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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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Kinase-defective Vps15p is unable to form Vps30p complexes. AKY109 (Δvps34)/pKHR54 (VPS34) (lane 1), AKY109/pKHR60 (vps34-N736K) (lane 2), AKY115 (Δvps15)/pKHR55 (VPS15) (lane 3), and AKY115/pKHR59 (vps15-E200R) (lane 4) cells were grown to mid-log phase in SC medium lacking uracil at 28°C. Total lysates were solubilized with Triton X-100 and incubated with protein A–immobilized anti-Vps30p antibodies. Bound proteins were washed and eluted with 100 mM glycine-HCl, pH 2.5. Proteins were separated by SDS-PAGE, followed by detection by immunoblotting with anti-Vps30p, anti-Apg14p, anti-Vps34p, anti-Vps15p, and anti-Vps38p antibodies.
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Figure 7: Kinase-defective Vps15p is unable to form Vps30p complexes. AKY109 (Δvps34)/pKHR54 (VPS34) (lane 1), AKY109/pKHR60 (vps34-N736K) (lane 2), AKY115 (Δvps15)/pKHR55 (VPS15) (lane 3), and AKY115/pKHR59 (vps15-E200R) (lane 4) cells were grown to mid-log phase in SC medium lacking uracil at 28°C. Total lysates were solubilized with Triton X-100 and incubated with protein A–immobilized anti-Vps30p antibodies. Bound proteins were washed and eluted with 100 mM glycine-HCl, pH 2.5. Proteins were separated by SDS-PAGE, followed by detection by immunoblotting with anti-Vps30p, anti-Apg14p, anti-Vps34p, anti-Vps15p, and anti-Vps38p antibodies.

Mentions: Stack et al. 1995 reported that Vps15 kinase domain mutants are unable to interact with Vps34p, suggesting that Vps15p autophosphorylation or Vps15p-mediated phosphorylation of Vps34p may be involved in Vps15p–Vps34p complex formation. Alternatively, a Vps34p PtdIns 3–kinase domain mutant was able to associate with Vps15p in a manner indistinguishable from wild-type Vps34p (Stack et al. 1995). We examined the ability of the Vps15 kinase domain mutant and the Vps34 PtdIns 3–kinase domain mutant to form complexes I and II. The glutamic acid at position 200 of Vps15p is a highly conserved residue among protein kinases, and its mutation to arginine abolishes the in vivo phosphorylation of Vps15p, resulting in a temperature-sensitive growth defect and missorting of CPY (Herman et al. 1991a). The asparagine at position 736 of Vps34p is part of the catalytic loop region (DXHXXN) of proteins functioning in ATP binding and phosphate transfer (Knighton et al. 1991) and is conserved among protein kinases and lipid kinases. The N736K mutation results in a dramatic decrease in PtdIns 3–kinase activity and a severe defect in vacuolar protein sorting (Schu et al. 1993). Coimmunoprecipitation experiments with anti-Vps30p antibodies were performed using a Δvps15 strain containing either wild-type or vps15-E200R allele on a low copy plasmid (Fig. 7, lanes 3 and 4). The effect of the kinase-negative vps15 mutant was similar to that of deletion of the VPS15 gene; the amount of Apg14p, Vps34p, and Vps15-E200R, but not Vps38p, precipitated with anti-Vps30p antibodies was severely reduced (Fig. 7, lane 4). Vps15p was not detected in the absence of Vps34p (Fig. 6 B, lane 5). We also found that Vps15-E200R, as well as Apg14p, was not detected in vps15-E200R cells (data not shown). Thus, Vps15p-mediated autophosphorylation of Vps15p or phosphorylation of Vps34p may be required for the interaction between Vps15p and Vps34p and for stabilization of Vps15p. In addition, Vps34p uncomplexed with Vps15p or unphosphorylated form of Vps34p does not complex with Vps30p, that is, does not bind to Vps38p for the reason described above. In contrast, all the interactions were normal in a Δvps34 strain expressing the Vps34-N736K mutant protein (Fig. 7, lane 2). These results suggest that production of PtdIns(3)P is not required for these protein–protein interactions.


