The pre-autophagosomal structure organized by concerted functions of APG genes is essential for autophagosome formation
2001; Springer Nature; Volume: 20; Issue: 21 Linguagem: Inglês
10.1093/emboj/20.21.5971
ISSN1460-2075
Autores Tópico(s)Endoplasmic Reticulum Stress and Disease
ResumoArticle1 November 2001free access The pre-autophagosomal structure organized by concerted functions of APG genes is essential for autophagosome formation Kuninori Suzuki Kuninori Suzuki Department of Cell Biology, National Institute for Basic Biology, Nishigonaka 38, Myodaiji-cho, Okazaki, 444-8585 Japan Department of Molecular Biomechanics, School of Life Science, The Graduate University for Advanced Studies, Japan Search for more papers by this author Takayoshi Kirisako Takayoshi Kirisako Department of Cell Biology, National Institute for Basic Biology, Nishigonaka 38, Myodaiji-cho, Okazaki, 444-8585 Japan Department of Molecular Biomechanics, School of Life Science, The Graduate University for Advanced Studies, Japan Search for more papers by this author Yoshiaki Kamada Yoshiaki Kamada Department of Cell Biology, National Institute for Basic Biology, Nishigonaka 38, Myodaiji-cho, Okazaki, 444-8585 Japan Department of Molecular Biomechanics, School of Life Science, The Graduate University for Advanced Studies, Japan Search for more papers by this author Noboru Mizushima Noboru Mizushima Department of Cell Biology, National Institute for Basic Biology, Nishigonaka 38, Myodaiji-cho, Okazaki, 444-8585 Japan PRESTO, Japan Science and Technology Corporation, Japan Search for more papers by this author Takeshi Noda Takeshi Noda Department of Cell Biology, National Institute for Basic Biology, Nishigonaka 38, Myodaiji-cho, Okazaki, 444-8585 Japan Department of Molecular Biomechanics, School of Life Science, The Graduate University for Advanced Studies, Japan Search for more papers by this author Yoshinori Ohsumi Corresponding Author Yoshinori Ohsumi Department of Cell Biology, National Institute for Basic Biology, Nishigonaka 38, Myodaiji-cho, Okazaki, 444-8585 Japan Department of Molecular Biomechanics, School of Life Science, The Graduate University for Advanced Studies, Japan Search for more papers by this author Kuninori Suzuki Kuninori Suzuki Department of Cell Biology, National Institute for Basic Biology, Nishigonaka 38, Myodaiji-cho, Okazaki, 444-8585 Japan Department of Molecular Biomechanics, School of Life Science, The Graduate University for Advanced Studies, Japan Search for more papers by this author Takayoshi Kirisako Takayoshi Kirisako Department of Cell Biology, National Institute for Basic Biology, Nishigonaka 38, Myodaiji-cho, Okazaki, 444-8585 Japan Department of Molecular Biomechanics, School of Life Science, The Graduate University for Advanced Studies, Japan Search for more papers by this author Yoshiaki Kamada Yoshiaki Kamada Department of Cell Biology, National Institute for Basic Biology, Nishigonaka 38, Myodaiji-cho, Okazaki, 444-8585 Japan Department of Molecular Biomechanics, School of Life Science, The Graduate University for Advanced Studies, Japan Search for more papers by this author Noboru Mizushima Noboru Mizushima Department of Cell Biology, National Institute for Basic Biology, Nishigonaka 38, Myodaiji-cho, Okazaki, 444-8585 Japan PRESTO, Japan Science and Technology Corporation, Japan Search for more papers by this author Takeshi Noda Takeshi Noda Department of Cell Biology, National Institute for Basic Biology, Nishigonaka 38, Myodaiji-cho, Okazaki, 444-8585 Japan Department of Molecular Biomechanics, School of Life Science, The Graduate University for Advanced Studies, Japan Search for more papers by this author Yoshinori