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Two distinct Vps34 phosphatidylinositol 3-kinase complexes function in autophagy and carboxypeptidase Y sorting in Saccharomyces cerevisiae.

Kihara A, Noda T, Ishihara N, Ohsumi Y - J. Cell Biol. (2001)

Bottom Line: We found that two proteins copurify with Vps30p.These results indicate that Vps30p functions as a subunit of a Vps34 PtdIns 3-kinase complex(es).We propose that multiple Vps34p-Vps15p complexes associated with specific regulatory proteins might fulfill their membrane trafficking events at different sites.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, National Institute for Basic Biology, Okazaki, 444-8585, Japan.

ABSTRACT
Vps30p/Apg6p is required for both autophagy and sorting of carboxypeptidase Y (CPY). Although Vps30p is known to interact with Apg14p, its precise role remains unclear. We found that two proteins copurify with Vps30p. They were identified by mass spectrometry to be Vps38p and Vps34p, a phosphatidylinositol (PtdIns) 3-kinase. Vps34p, Vps38p, Apg14p, and Vps15p, an activator of Vps34p, were coimmunoprecipitated with Vps30p. These results indicate that Vps30p functions as a subunit of a Vps34 PtdIns 3-kinase complex(es). Phenotypic analyses indicated that Apg14p and Vps38p are each required for autophagy and CPY sorting, respectively, whereas Vps30p, Vps34p, and Vps15p are required for both processes. Coimmunoprecipitation using anti-Apg14p and anti-Vps38p antibodies and pull-down experiments showed that two distinct Vps34 PtdIns 3-kinase complexes exist: one, containing Vps15p, Vps30p, and Apg14p, functions in autophagy and the other containing Vps15p, Vps30p, and Vps38p functions in CPY sorting. The vps34 and vps15 mutants displayed additional phenotypes such as defects in transport of proteinase A and proteinase B, implying the existence of another PtdIns 3-kinase complex(es). We propose that multiple Vps34p-Vps15p complexes associated with specific regulatory proteins might fulfill their membrane trafficking events at different sites.

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Purification of Vps30p complexes. (A) AKY73 (wild type; lane 1) and AKY76 (Δvps30; lane 2) cells grown in SC medium lacking methionine at 30°C were labeled with Express™ [35S]methionine/cysteine protein labeling mix (NEN Life Science Products) for 1 h. The labeled cells were converted to spheroplasts and lysed by extrusion through a polycarbonate filter with 3 μm pores. Thus, obtained lysates were solubilized with Triton X-100 and incubated with anti-Vps30p antibodies and protein A–Sepharose beads at 4°C for 2 h. Bound proteins were eluted, separated by SDS-PAGE, and detected by autoradiography using a PhosphorImager BAS2000 (Fuji Film). (B) Protein profiles during the purification of the Vps30p complexes. Total lysates prepared from AKY76 cells harboring pKHR25 (His6–Myc–VPS30; 2 μm) were solubilized with Triton X-100 and subjected to Ni-NTA agarose chromatography (load, lane 1; flow-through, lane 2). Proteins bound to Ni-NTA agarose were washed (lane 3) and eluted with 250 mM imidazole (lane 4). The eluates were then incubated with protein A–immobilized anti-Vps30p antibodies (flow-through, lane 5). The column was washed, and retained proteins were eluted with 100 mM glycine-HCl, pH 2.5 (lane 6; and C, lane 1). Proteins were separated by SDS-PAGE and visualized by Coomassie brilliant blue staining. (C) For control, a purification procedure as described in B was applied to lysates prepared from AKY76 cells harboring an empty pRS424 plasmid (lane 2). Proteins were analyzed by SDS-PAGE and visualized by silver staining. (D) Cells of AKY106 (wild type; lane 1) and AKY111 (Δvps30; lane 2) were grown in YPD at 28°C. Total lysates were solubilized with Triton X-100 and incubated with protein A–immobilized anti-Vps30p antibodies. Retained proteins were eluted, separated by SDS-PAGE, and detected by immunoblotting with anti-Vps30p, anti-Vps34p, anti-Vps38p, anti-Apg14p, and anti-Vps15p antibodies.
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Figure 1: Purification of Vps30p complexes. (A) AKY73 (wild type; lane 1) and AKY76 (Δvps30; lane 2) cells grown in SC medium lacking methionine at 30°C were labeled with Express™ [35S]methionine/cysteine protein labeling mix (NEN Life Science Products) for 1 h. The labeled cells were converted to spheroplasts and lysed by extrusion through a polycarbonate filter with 3 μm pores. Thus, obtained lysates were solubilized with Triton X-100 and incubated with anti-Vps30p antibodies and protein A–Sepharose beads at 4°C for 2 h. Bound proteins were eluted, separated by SDS-PAGE, and detected by autoradiography using a PhosphorImager BAS2000 (Fuji Film). (B) Protein profiles during the purification of the Vps30p complexes. Total lysates prepared from AKY76 cells harboring pKHR25 (His6–Myc–VPS30; 2 μm) were solubilized with Triton X-100 and subjected to Ni-NTA agarose chromatography (load, lane 1; flow-through, lane 2). Proteins bound to Ni-NTA agarose were washed (lane 3) and eluted with 250 mM imidazole (lane 4). The eluates were then incubated with protein A–immobilized anti-Vps30p antibodies (flow-through, lane 5). The column was washed, and retained proteins were eluted with 100 mM glycine-HCl, pH 2.5 (lane 6; and C, lane 1). Proteins were separated by SDS-PAGE and visualized by Coomassie brilliant blue staining. (C) For control, a purification procedure as described in B was applied to lysates prepared from AKY76 cells harboring an empty pRS424 plasmid (lane 2). Proteins were analyzed by SDS-PAGE and visualized by silver staining. (D) Cells of AKY106 (wild type; lane 1) and AKY111 (Δvps30; lane 2) were grown in YPD at 28°C. Total lysates were solubilized with Triton X-100 and incubated with protein A–immobilized anti-Vps30p antibodies. Retained proteins were eluted, separated by SDS-PAGE, and detected by immunoblotting with anti-Vps30p, anti-Vps34p, anti-Vps38p, anti-Apg14p, and anti-Vps15p antibodies.

