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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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Subcellular fractionation of Vps30p, Vps38p, and Vps34p. AKY106 (wild type), AKY110 (Δvps34), AKY111 (Δvps30), AKY112 (Δapg14), and AKY126 (Δvps15) cells, each bearing pKHR65 (VPS38-3xHA), and AKY113 (Δvps38) cells bearing pRS313 (empty vector) were grown in SC without histidine at 28°C. Cell lysates were subjected to subcellular fractionation by differential centrifugation as described in Materials and Methods. Total cell lysate (lane 1), LSP (lane 2), HSP (lane 3), and HSS (lane 4) fractions were subjected to SDS-PAGE, followed by immunoblotting with anti-Vps30p (A), anti-HA (16B12) (B), anti-Vps34p (C), or anti-Pho8p (D) antibodies. Relative amounts of each fraction were indicated. The values in A and C were averages of three independent experiments.
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Figure 8: Subcellular fractionation of Vps30p, Vps38p, and Vps34p. AKY106 (wild type), AKY110 (Δvps34), AKY111 (Δvps30), AKY112 (Δapg14), and AKY126 (Δvps15) cells, each bearing pKHR65 (VPS38-3xHA), and AKY113 (Δvps38) cells bearing pRS313 (empty vector) were grown in SC without histidine at 28°C. Cell lysates were subjected to subcellular fractionation by differential centrifugation as described in Materials and Methods. Total cell lysate (lane 1), LSP (lane 2), HSP (lane 3), and HSS (lane 4) fractions were subjected to SDS-PAGE, followed by immunoblotting with anti-Vps30p (A), anti-HA (16B12) (B), anti-Vps34p (C), or anti-Pho8p (D) antibodies. Relative amounts of each fraction were indicated. The values in A and C were averages of three independent experiments.

Mentions: Although Vps30p has no apparent transmembrane domains or sites for lipid modification, Vps30p was found in the membrane fraction, in addition to the soluble fraction (Seaman et al. 1997). Membrane-associated Vps30p could be solubilized by salt or urea (Seaman et al. 1997; Kametaka et al. 1998). It is possible that the membrane association of Vps30p is mediated by a protein–protein interaction, with Vps15p, Vps34p, Vps38p, and Apg14p being candidates for a postulated membrane anchor. Therefore, we examined the subcellular location of Vps30p in these mutant cells. Total cell lysates were separated by sequential centrifugation at 13,000 and 100,000 g into LSP, HSP, and HSS fractions. In wild-type cells, most Vps30p was found in the LSP (60%) and HSS (34%), whereas only 6% of Vps30p was separated to the HSP (Fig. 8 A). A dramatic shift of Vps30p into the HSS fraction was observed in Δvps38, Δvps34, and Δvps15 cells (Fig. 8 A). These results indicated that Vps15p, Vps34p, and Vps38p are required for recruitment of Vps30p to the membrane. Again, the absence of Apg14p had no effect on the subcellular location of Vps30p (Fig. 8 A), probably because complex I is in low abundance. Control experiments showed that ALP, a vacuolar membrane protein, was mainly localized to the LSP fraction (Fig. 8 D) and alcohol dehydrogenase was localized to the HSS fraction exclusively (data not shown) in all mutants tested. We also examined the subcellular distribution of Vps38p. The distribution pattern of Vps38p in wild-type cells was similar to that of Vps30p: 58, 8, and 34% of Vps38p was found in the LSP, HSP, and HSS, respectively (Fig. 8 B). We could not determine the subcellular location of Vps38p in Δvps30 cells because Vps38p was scarcely detected in the absence of Vps30p (data not shown; Fig. 6 E, lane 2). The pattern was not changed in Δapg14 cells (Fig. 8 B). Redistribution of Vps38p into the HSS fraction was observed both in Δvps34 and Δvps15 cells (Fig. 8 B). These results indicate that Vps38p is released to the cytoplasm together with Vps30p in Δvps34 and Δvps15 cells. A previous study indicated that Vps34p exists in both membrane and soluble fractions and that Vps15p is required for the membrane association of Vps34p (Stack et al. 1993). In our assay conditions, most Vps34p was found in the LSP (57%) and HSP (35%), and only 8% of Vps34p was present in the HSS fraction in wild-type cells (Fig. 8 C). In Δvps15 cells, 35% of Vps34p was released into the HSS fraction, whereas about half of Vps34p was detected in the HSP fraction (Fig. 8 C). Distribution of Vps34p was not changed in Δapg14 cells (Fig. 8 C). In Δvps30 and Δvps38 cells, some shift of Vps34p to the HSP and HSS fractions was observed (Fig. 8 C). These results indicate that complex II resides on membranes in the LSP fraction, and disruption of the complex causes redistribution of Vps34p to the HSP membranes and the cytoplasm and a shift of Vps30p–Vps38p to the cytoplasm.