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)

Kinase-defective Vps15p is unable to form Vps30p complexes. AKY109 (Δvps34)/pKHR54 (VPS34) (lane 1), AKY109/pKHR60 (vps34-N736K) (lane 2), AKY115 (Δvps15)/pKHR55 (VPS15) (lane 3), and AKY115/pKHR59 (vps15-E200R) (lane 4) cells were grown to mid-log phase in SC medium lacking uracil at 28°C. Total lysates were solubilized with Triton X-100 and incubated with protein A–immobilized anti-Vps30p antibodies. Bound proteins were washed and eluted with 100 mM glycine-HCl, pH 2.5. Proteins were separated by SDS-PAGE, followed by detection by immunoblotting with anti-Vps30p, anti-Apg14p, anti-Vps34p, anti-Vps15p, and anti-Vps38p antibodies.
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Figure 7: Kinase-defective Vps15p is unable to form Vps30p complexes. AKY109 (Δvps34)/pKHR54 (VPS34) (lane 1), AKY109/pKHR60 (vps34-N736K) (lane 2), AKY115 (Δvps15)/pKHR55 (VPS15) (lane 3), and AKY115/pKHR59 (vps15-E200R) (lane 4) cells were grown to mid-log phase in SC medium lacking uracil at 28°C. Total lysates were solubilized with Triton X-100 and incubated with protein A–immobilized anti-Vps30p antibodies. Bound proteins were washed and eluted with 100 mM glycine-HCl, pH 2.5. Proteins were separated by SDS-PAGE, followed by detection by immunoblotting with anti-Vps30p, anti-Apg14p, anti-Vps34p, anti-Vps15p, and anti-Vps38p antibodies.
Mentions: Stack et al. 1995 reported that Vps15 kinase domain mutants are unable to interact with Vps34p, suggesting that Vps15p autophosphorylation or Vps15p-mediated phosphorylation of Vps34p may be involved in Vps15p–Vps34p complex formation. Alternatively, a Vps34p PtdIns 3–kinase domain mutant was able to associate with Vps15p in a manner indistinguishable from wild-type Vps34p (Stack et al. 1995). We examined the ability of the Vps15 kinase domain mutant and the Vps34 PtdIns 3–kinase domain mutant to form complexes I and II. The glutamic acid at position 200 of Vps15p is a highly conserved residue among protein kinases, and its mutation to arginine abolishes the in vivo phosphorylation of Vps15p, resulting in a temperature-sensitive growth defect and missorting of CPY (Herman et al. 1991a). The asparagine at position 736 of Vps34p is part of the catalytic loop region (DXHXXN) of proteins functioning in ATP binding and phosphate transfer (Knighton et al. 1991) and is conserved among protein kinases and lipid kinases. The N736K mutation results in a dramatic decrease in PtdIns 3–kinase activity and a severe defect in vacuolar protein sorting (Schu et al. 1993). Coimmunoprecipitation experiments with anti-Vps30p antibodies were performed using a Δvps15 strain containing either wild-type or vps15-E200R allele on a low copy plasmid (Fig. 7, lanes 3 and 4). The effect of the kinase-negative vps15 mutant was similar to that of deletion of the VPS15 gene; the amount of Apg14p, Vps34p, and Vps15-E200R, but not Vps38p, precipitated with anti-Vps30p antibodies was severely reduced (Fig. 7, lane 4). Vps15p was not detected in the absence of Vps34p (Fig. 6 B, lane 5). We also found that Vps15-E200R, as well as Apg14p, was not detected in vps15-E200R cells (data not shown). Thus, Vps15p-mediated autophosphorylation of Vps15p or phosphorylation of Vps34p may be required for the interaction between Vps15p and Vps34p and for stabilization of Vps15p. In addition, Vps34p uncomplexed with Vps15p or unphosphorylated form of Vps34p does not complex with Vps30p, that is, does not bind to Vps38p for the reason described above. In contrast, all the interactions were normal in a Δvps34 strain expressing the Vps34-N736K mutant protein (Fig. 7, lane 2). These results suggest that production of PtdIns(3)P is not required for these protein–protein interactions.

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.

Show MeSH