Ohsumi Corresponding Author Yoshinori Ohsumi Department of Cell Biology, National Institute for Basic Biology, Nishigonaka 38, Myodaiji-cho, Okazaki, 444-8585 Japan Department of Molecular Biomechanics, School of Life Science, The Graduate University for Advanced Studies, Japan Search for more papers by this author Author Information Kuninori Suzuki1,2, Takayoshi Kirisako1,2, Yoshiaki Kamada1,2, Noboru Mizushima1,3, Takeshi Noda1,2 and Yoshinori Ohsumi 1,2 1Department of Cell Biology, National Institute for Basic Biology, Nishigonaka 38, Myodaiji-cho, Okazaki, 444-8585 Japan 2Department of Molecular Biomechanics, School of Life Science, The Graduate University for Advanced Studies, Japan 3PRESTO, Japan Science and Technology Corporation, Japan *Corresponding author. E-mail: [email protected] The EMBO Journal (2001)20:5971-5981https://doi.org/10.1093/emboj/20.21.5971 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Macroautophagy is a bulk degradation process induced by starvation in eukaryotic cells. In yeast, 15 Apg proteins coordinate the formation of autophagosomes. Several key reactions performed by these proteins have been described, but a comprehensive understanding of the overall network is still lacking. Based on Apg protein localization, we have identified a novel structure that functions in autophagosome formation. This pre-autophagosomal structure, containing at least five Apg proteins, i.e. Apg1p, Apg2p, Apg5p, Aut7p/Apg8p and Apg16p, is localized in the vicinity of the vacuole. Analysis of apg mutants revealed that the formation of both a phosphatidylethanolamine-conjugated Aut7p and an Apg12p–Apg5p conjugate is essential for the localization of Aut7p to the pre-autophagosomal structure. Vps30p/Apg6p and Apg14p, components of an autophagy- specific phosphatidylinositol 3-kinase complex, Apg9p and Apg16p are all required for the localization of Apg5p and Aut7p to the structure. The Apg1p protein kinase complex functions in the late stage of autophagosome formation. Here, we present the classification of Apg proteins into three groups that reflect each step of autophagosome formation. Introduction During macroautophagy in yeast, a portion of the cytoplasm is non-selectively sequestered into double-membrane structures (autophagosomes) and delivered to the vacuole for degradation. Electron microscopic analysis revealed that autophagosomes fuse with the vacuolar membrane to release the inner membrane structure into the lumen (Baba et al., 1994). Autophagosomes are generated when a cup-shaped membrane sac (isolation membrane) extends to enwrap and seal cytoplasmic materials. This elongation process, accompanied by the assembly of membranous structures (Kirisako et al., 1999), is currently not well characterized. In mammalian cells, autophagosomes are generated in a similar manner. In rat hepatocytes, osmiophilic membranes designated as phagophores were observed under an electron microscope (Seglen, 1987; Fengsrud et al., 1995, 2000). In mouse embryonic stem cells, similar isolation membranes were identified by electron microscopy as precursors of autophagosomes (Mizushima et al., 2001). To understand the molecular mechanism controlling autophagy, we isolated 15 mutants defective in autophagy (apg) in yeast (Tsukada and Ohsumi, 1993) with disorders of bulk protein degradation, sporulation and the maintenance of viability under starvation conditions (Klionsky and Ohsumi, 1999). A vacuolar hydrolase aminopeptidase I (API) was constitutively transported to the vacuole via small double-membrane vesicles (Scott et al., 1997). This cytosol-to-vacuole targeting (Cvt) pathway runs under growing conditions. Extensive overlaps have been found