Mentions: Although Δvps30 cells are defective in both autophagy and CPY sorting, Δapg14 cells only have a defect in autophagy (Kametaka et al. 1998). Moreover, overproduction of Apg14p partially suppresses the autophagic defect caused by the apg6-1 mutation, which resulted in an ∼50% COOH-terminal truncation of Vps30p but not the CPY transport defect (Kametaka et al. 1998). These results prompted us to hypothesize that Vps30p/Apg6p may compose large protein complexes functioning in different processes. To test this, we first examined whether Vps30p interacts with proteins other than Apg14p. Wild-type or Δvps30 cells were labeled with [35S]methionine/cysteine. Total lysates prepared from the wild-type or Δvps30 cells were solubilized with Triton X-100 and subjected to immunoprecipitation using anti-Vps30p antibodies. In the immunoprecipitates from wild-type cells, many protein bands together with Vps30p were detected (Fig. 1 A, lane 1). Most of them were nonspecific backgrounds because they were also present in the immunoprecipitates from Δvps30 cells (Fig. 1 A, lane 2). However, three bands, termed p160, p90, and p50, were present only in the immunoprecipitates from wild-type cells, indicating that these proteins specifically interact with Vps30p. To identify them, we constructed pKHR25, which carried His6–Myc–VPS30, a fusion gene encoding Vps30p with attached NH2-terminal His6 and Myc tag sequences. pKHR25 is a multicopy plasmid (2 μm) that expresses His6–Myc–Vps30p at levels 30-fold higher than chromosomally expressed Vps30p. Total lysates prepared from cells of AKY76 carrying pKHR25 were solubilized with Triton X-100, and the detergent extracts were loaded on a Ni-NTA agarose column (Fig. 1 B, lanes 1–4). The elutes were further purified by incubation with an immunoaffinity column prepared by covalently attaching anti-Vps30p antibodies to protein A–Sepharose beads (Fig. 1 B, lanes 5 and 6). Several proteins were eluted from the column along with His6–Myc–Vps30p (Fig. 1 B, lane 6). To discriminate bands that specifically interacted with His6–Myc–Vps30p from nonspecific background binding, the same purification procedure was repeated using the lysates of AKY76 carrying a plasmid that did not express His6–Myc–Vps30p. p90 and p50 but not p160 were specifically present in the purified fraction from His6–Myc–Vps30p-expressing cells (Fig. 1 C). Bands of p90 and p50 were excised from the gel, and the protein samples were digested with trypsin in the gel matrix. Extracted peptide mixtures were analyzed by matrix-assisted laser desorption/ionization mass spectrometry. The peptide mass maps were used to query a comprehensive sequence database for unambiguous protein identification. The results indicated that p90 and p50 were Vps34p and Vps38p, respectively. Vps34p is a PtdIns 3–kinase required for proper sorting of a subset of vacuolar proteins (Robinson et al. 1988; Herman and Emr 1990). Vps38p is involved in the targeting of CPY and mutant Pma1p to the vacuole (Luo and Chang 1997), although its precise role in these processes is unclear. To confirm that Vps34p and Vps38p indeed interact with Vps30p, coimmunoprecipitation experiments were performed using anti-Vps30p antibodies. Immunoblotting showed that Vps30p, Vps34p, and Vps38p were present in immunoprecipitates prepared from wild-type yeast, (Fig. 1 D, lane 1) but not in those from the Δvps30 strain (Fig. 1 D, lane 2), indicating that Vps34p and Vps38p were precipitated specifically through the interaction with Vps30p. As reported previously, Apg14p also coimmunoprecipitated with Vps30p (Fig. 1 D, lane 1). Vps15p, a serine/threonine kinase, has been shown to interact with Vps34p (Stack et al. 1993). Therefore, Vps15p was the best candidate for p160. We next examined whether Vps15p existed in the immunoprecipitates obtained using anti-Vps30p antibodies. Immunoblotting using anti-Vps15p antibodies showed that Vps15p was also present (Fig. 1 D, lane 1). These results indicate that Vps30p form a complex(es) with Apg14p, Vps15p, Vps34p, and Vps38p. Until now, the role of Vps30p in autophagy and CPY sorting has remained unclear. We demonstrate here that Vps30p functions as a subunit of a Vps34 PtdIns 3–kinase complex(es). The reason why we failed to detect Apg14p in Fig. 1A and Fig. C, was probably due to its low abundance (see below). We could not detect Vps15p in Fig. 1 C. Vps15p might be concealed by nonspecific backgrounds because p160 was very close to nonspecific backgrounds in Fig. 1 A. Alternatively, Vps15p was degraded during the purification procedure due to its instability (see Discussion).


