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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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Model for two distinct PtdIns 3–kinase complexes. Vps15p is anchored to membrane by myristic acid attached to the NH2 terminus of Vps15p (Herman et al. 1991b). Apg14p and Vps38p act as connectors between Vps30p and Vps34p. Phosphorylation of Vps34p by Vps15p is required for Vps34p–Vps15p and Vps34p–Apg14p/Vps38p interactions. White thick lines indicate sites of the interactions essential for the in vivo protein stabilization.
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Figure 9: Model for two distinct PtdIns 3–kinase complexes. Vps15p is anchored to membrane by myristic acid attached to the NH2 terminus of Vps15p (Herman et al. 1991b). Apg14p and Vps38p act as connectors between Vps30p and Vps34p. Phosphorylation of Vps34p by Vps15p is required for Vps34p–Vps15p and Vps34p–Apg14p/Vps38p interactions. White thick lines indicate sites of the interactions essential for the in vivo protein stabilization.

Mentions: Vps38p could be coimmunoprecipitated with Vps30p in the absence of other factors (Fig. 5 E). Only Vps30p is required for stabilization of Vps38p (Fig. 6 E). These results suggest that Vps38p binds directly to Vps30p (Fig. 9). In contrast, although Vps15p and Vps34p were present in the Δvps38 mutant (Fig. 6B and Fig. C), they could not be coprecipitated with Vps30p (Fig. 5B and Fig. C). Therefore, it seems that the interaction between Vps30p and the Vps34p–Vps15p core is not direct but mediated by Vps38p in complex II (Fig. 9). Apg14p is unstable in Δvps30, Δvps15, and Δvps34 cells. These results suggest that both Vps30p and Vps15p–Vps34p may directly bind to Apg14p and conceal recognition sites for proteases (proteolytic systems) in Apg14p or induce Apg14p to adopt a protease-resistant conformation. Thus, Apg14p and Vps38p may act as connectors between Vps30p and Vps15p–Vps34p in complexes I and II, respectively (Fig. 9). However, deletion of APG14 appeared to have no effects on the interaction between Vps30p and Vps15p–Vps34p (Fig. 5B and Fig. C) and on the subcellular distribution of Vps30p (Fig. 8 A) and Vps34p (Fig. 8 C), whereas deletion of VPS38 had dramatic effects on them (Fig. 5B and Fig. C; Fig. 8A and Fig. C). These results suggest that complex I may represent only a minor population of PtdIns 3–kinase. In fact, the overall cellular amount of Apg14p is very low; we estimated that wild-type yeast cells contain ∼15-fold less Apg14p than Vps30p (data not shown). Moreover, the PtdIns 3–kinase activity of complex I was lower by ∼10-fold than that of complex II (Fig. 4 B). Therefore, the effects of the absence of Apg14p might be hidden by the abundant complex II. Although Apg14p and Vps38p have no significant sequence similarities, PairCoil (Berger et al. 1995) predicted that both proteins have potential coiled coil structures, which often mediate 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)

Model for two distinct PtdIns 3–kinase complexes. Vps15p is anchored to membrane by myristic acid attached to the NH2 terminus of Vps15p (Herman et al. 1991b). Apg14p and Vps38p act as connectors between Vps30p and Vps34p. Phosphorylation of Vps34p by Vps15p is required for Vps34p–Vps15p and Vps34p–Apg14p/Vps38p interactions. White thick lines indicate sites of the interactions essential for the in vivo protein stabilization.
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Related In: Results  -  Collection

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Figure 9: Model for two distinct PtdIns 3–kinase complexes. Vps15p is anchored to membrane by myristic acid attached to the NH2 terminus of Vps15p (Herman et al. 1991b). Apg14p and Vps38p act as connectors between Vps30p and Vps34p. Phosphorylation of Vps34p by Vps15p is required for Vps34p–Vps15p and Vps34p–Apg14p/Vps38p interactions. White thick lines indicate sites of the interactions essential for the in vivo protein stabilization.
Mentions: Vps38p could be coimmunoprecipitated with Vps30p in the absence of other factors (Fig. 5 E). Only Vps30p is required for stabilization of Vps38p (Fig. 6 E). These results suggest that Vps38p binds directly to Vps30p (Fig. 9). In contrast, although Vps15p and Vps34p were present in the Δvps38 mutant (Fig. 6B and Fig. C), they could not be coprecipitated with Vps30p (Fig. 5B and Fig. C). Therefore, it seems that the interaction between Vps30p and the Vps34p–Vps15p core is not direct but mediated by Vps38p in complex II (Fig. 9). Apg14p is unstable in Δvps30, Δvps15, and Δvps34 cells. These results suggest that both Vps30p and Vps15p–Vps34p may directly bind to Apg14p and conceal recognition sites for proteases (proteolytic systems) in Apg14p or induce Apg14p to adopt a protease-resistant conformation. Thus, Apg14p and Vps38p may act as connectors between Vps30p and Vps15p–Vps34p in complexes I and II, respectively (Fig. 9). However, deletion of APG14 appeared to have no effects on the interaction between Vps30p and Vps15p–Vps34p (Fig. 5B and Fig. C) and on the subcellular distribution of Vps30p (Fig. 8 A) and Vps34p (Fig. 8 C), whereas deletion of VPS38 had dramatic effects on them (Fig. 5B and Fig. C; Fig. 8A and Fig. C). These results suggest that complex I may represent only a minor population of PtdIns 3–kinase. In fact, the overall cellular amount of Apg14p is very low; we estimated that wild-type yeast cells contain ∼15-fold less Apg14p than Vps30p (data not shown). Moreover, the PtdIns 3–kinase activity of complex I was lower by ∼10-fold than that of complex II (Fig. 4 B). Therefore, the effects of the absence of Apg14p might be hidden by the abundant complex II. Although Apg14p and Vps38p have no significant sequence similarities, PairCoil (Berger et al. 1995) predicted that both proteins have potential coiled coil structures, which often mediate 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