in genes involved in the APG, AUT (autophagy) and CVT pathways (Harding et al., 1996). All apg mutants except apg17 are known to be defective in the Cvt pathway. Recent characterization of the Apg proteins has identified two ubiquitin-like systems utilizing approximately half of the Apg proteins. Apg12p, a ubiquitin-like protein, is covalently attached to Apg5p to form an Apg12p–Apg5p conjugate controlled by the serial action of Apg7p and Apg10p (Mizushima et al., 1998). Apg7p, a member of the E1-like activating enzymes, forms a thioester linkage with Apg12p (Kim et al., 1999; Tanida et al., 1999; Yuan et al., 1999). Apg12p subsequently forms a thioester intermediate with Apg10p, an E2 conjugating enzyme (Shintani et al., 1999). Apg12p is then linked to Lys149 of Apg5p through an isopeptide bond with the C-terminal glycine. Apg16p then interacts through its C-terminal coiled-coil region to form homo-oligomers following direct interaction with the Apg12p–Apg5p conjugate (Mizushima et al., 1999). In mammalian cells, a homologue of the Apg12p–Apg5p conjugate transiently associates with the membranes of precursor autophagosomes (Mizushima et al., 2001). Aut7p/Apg8p, a second ubiquitin-like protein involved in autophagy, is conjugated to phosphatidylethanolamine (PE; Aut7p–PE) by the serial action of three Apg proteins, Aut2p/Apg4p, Apg7p and Aut1p/Apg3p (Ichimura et al., 2000; Kirisako et al., 2000). The C-terminal Arg117 of Aut7p is removed through the action of Aut2p, a cysteine protease (Kirisako et al., 2000), to expose Gly116. Following activation by Apg7p (E1), the processed Aut7p is transferred to Aut1p (E2). Aut7p is then covalently linked to PE. Aut2p further cleaves Aut7p–PE, releasing Aut7p, an essential step in the normal progression of autophagy (Kirisako et al., 2000). Aut7p is the first protein to localize to autophagosomes and intermediate structures (Kirisako et al., 1999; Huang et al., 2000); this characteristic of Aut7p allows us to use Aut7p to monitor autophagosome formation. LC3, the mammalian homologue of Aut7p, is also localized on the membranes of autophagosomal and precursor membranes (Kabeya et al., 2000; Mizushima et al., 2001). Additional Apg proteins, not known to participate in ubiquitin-like systems, are also required for autophagy. Vps30p/Apg6p, in addition to a role in autophagy, functions in vacuolar protein sorting (Kametaka et al., 1998). Vps30p forms a specific phosphatidylinositol 3-kinase (PI3-kinase) complex required for autophagy, consisting of Vps30p, Apg14p, Vps34p and Vps15p. These data suggest that the dynamic membrane events mediated by the PI3-kinase complex are necessary for autophagy (Kihara et al., 2001a). A class of PI3-kinase and beclin, a human homologue of Vps30p, are also essential for autophagy in human cells (Liang et al., 1999; Petiot et al., 2000). Beclin physically interacts with the PI3-kinase complex on the trans-Golgi network (Kihara et al., 2001b). Tor kinase negatively regulates the induction of autophagy; this kinase activity is inhibited by rapamycin (Noda and Ohsumi, 1998). As the inactivation of Tor activity induces autophagy, treatment with rapamycin mimics the starvation response. The inactivation of Tor activity causes a rapid dephospholylation of Apg13p (Abeliovich et al., 2000; Kamada et al., 2000). Apg13p and Apg17p associate with Apg1p protein kinase (Matsuura et al., 1997) to form the Apg1p protein kinase complex, an essential component of autophagy. Binding of dephosphorylated Apg13p to this complex enhances the kinase activity of Apg1p (Kamada et al., 2000). The characteristics of each Apg protein are gradually being uncovered; their functional interrelationship, however, is