Two distinct Vps34 phosphatidylinositol 3-kinase complexes function in autophagy and carboxypeptidase Y sorting in Saccharomyces cerevisiae.

Kihara A, Noda T, Ishihara N, Ohsumi Y - J. Cell Biol. (2001)

Purification of Vps30p complexes. (A) AKY73 (wild type; lane 1) and AKY76 (Δvps30; lane 2) cells grown in SC medium lacking methionine at 30°C were labeled with Express™ [35S]methionine/cysteine protein labeling mix (NEN Life Science Products) for 1 h. The labeled cells were converted to spheroplasts and lysed by extrusion through a polycarbonate filter with 3 μm pores. Thus, obtained lysates were solubilized with Triton X-100 and incubated with anti-Vps30p antibodies and protein A–Sepharose beads at 4°C for 2 h. Bound proteins were eluted, separated by SDS-PAGE, and detected by autoradiography using a PhosphorImager BAS2000 (Fuji Film). (B) Protein profiles during the purification of the Vps30p complexes. Total lysates prepared from AKY76 cells harboring pKHR25 (His6–Myc–VPS30; 2 μm) were solubilized with Triton X-100 and subjected to Ni-NTA agarose chromatography (load, lane 1; flow-through, lane 2). Proteins bound to Ni-NTA agarose were washed (lane 3) and eluted with 250 mM imidazole (lane 4). The eluates were then incubated with protein A–immobilized anti-Vps30p antibodies (flow-through, lane 5). The column was washed, and retained proteins were eluted with 100 mM glycine-HCl, pH 2.5 (lane 6; and C, lane 1). Proteins were separated by SDS-PAGE and visualized by Coomassie brilliant blue staining. (C) For control, a purification procedure as described in B was applied to lysates prepared from AKY76 cells harboring an empty pRS424 plasmid (lane 2). Proteins were analyzed by SDS-PAGE and visualized by silver staining. (D) Cells of AKY106 (wild type; lane 1) and AKY111 (Δvps30; lane 2) were grown in YPD at 28°C. Total lysates were solubilized with Triton X-100 and incubated with protein A–immobilized anti-Vps30p antibodies. Retained proteins were eluted, separated by SDS-PAGE, and detected by immunoblotting with anti-Vps30p, anti-Vps34p, anti-Vps38p, anti-Apg14p, and anti-Vps15p antibodies.
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Figure 1: Purification of Vps30p complexes. (A) AKY73 (wild type; lane 1) and AKY76 (Δvps30; lane 2) cells grown in SC medium lacking methionine at 30°C were labeled with Express™ [35S]methionine/cysteine protein labeling mix (NEN Life Science Products) for 1 h. The labeled cells were converted to spheroplasts and lysed by extrusion through a polycarbonate filter with 3 μm pores. Thus, obtained lysates were solubilized with Triton X-100 and incubated with anti-Vps30p antibodies and protein A–Sepharose beads at 4°C for 2 h. Bound proteins were eluted, separated by SDS-PAGE, and detected by autoradiography using a PhosphorImager BAS2000 (Fuji Film). (B) Protein profiles during the purification of the Vps30p complexes. Total lysates prepared from AKY76 cells harboring pKHR25 (His6–Myc–VPS30; 2 μm) were solubilized with Triton X-100 and subjected to Ni-NTA agarose chromatography (load, lane 1; flow-through, lane 2). Proteins bound to Ni-NTA agarose were washed (lane 3) and eluted with 250 mM imidazole (lane 4). The eluates were then incubated with protein A–immobilized anti-Vps30p antibodies (flow-through, lane 5). The column was washed, and retained proteins were eluted with 100 mM glycine-HCl, pH 2.5 (lane 6; and C, lane 1). Proteins were separated by SDS-PAGE and visualized by Coomassie brilliant blue staining. (C) For control, a purification procedure as described in B was applied to lysates prepared from AKY76 cells harboring an empty pRS424 plasmid (lane 2). Proteins were analyzed by SDS-PAGE and visualized by silver staining. (D) Cells of AKY106 (wild type; lane 1) and AKY111 (Δvps30; lane 2) were grown in YPD at 28°C. Total lysates were solubilized with Triton X-100 and incubated with protein A–immobilized anti-Vps30p antibodies. Retained proteins were eluted, separated by SDS-PAGE, and detected by immunoblotting with anti-Vps30p, anti-Vps34p, anti-Vps38p, anti-Apg14p, and anti-Vps15p antibodies.