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)

Subcellular fractionation of Vps30p, Vps38p, and Vps34p. AKY106 (wild type), AKY110 (Δvps34), AKY111 (Δvps30), AKY112 (Δapg14), and AKY126 (Δvps15) cells, each bearing pKHR65 (VPS38-3xHA), and AKY113 (Δvps38) cells bearing pRS313 (empty vector) were grown in SC without histidine at 28°C. Cell lysates were subjected to subcellular fractionation by differential centrifugation as described in Materials and Methods. Total cell lysate (lane 1), LSP (lane 2), HSP (lane 3), and HSS (lane 4) fractions were subjected to SDS-PAGE, followed by immunoblotting with anti-Vps30p (A), anti-HA (16B12) (B), anti-Vps34p (C), or anti-Pho8p (D) antibodies. Relative amounts of each fraction were indicated. The values in A and C were averages of three independent experiments.
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Figure 8: Subcellular fractionation of Vps30p, Vps38p, and Vps34p. AKY106 (wild type), AKY110 (Δvps34), AKY111 (Δvps30), AKY112 (Δapg14), and AKY126 (Δvps15) cells, each bearing pKHR65 (VPS38-3xHA), and AKY113 (Δvps38) cells bearing pRS313 (empty vector) were grown in SC without histidine at 28°C. Cell lysates were subjected to subcellular fractionation by differential centrifugation as described in Materials and Methods. Total cell lysate (lane 1), LSP (lane 2), HSP (lane 3), and HSS (lane 4) fractions were subjected to SDS-PAGE, followed by immunoblotting with anti-Vps30p (A), anti-HA (16B12) (B), anti-Vps34p (C), or anti-Pho8p (D) antibodies. Relative amounts of each fraction were indicated. The values in A and C were averages of three independent experiments.
Mentions: Although Vps30p has no apparent transmembrane domains or sites for lipid modification, Vps30p was found in the membrane fraction, in addition to the soluble fraction (Seaman et al. 1997). Membrane-associated Vps30p could be solubilized by salt or urea (Seaman et al. 1997; Kametaka et al. 1998). It is possible that the membrane association of Vps30p is mediated by a protein–protein interaction, with Vps15p, Vps34p, Vps38p, and Apg14p being candidates for a postulated membrane anchor. Therefore, we examined the subcellular location of Vps30p in these mutant cells. Total cell lysates were separated by sequential centrifugation at 13,000 and 100,000 g into LSP, HSP, and HSS fractions. In wild-type cells, most Vps30p was found in the LSP (60%) and HSS (34%), whereas only 6% of Vps30p was separated to the HSP (Fig. 8 A). A dramatic shift of Vps30p into the HSS fraction was observed in Δvps38, Δvps34, and Δvps15 cells (Fig. 8 A). These results indicated that Vps15p, Vps34p, and Vps38p are required for recruitment of Vps30p to the membrane. Again, the absence of Apg14p had no effect on the subcellular location of Vps30p (Fig. 8 A), probably because complex I is in low abundance. Control experiments showed that ALP, a vacuolar membrane protein, was mainly localized to the LSP fraction (Fig. 8 D) and alcohol dehydrogenase was localized to the HSS fraction exclusively (data not shown) in all mutants tested. We also examined the subcellular distribution of Vps38p. The distribution pattern of Vps38p in wild-type cells was similar to that of Vps30p: 58, 8, and 34% of Vps38p was found in the LSP, HSP, and HSS, respectively (Fig. 8 B). We could not determine the subcellular location of Vps38p in Δvps30 cells because Vps38p was scarcely detected in the absence of Vps30p (data not shown; Fig. 6 E, lane 2). The pattern was not changed in Δapg14 cells (Fig. 8 B). Redistribution of Vps38p into the HSS fraction was observed both in Δvps34 and Δvps15 cells (Fig. 8 B). These results indicate that Vps38p is released to the cytoplasm together with Vps30p in Δvps34 and Δvps15 cells. A previous study indicated that Vps34p exists in both membrane and soluble fractions and that Vps15p is required for the membrane association of Vps34p (Stack et al. 1993). In our assay conditions, most Vps34p was found in the LSP (57%) and HSP (35%), and only 8% of Vps34p was present in the HSS fraction in wild-type cells (Fig. 8 C). In Δvps15 cells, 35% of Vps34p was released into the HSS fraction, whereas about half of Vps34p was detected in the HSP fraction (Fig. 8 C). Distribution of Vps34p was not changed in Δapg14 cells (Fig. 8 C). In Δvps30 and Δvps38 cells, some shift of Vps34p to the HSP and HSS fractions was observed (Fig. 8 C). These results indicate that complex II resides on membranes in the LSP fraction, and disruption of the complex causes redistribution of Vps34p to the HSP membranes and the cytoplasm and a shift of Vps30p–Vps38p to the cytoplasm.

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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