poorly understood. This is due to a lack of markers allowing the identification of intermediate steps occurring before or during autophagosome formation. To obtain further insights into the functional relationship between the Apg proteins, we observed the behaviour of Apg proteins fused to fluorescent proteins utilizing a sensitive imaging system. We examined the dynamics of the Apg proteins involved in the two ubiquitin-like systems, Apg5p and Aut7p. Based on their modification and localization in apg mutants, we have defined a pre-autophagosomal structure containing Apg1p, Apg2p, Apg5p, Aut7p and Apg16p. This structure, involved in the production of autophagosomes, provides a clue for investigating the steps of autophagosome formation. Results A punctate structure on which Apg1p, Apg5p, Aut7p and Apg16p are colocalized Apg proteins are involved in several essential reactions in autophagy, such as ubiquitin-like systems or phosphorylation reactions. The interrelationship between these reactions in autophagosome formation, however, remains unknown. It is critical to know the intracellular localization. Several Apg proteins, e.g. Apg5p (George et al., 2000), Aut7p/Apg8p (Kim et al., 2001) and Apg9p (Noda et al., 2000), are localized to perivacuolar punctate structures. To analyse the localization of Apg proteins to these punctate structures, we visualized green fluorescent protein (GFP)-fused Apg proteins using a sensitive microscope system. GFP fusions were expressed from centromeric plasmids under the control of natural promoters. In Δaut7 cells expressing physiological levels of a GFP–Aut7p fusion protein (Figure 1A), GFP–Aut7p complemented the defect of autophagy (Figure 1C–E). These molecules were localized to punctate structures, proximal to the vacuole (Figure 1H and I). Apg5p–GFP retained the ability to conjugate with Apg12p (Figure 1B); when introduced into deficient cells, Apg5p–GFP restored proper autophagic function (Figure 1C, F and G). The additional chimeric proteins used in this study were also functional in the autophagic process (data not shown). Figure 1.Functional GFP–Aut7p and Apg5p–GFP are expressed from natural promoters at physiological levels. (A) The expression level of GFP–Aut7p under growing conditions. Cell lysates were prepared as described in Materials and methods. (1) Wild-type (KA311A), (2) Δaut7 (YYK218) and (3) Δaut7 (YYK218) expressing GFP–Aut7p. (B) The Apg12p–Apg5p conjugate was produced normally in Δapg5 cells expressing Apg5p–GFP. Cell lysates were prepared as described in Materials and methods. (1) Wild-type (KA311A), (2) Δapg5 (GYS59) and (3) Δapg5 (GYS59) expressing Apg5p–GFP. (C–G) The accumulation of autophagic bodies was examined under a light microscope. Cells were incubated for 6 h in 0.17% yeast nitrogen base w/o amino acid and ammonium sulfate containing 1 mM PMSF. Nomarski images of (C) wild-type (KA311A), (D) Δaut7 (YYK218), (E) Δaut7 (YYK218) expressing GFP–Aut7p, (F) Δapg5 (GYS59) and (G) Δapg5 (GYS59) expressing Apg5p–GFP. (H–I) GFP–Aut7p visualized in Δaut7 cells (KVY5) under growing conditions. A punctate structure containing GFP–Aut7p is detected close to the vacuole, identified by FM4-64 labelling. (H) Fluorescence of GFP–Aut7p (green) and a FM4-64-labelled vacuole (red). (I) The Nomarski image is overlaid with GFP–Aut7p and FM4-64 fluorescence. The punctate structures, close to the vacuole, were detected in 43 of 206 cells (21%). Bar: 5 μm. Download figure Download PowerPoint Next, we fused cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) to several different Apg proteins and examined their colocalization. Fluorescent Aut7p served as a marker of the punctate