Mentions: Although Δvps30 cells are defective in both autophagy and CPY sorting, Δapg14 cells only have a defect in autophagy (Kametaka et al. 1998). Moreover, overproduction of Apg14p partially suppresses the autophagic defect caused by the apg6-1 mutation, which resulted in an ∼50% COOH-terminal truncation of Vps30p but not the CPY transport defect (Kametaka et al. 1998). These results prompted us to hypothesize that Vps30p/Apg6p may compose large protein complexes functioning in different processes. To test this, we first examined whether Vps30p interacts with proteins other than Apg14p. Wild-type or Δvps30 cells were labeled with [35S]methionine/cysteine. Total lysates prepared from the wild-type or Δvps30 cells were solubilized with Triton X-100 and subjected to immunoprecipitation using anti-Vps30p antibodies. In the immunoprecipitates from wild-type cells, many protein bands together with Vps30p were detected (Fig. 1 A, lane 1). Most of them were nonspecific backgrounds because they were also present in the immunoprecipitates from Δvps30 cells (Fig. 1 A, lane 2). However, three bands, termed p160, p90, and p50, were present only in the immunoprecipitates from wild-type cells, indicating that these proteins specifically interact with Vps30p. To identify them, we constructed pKHR25, which carried His6–Myc–VPS30, a fusion gene encoding Vps30p with attached NH2-terminal His6 and Myc tag sequences. pKHR25 is a multicopy plasmid (2 μm) that expresses His6–Myc–Vps30p at levels 30-fold higher than chromosomally expressed Vps30p. Total lysates prepared from cells of AKY76 carrying pKHR25 were solubilized with Triton X-100, and the detergent extracts were loaded on a Ni-NTA agarose column (Fig. 1 B, lanes 1–4). The elutes were further purified by incubation with an immunoaffinity column prepared by covalently attaching anti-Vps30p antibodies to protein A–Sepharose beads (Fig. 1 B, lanes 5 and 6). Several proteins were eluted from the column along with His6–Myc–Vps30p (Fig. 1 B, lane 6). To discriminate bands that specifically interacted with His6–Myc–Vps30p from nonspecific background binding, the same purification procedure was repeated using the lysates of AKY76 carrying a plasmid that did not express His6–Myc–Vps30p. p90 and p50 but not p160 were specifically present in the purified fraction from His6–Myc–Vps30p-expressing cells (Fig. 1 C). Bands of p90 and p50 were excised from the gel, and the protein samples were digested with trypsin in the gel matrix. Extracted peptide mixtures were analyzed by matrix-assisted laser desorption/ionization mass spectrometry. The peptide mass maps were used to query a comprehensive sequence database for unambiguous protein identification. The results indicated that p90 and p50 were Vps34p and Vps38p, respectively. Vps34p is a PtdIns 3–kinase required for proper sorting of a subset of vacuolar proteins (Robinson et al. 1988; Herman and Emr 1990). Vps38p is involved in the targeting of CPY and mutant Pma1p to the vacuole (Luo and Chang 1997), although its precise role in these processes is unclear. To confirm that Vps34p and Vps38p indeed interact with Vps30p, coimmunoprecipitation experiments were performed using anti-Vps30p antibodies. Immunoblotting showed that Vps30p, Vps34p, and Vps38p were present in immunoprecipitates prepared from wild-type yeast, (Fig. 1 D, lane 1) but not in those from the Δvps30 strain (Fig. 1 D, lane 2), indicating that Vps34p and Vps38p were precipitated specifically through the interaction with Vps30p. As reported previously, Apg14p also coimmunoprecipitated with Vps30p (Fig. 1 D, lane 1). Vps15p, a serine/threonine kinase, has been shown to interact with Vps34p (Stack et al. 1993). Therefore, Vps15p was the best candidate for p160. We next examined whether Vps15p existed in the immunoprecipitates obtained using anti-Vps30p antibodies. Immunoblotting using anti-Vps15p antibodies showed that Vps15p was also present (Fig. 1 D, lane 1). These results indicate that Vps30p form a complex(es) with Apg14p, Vps15p, Vps34p, and Vps38p. Until now, the role of Vps30p in autophagy and CPY sorting has remained unclear. We demonstrate here that Vps30p functions as a subunit of a Vps34 PtdIns 3–kinase complex(es). The reason why we failed to detect Apg14p in Fig. 1A and Fig. C, was probably due to its low abundance (see below). We could not detect Vps15p in Fig. 1 C. Vps15p might be concealed by nonspecific backgrounds because p160 was very close to nonspecific backgrounds in Fig. 1 A. Alternatively, Vps15p was degraded during the purification procedure due to its instability (see Discussion).

