg17 double mutant (Suzuki et al., 2007).

Atg1, Atg13, and Atg17 associate with each other regardless of Atg1 kinase activity, and the affinity of these interactions seems to increase upon starvation (Kamada et al., 2000; Kabeya et al., 2005). Accordingly we examined whether the interaction among Atg1, Atg13, and Atg17 affects PAS formation under starvation conditions. We mapped an interaction site for Atg13 in the Atg1 C terminus (Figure 9). Deletion of the C-terminal 20 amino acids, or point mutations within this region strongly, but not completely, abolished interaction with Atg13; thus, there may be additional sites in Atg1 that mediate this interaction. Similar to the results with the atg1 and atg13 strains, we found that Atg1