structures. Apg1p, Apg5p and Apg16p colocalized with Aut7p on a single punctate structure located close to the vacuole (Figure 2). After the addition of rapamycin, CFP–Aut7p was delivered to vacuoles in a time-dependent manner (Figure 2A, D and G). YFP–Apg1p was delivered to vacuoles after lengthy treatment with rapamycin (data not shown), whereas Apg5p–YFP and Apg16p–YFP were never transported to vacuoles (Figure 2B and H). Apg2p colocalizes with Aut7p on a perivacuolar punctate structure (Shintani et al., 2001). Consequently, the punctate structure is shown to contain Apg1p, Apg2p, Apg5p, Aut7p and Apg16p. Apg12p is likely to be colocalized with Apg5p and Apg16p as a component of the AApg12p–Apg5p–Apg16p complex. As Apg13p and Apg17p interact physically with Apg1p (Kamada et al., 2000), it is probable that these molecules also reside on the punctate structure. This is the first report demonstrating the colocalization of multiple Apg proteins. Figure 2.Colocalization of Apg1p, Apg5p, Apg16p and Aut7p on a punctate structure close to the vacuole. (A–C) Δapg5 cells (YNM119) expressing Apg5p–YFP and CFP–Aut7p were treated with rapamycin for 3 h: (A) Apg5p–YFP, (B) CFP–Aut7p, (C) merged image of Apg5p–YFP (green) and CFP–Aut7p (red). (D–F) Δapg1 cells (NNY20) expressing CFP–Aut7p and YFP–Apg1p were treated with rapamycin for 30 min: (D) CFP–Aut7p, (E) YFP–Apg1p, (F) merged image of CFP–Aut7p (red) and YFP–Apg1p (green). (G–I) Δapg16 cells (KVY117) expressing CFP–Aut7p and YFP–Apg16p were treated with rapamycin for 6 h: (G) CFP–Aut7p, (H) YFP–Apg16p, (I) merged image of CFP–Aut7p (red) and YFP–Apg16p (green). Bar: 5 μm. Download figure Download PowerPoint Apg9p is a putative membrane protein and is not cofractionated with typical endomembrane marker proteins, autophagosomes or Cvt vesicles (Noda et al., 2000). Apg9p–GFP exhibited an uneven, cytosolic distribution in addition to its localization on several punctate structures (Noda et al., 2000; Figure 3A and B). This pattern was not altered in other apg mutants (Figure 3C and D; data not shown). To examine the use of this protein as a punctate structure marker, we compared the localization of Apg9p–GFP and Aut7p by immunofluorescence microscopy. The punctate structures labelled with Aut7p seldom colocalized with those labelled by Apg9p–GFP (Figure 3E–L), although the two structures were occasionally in close proximity (arrows in Figure 3K). This pattern of Apg9p indicates that it is not a suitable marker for the punctate structure. Figure 3.Localization of Aut7p and Apg9p–GFP. (A and B) Apg9p–GFP was visualized in Δapg9 cells (CTD1) under growing conditions: (A) Apg9p–GFP, (B) the Nomarski image is overlaid with the fluorescence of Apg9p–GFP. (C and D) Apg9p–GFP in Δapg9Δapg14 cells (GYS29) was identified under growing conditions: (C) Apg9p–GFP, (D) the Nomarski image is overlaid with the fluorescence of Apg9p–GFP. (E–L) Δapg9 cells (CTD1) expressing Apg9p–GFP were treated with rapamycin for 2 h. Immunofluorescence microscopy was performed as described in Materials and methods. (E and I) Apg9p–GFP, (F and J) Aut7p, (G and K) merged images of Apg9p–GFP (green) and Aut7p (red) and (H and L) Nomarski images. Apg9p and Aut7p were occasionally found in close proximity (arrows in K). Bar: 2 μm. Download figure Download PowerPoint Electron microscopic analysis established that autophagosomes accumulate in the Δypt7 mutant (Kirisako et al., 1999). We therefore examined the localization of Apg5p–YFP and CFP–Aut7p in Δypt7 cells. Following rapamycin treatment of these cells, autophagosomes labelled with CFP–Aut7p appeared as dim dots in the cytosol (Figure 4A), whereas Apg5p–YFP was detected as a punctate pattern (arrow in Figure 4B). Every punctate structure labelled with Apg5p–YFP was associated with CFP–Aut7p (arrow in Figure 4C). CFP–Aut7p staining of the structure was of a greater intensity than the other CFP–Aut7p dots (arrow in Figure 4A). These observations suggest that this punctate structure containing several colocalized Apg proteins is not an autophagosome. Figure 4.Localization of Aut7p and Apg5p in Δypt7 cells. Δypt7Δapg5 cells (YAK3) expressing CFP–Aut7p and Apg5p–YFP were treated with rapamycin for 5 h. (A) Autophagosomes stained with CFP–Aut7p; (B) Apg5p–YFP; (C) merged image of CFP–Aut7p (red) and Apg5p–YFP (green); (D) Nomarski image: CFP–Aut7p and Apg5p–YFP colocalized on a punctate structure (arrows). Bar: 5 μm. Download figure Download PowerPoint To investigate the involvement of the endocytic pathway in punctate structure formation, we examined the class E compartment, an exaggerated prevacuolar compartment in class E vacuolar protein sorting (vps) mutants (Raymond et al., 1992). The class E compartment is labelled with FM4-64, a lipophilic stylyl dye, as an intensely stained membrane-enclosed structure (Vida and Emr, 1995) and morphologically similar to the punctate structure labelled with Aut7p. We observed the localization of GFP–Aut7p in a class E vps mutant, Δvps4 (Raymond et al., 1992). The GFP–Aut7p punctate structure did not colocalize with the class E compartment in this mutant (Figure 5). These data suggest that the punctate structure is not organized through the endocytic pathway. Therefore, this punctate structure is a novel structure localized proximal to the vacuole. Figure 5.Localization of Aut7p and the class E compartment. The class E compartment of Δvps4 cells (MBY3) expressing GFP–Aut7p was labelled with FM4-64 as described in Materials and methods, following treatment with rapamycin for 2 h. (A and E) GFP–Aut7p. (B and F) The class E compartments stained with FM4-64 (arrows). (C and G) Merged images of GFP–Aut7p (green) and FM4-64 (red). (D and H) Nomarski images. Bar: 2 μm. Download figure Download PowerPoint Genetic analysis of APG genes required for the organization of the punctate structure We have found a novel punctate structure in which at least five Apg proteins are concentrated. We examined the organization within this punctate structure by studying the localization of Apg5p–GFP and GFP–Aut7p in apg mutants (Figure 6; summarized in Table I). We also assessed the quantities of PE-conjugated Aut7p (Ichimura et al., 2000; Kirisako et al., 2000) in each apg mutant by SDS–PAGE in the presence of 6 M urea (Figure 7). Based on these observations, we grouped the apg mutants into three categories. These classes corresponded well to defined functional units, including the Apg1p protein kinase complex, the two ubiquitin-like systems and the PI3-kinase complex necessary for autophagy. This classification provides several new insights into the interaction between these classes, detailed in the following sections. Figure 6.Localization of GFP–Aut7p and Apg5p–GFP in the apg mutants grown in SD + CA medium. (A) Δapg1 cells (NNY20). GFP–Aut7p was detected on a punctate structure close to the vacuole in 30 of 161 cells (18%). Δapg13 (C; TFD13W2), Δapg17 (YYK111), apg2 (MT2-4-4) cells exhibited an identical phenotype. (B) Δapg1 cells (YYK36) expressing Apg5p–GFP. Punctate structures were detected in 11 of 91 cells (12%). Δapg13 (D; TFD13W2), Δapg17 (YYK111) and apg2 (MT2-4-4) cells demonstrated an identical phenotype. (E) This image of a Δaut2 cell (GYS6) expressing GFP–Aut7p is representative of the Aut7 system. The punctate structures were detected in one of 78 cells (1%). Δapg7 (GYS9) and Δaut1 (GYS5) cells exhibited