Bottom Line: We found that two proteins copurify with Vps30p.These results indicate that Vps30p functions as a subunit of a Vps34 PtdIns 3-kinase complex(es).We propose that multiple Vps34p-Vps15p complexes associated with specific regulatory proteins might fulfill their membrane trafficking events at different sites.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, National Institute for Basic Biology, Okazaki, 444-8585, Japan.

ABSTRACT
Vps30p/Apg6p is required for both autophagy and sorting of carboxypeptidase Y (CPY). Although Vps30p is known to interact with Apg14p, its precise role remains unclear. We found that two proteins copurify with Vps30p. They were identified by mass spectrometry to be Vps38p and Vps34p, a phosphatidylinositol (PtdIns) 3-kinase. Vps34p, Vps38p, Apg14p, and Vps15p, an activator of Vps34p, were coimmunoprecipitated with Vps30p. These results indicate that Vps30p functions as a subunit of a Vps34 PtdIns 3-kinase complex(es). Phenotypic analyses indicated that Apg14p and Vps38p are each required for autophagy and CPY sorting, respectively, whereas Vps30p, Vps34p, and Vps15p are required for both processes. Coimmunoprecipitation using anti-Apg14p and anti-Vps38p antibodies and pull-down experiments showed that two distinct Vps34 PtdIns 3-kinase complexes exist: one, containing Vps15p, Vps30p, and Apg14p, functions in autophagy and the other containing Vps15p, Vps30p, and Vps38p functions in CPY sorting. The vps34 and vps15 mutants displayed additional phenotypes such as defects in transport of proteinase A and proteinase B, implying the existence of another PtdIns 3-kinase complex(es). We propose that multiple Vps34p-Vps15p complexes associated with specific regulatory proteins might fulfill their membrane trafficking events at different sites.

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