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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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PtdIns 3–kinase activity in mutants lacking a component of Vps30p complexes. TN125 (wild type; lane 1), AKY15 (Δvps30; lane 2), AKY13 (Δapg14; lane 3), AKY114 (Δvps38; lane 4), AKY109 (Δvps34; lane 5), and AKY115 (Δvps15; lane 6) cells were grown in YPD medium at 28°C. Total lysates were incubated with soybean PtdIns, [γ-32P]ATP, and 60 μM cold ATP in buffer L (20 mM Hepes-NaOH, pH 7.5, 10 mM MgCl2) for 5 min at 25°C. Lipids were extracted using chloroform–methanol, and samples were separated on Silica gel 60 TLC plates (Merck) with the borate system (Walsh et al. 1991) and detected by autoradiography using BAS2000.
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Figure 2: PtdIns 3–kinase activity in mutants lacking a component of Vps30p complexes. TN125 (wild type; lane 1), AKY15 (Δvps30; lane 2), AKY13 (Δapg14; lane 3), AKY114 (Δvps38; lane 4), AKY109 (Δvps34; lane 5), and AKY115 (Δvps15; lane 6) cells were grown in YPD medium at 28°C. Total lysates were incubated with soybean PtdIns, [γ-32P]ATP, and 60 μM cold ATP in buffer L (20 mM Hepes-NaOH, pH 7.5, 10 mM MgCl2) for 5 min at 25°C. Lipids were extracted using chloroform–methanol, and samples were separated on Silica gel 60 TLC plates (Merck) with the borate system (Walsh et al. 1991) and detected by autoradiography using BAS2000.

Mentions: Vps15 serine/threonine kinase is known to be essential for activation of Vps34p (Stack et al. 1993). One possible role of Vps30p, Apg14p, or Vps38p is a coactivator of Vps34p. To test this, total lysates prepared from each of the deletion strains were assayed for PtdIns 3–kinase activity by incubating with PtdIns and [γ-32P]ATP. Lipids were extracted and separated by TLC on Silica gel 60 plates (Walsh et al. 1991). Wild-type cells produced both PtdIns 4–phosphate, which was synthesized by PtdIns 4–kinases such as Stt4p and Pik1p, and PtdIns 3–phosphate [PtdIns(3)P] (Fig. 2, lane 1). As shown previously (Schu et al. 1993; Stack et al. 1993), Δvps34 cells have no PtdIns 3–kinase activity at all (Fig. 2, lane 5), and Δvps15 cells exhibit a very low but detectable level of PtdIns 3–kinase activity (Fig. 2, lane 6). Δvps30 and Δvps38 cells showed only a slight decrease in PtdIns 3–kinase activity (∼80% of wild-type cells) (Fig. 2, lanes 2 and 4). Δapg14 cells exhibited an equivalent level of PtdIns 3–kinase activity to wild-type cells (Fig. 2, lane 3). These results indicate that Vps30p, Apg14p, and Vps38p are not essential for the PtdIns 3–kinase activity of Vps34p.


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)

PtdIns 3–kinase activity in mutants lacking a component of Vps30p complexes. TN125 (wild type; lane 1), AKY15 (Δvps30; lane 2), AKY13 (Δapg14; lane 3), AKY114 (Δvps38; lane 4), AKY109 (Δvps34; lane 5), and AKY115 (Δvps15; lane 6) cells were grown in YPD medium at 28°C. Total lysates were incubated with soybean PtdIns, [γ-32P]ATP, and 60 μM cold ATP in buffer L (20 mM Hepes-NaOH, pH 7.5, 10 mM MgCl2) for 5 min at 25°C. Lipids were extracted using chloroform–methanol, and samples were separated on Silica gel 60 TLC plates (Merck) with the borate system (Walsh et al. 1991) and detected by autoradiography using BAS2000.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2196002&req=5

Figure 2: PtdIns 3–kinase activity in mutants lacking a component of Vps30p complexes. TN125 (wild type; lane 1), AKY15 (Δvps30; lane 2), AKY13 (Δapg14; lane 3), AKY114 (Δvps38; lane 4), AKY109 (Δvps34; lane 5), and AKY115 (Δvps15; lane 6) cells were grown in YPD medium at 28°C. Total lysates were incubated with soybean PtdIns, [γ-32P]ATP, and 60 μM cold ATP in buffer L (20 mM Hepes-NaOH, pH 7.5, 10 mM MgCl2) for 5 min at 25°C. Lipids were extracted using chloroform–methanol, and samples were separated on Silica gel 60 TLC plates (Merck) with the borate system (Walsh et al. 1991) and detected by autoradiography using BAS2000.
Mentions: Vps15 serine/threonine kinase is known to be essential for activation of Vps34p (Stack et al. 1993). One possible role of Vps30p, Apg14p, or Vps38p is a coactivator of Vps34p. To test this, total lysates prepared from each of the deletion strains were assayed for PtdIns 3–kinase activity by incubating with PtdIns and [γ-32P]ATP. Lipids were extracted and separated by TLC on Silica gel 60 plates (Walsh et al. 1991). Wild-type cells produced both PtdIns 4–phosphate, which was synthesized by PtdIns 4–kinases such as Stt4p and Pik1p, and PtdIns 3–phosphate [PtdIns(3)P] (Fig. 2, lane 1). As shown previously (Schu et al. 1993; Stack et al. 1993), Δvps34 cells have no PtdIns 3–kinase activity at all (Fig. 2, lane 5), and Δvps15 cells exhibit a very low but detectable level of PtdIns 3–kinase activity (Fig. 2, lane 6). Δvps30 and Δvps38 cells showed only a slight decrease in PtdIns 3–kinase activity (∼80% of wild-type cells) (Fig. 2, lanes 2 and 4). Δapg14 cells exhibited an equivalent level of PtdIns 3–kinase activity to wild-type cells (Fig. 2, lane 3). These results indicate that Vps30p, Apg14p, and Vps38p are not essential for the PtdIns 3–kinase activity of Vps34p.

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