the same phenotype. (F) Δaut2 cells (GYS6) expressing Apg5p–GFP. A single punctate structure was detected in 11 of 52 cells (21%). Δaut1 (GYS5) and Δaut7 (YYK218) cells demonstrated a similar phenotype. (G) This Δapg12 cell (GYS13) expressing GFP–Aut7p is representative of mutants of the Apg12 system. Punctate structures were detected in one of 84 cells (1%). Δapg10 (TFD10-L1) and Δapg5 (SKD5-1D) cells possessed an identical phenotype. (H) A Δapg5Δapg12 cell (YNM117) expressing Apg5p–GFP. A single punctate structure was detected in 16 of 104 cells (15%). Δapg7 (GYS9), Δapg10 (TFD10-L1) and Δapg5 (SKD5-1D) cells all exhibited an identical phenotype. (I) A Δapg14 cell (SKD14–1C) expressing GFP–Aut7p. None of the 51 cells displayed a punctate structure. Δvps30 cells (SKD6-1D) and Δapg9 cells (CTD1) possessed an identical phenotype. (J) A Δapg14 cell (AKY12) expressing Apg5p–GFP under growing conditions. Punctate structures were observed in one of 248 cells (<1%). Δvps30 cells (AKY74) and Δapg9 cells (CTD1) exhibited an identical phenotype. (K) A Δapg16 cell (KVY117) expressing GFP–Aut7p. No punctate structures were observed in any of the 77 cells. (L) A Δapg5Δapg16 cell (YNM126) expressing Apg5p–GFP under growing conditions. No punctate structures were observed in the 184 cells examined. Bar: 5 μm. Download figure Download PowerPoint Figure 7.Levels of Aut7p–PE present in each apg mutant. Lysates were prepared by glass bead disruption prior to SDS–PAGE as described in Materials and methods. (A) Cells in vegetative growth. (B) Cells following a 4.5 h starvation in SD (−N) medium. We examined the phenotype of wild type (SEY6210), Δapg1 (GYS102), apg2 (MT2-4-4), Δaut1 (KVY113), Δaut2 (KVY13), Δapg5 (KVY142), Δvps30 (KVY135), Δapg7 (KVY118), Δaut7 (KVY5), Δapg9 (KVY114), Δapg10 (KVY136), Δapg12 (KVY115), ΔΔapg13 (KVY116), Δapg14 (GYS115), Δapg16 (KVY117) and Δapg17 (YYK111) cells. Download figure Download PowerPoint Table 1. Localization of GFP–Aut7p and Apg5p–GFP on the punctate structure in apg mutants Genotype GFP–Aut7p Apg5p–GFP Δapg1 + + apg2 + + Δaut1/Δapg3 − + Δaut2/Δapg4 − + Δapg5 − + (WT) Δvps30/Δapg6 − − Δapg7 − + Δaut7/Δapg8 + (WT) + Δapg9 − − Δapg10 − + Δapg12 − + Δapg13 + + Δapg14 − − Δapg16 − − Δapg17 + + Δapg1Δapg7 − not examined Δapg1Δvps30 − not examined Δapg1Δapg9 − not examined Δaut2Δapg10 expressing GFP–Aut7FGp − not examined Δaut2Δapg12 expressing GFP–Aut7FGp − not examined Δvps38 + not examined +: GFP–Aut7p or Apg5p–GFP is localized to the punctate structure. −: GFP–Aut7p or Apg5p–GFP shows a diffuse cytoplasmic staining pattern. WT: wild type. Analyses of class A apg mutants provide evidence that the punctate structure is involved in autophagosome formation The class A mutants possess both Apg5p–GFP and GFP–Aut7p localized to the punctate structure as in wild-type cells (Table I). The class A genes consist of APG1, APG2, APG13 and APG17. Apg13p and Apg17p are regulatory factors of the Apg1p protein kinase. As the class A mutants demonstrate normal punctate structures, the Apg1p protein kinase complex is unlikely to be involved in the formation of the punctate structure. The class A mutants all possessed normal levels of Aut7p–PE (Figure 7). To examine whether the punctate structure is physiologically functional, we generated an apg1ts allele by polymerase chain reaction (PCR) mutagenesis. Auto phagic activities were measured by monitoring the alkaline phosphatase (ALP) activity in cells expressing a cytosolic proform of the ALP (Pho8Δ60p), which is transported to vacuoles and becomes mature (Noda et al., 1995). We estimated the temperature sensitivity of this allele from the maturation of API (Figure 8A) and the ALP assay (Figure 8B) using Δapg1 cells carrying the apg1ts plasmid (apg1ts cells). At 23°C, the apg1ts cells contained normal levels of mature API (Figure 8A) and possessed significant autophagic activity (Figure 8B). At 37°C, however, both the maturation of API and autophagic activity were completely blocked (Figure 8A and B). Autophagy in the apg1ts cells at 30°C occurred at the same levels as at 23°C. The activity of ALP increased linearly with time for 6 h following incubation in nitrogen-free medium at 23°C (Figure 8C). On transferring the cells to 37°C, autophagy was immediately blocked (Figure 8C). When the cells were transferred from 37°C to 23°C, the autophagic activity was recovered without a lag period (Figure 8C). Apg1tsp was detectable by immunoblot even after an overnight incubation at 37°C (Figure 8A), demonstrating that apg1ts is a reversible temperature-sensitive allele defective in autophagy. Figure 8.apg1ts mutant possessing functional temperature sensitivity. (A) Although temperature-sensitive Apg1p was detectable, the maturation of API was completely blocked at 37°C in apg1ts cells. The API proform (proAPI) is processed to mature API (mAPI) in an Apg1p activity-dependent manner. Wild-type cells (TN125), Δapg1 cells (YYK126) and Δapg1 cells (YYK126) carrying the apg1ts plasmid were grown in YEPD medium at 23°C or 37°C overnight. Apg1p and API were detected as described in Materials and methods. (B) Δapg1 cells (YYK126) carrying (1) the APG1 plasmid, (2) the vector or (3) the apg1ts plasmid were grown in YEPD medium at 23°C. They were then transferred into SD (−N) medium at 23°C or 37°C and incubated for 6 h before ALP activity was measured. (C) Δapg1 cells (YYK126) carrying the apg1ts plasmid were grown in YEPD medium at 23°C. The cells were then transferred into SD (−N) medium and incubated at 23°C or 37°C. Closed squares, cells starved continuously at 23°C. Open squares, cells incubated for 2 h at 23°C and then transferred to 37°C. Open lozenges, cells starved continuously at 37°C. Closed lozenges, cells incubated for 2 h at 37°C and then transferred to 23°C. Download figure Download PowerPoint On shifting the temperature from 37°C to 30°C, we followed the dynamics of GFP–Aut7p in apg1ts cells. At 30°C, GFP–Aut7p was visualized in the punctate structure and the vacuole after rapamycin treatment, as seen in wild-type cells (Figure 9A). At 37°C, the staining intensity of the punctate structure increased, accompanied by a lack of vacuolar staining after rapamycin treatment (Figure 9B, 4 min). Next, we examined the progression of GFP–Aut7p staining following temperature decrease in real time using time-lapse microscopy. Within 10 min, several dots labelled with GFP–Aut7p separated from the punctate structure (Figure 9B). During 30 min of incubation at 30°C, fluorescence intensity of vacuoles became brighter as these dots disappeared, suggesting that they are autophagosomes. We concluded that the punctate structure actually participates in the formation of autophagosomes. Figure 9.Time-lapse microscopy of apg1ts cells expressing GFP–Aut7p. Δapg1 cells (NNY20) carrying the apg1ts and GFP–Aut7p plasmids were used. (A) Cells treated with rapamycin for 3 h at 30°C. (B) Time-lapse images following temperature decrease. Cells were treated with rapamycin for 4 h at 37°C before decreasing the temperature to 30°C. (C) Cell incubated for 2 h at 30°C after temperature decrease. Bar: 5 μm. Download figure Download PowerPoint We next treated apg1ts cells with cycloheximide for 20 min, following a 4 h incubation at 37°C in the presence of rapamycin. On decreasing the temperature, similar dots
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