Limits...
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.

Show MeSH
Coimmunoprecipitaion with Vps30p. AKY112 (Δapg14; lane 1), AKY113 (Δvps38; lane 2), AKY110 (Δvps34; lane 3), and AKY116 (Δvps15; lane 4) cells 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 washed, eluted with 100 mM glycine-HCl, pH 2.5, precipitated with 5% TCA, and suspended in SDS sample buffer. Proteins were separated by SDS-PAGE and detected by immunoblotting with anti-Vps30p (A), anti-Apg14p (B), anti-Vps34p (C), anti-Vps15p (D), and anti-Vps38p (E) antibodies.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2196002&req=5

Figure 5: Coimmunoprecipitaion with Vps30p. AKY112 (Δapg14; lane 1), AKY113 (Δvps38; lane 2), AKY110 (Δvps34; lane 3), and AKY116 (Δvps15; lane 4) cells 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 washed, eluted with 100 mM glycine-HCl, pH 2.5, precipitated with 5% TCA, and suspended in SDS sample buffer. Proteins were separated by SDS-PAGE and detected by immunoblotting with anti-Vps30p (A), anti-Apg14p (B), anti-Vps34p (C), anti-Vps15p (D), and anti-Vps38p (E) antibodies.

Mentions: Previous studies suggested that Vps15p directly phosphorylates Vps34p (Stack et al. 1993). These proteins appear to form the cores of complexes I and II. To address the organization of complexes I and II, the coimmunoprecipitation experiments using anti-Vps30p antibodies, as shown in Fig. 1 D, were performed on Δapg14, Δvps38, Δvps34, and Δvps15 cells. Consistent with the results that Apg14p and Vps38p compose separate complexes, the interaction between Vps30p and Apg14p was not affected by deletion of VPS38 (Fig. 5 D, lane 2), and deletion of APG14 did not affect the Vps30p–Vps38p interaction (Fig. 5 E, lane 1). We also investigated their cellular amounts in the respective deletion strains by immunoblotting. The amounts of Apg14p and Vps38p were not changed compared with wild-type cells in Δvps38 (Fig. 6 D, lane 4) or Δapg14 (Fig. 6 E, lane 3) cells, respectively. The Vps30p–Vps38p interaction and the cellular amount of Vps38p were unchanged in Δvps34 and Δvps15 cells (Fig. 5 E, lanes 3 and 4; Fig. 6 E, lanes 5 and 6). However, Vps38p was not detected in Δvps30 cells (Fig. 6 E, lane 2). These results suggest that Vps30p directly binds to Vps38p and protects Vps38p from degradation. In contrast, although both Vps15p and Vps34p were present in Δvps38 cells (Fig. 6 B, lane 4; Fig. 6 C, lane 4), they could not interact with Vps30p in the absence of Vps38p (Fig. 5 B, lane 2; Fig. 5 C, lane 2). These results imply that the interaction between Vps30p and the Vps34p–Vps15p core complex is indirect and is mediated by Vps38p. Apg14p was barely detected in Δvps30, Δvps34, and Δvps15 cells (Fig. 6 D, lanes 2, 5, and 6). Accordingly, a reduced amount of Apg14p was coimmunoprecipitated with Vps30p in Δvps34 cells (Fig. 5 D, lane 3), and Apg14p was not detected in the immunoprecipitates from Δvps15 cells (Fig. 5 D, lane 4). These results suggest that direct binding of Vps30p and Vps34p–Vps15p to Apg14p may protect Apg14p from proteolysis by hindering recognition sites against proteases (proteolytic systems) or by inducing Apg14p to assume a tight conformation. Deletion of APG14 appears not to affect the interaction between Vps30p and Vps34p–Vps15p (Fig. 5 B, lane 1; Fig. 5 C, lane 1), although deletion of VPS38 leads to dramatic effects on it (Fig. 5 B, lane 2; Fig. 5 C, lane 2). These results suggest that complex I is much less abundant than complex II in yeast cells. In fact, the total amount of Apg14p is very low (data not shown).


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)

Coimmunoprecipitaion with Vps30p. AKY112 (Δapg14; lane 1), AKY113 (Δvps38; lane 2), AKY110 (Δvps34; lane 3), and AKY116 (Δvps15; lane 4) cells 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 washed, eluted with 100 mM glycine-HCl, pH 2.5, precipitated with 5% TCA, and suspended in SDS sample buffer. Proteins were separated by SDS-PAGE and detected by immunoblotting with anti-Vps30p (A), anti-Apg14p (B), anti-Vps34p (C), anti-Vps15p (D), and anti-Vps38p (E) antibodies.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2196002&req=5

Figure 5: Coimmunoprecipitaion with Vps30p. AKY112 (Δapg14; lane 1), AKY113 (Δvps38; lane 2), AKY110 (Δvps34; lane 3), and AKY116 (Δvps15; lane 4) cells 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 washed, eluted with 100 mM glycine-HCl, pH 2.5, precipitated with 5% TCA, and suspended in SDS sample buffer. Proteins were separated by SDS-PAGE and detected by immunoblotting with anti-Vps30p (A), anti-Apg14p (B), anti-Vps34p (C), anti-Vps15p (D), and anti-Vps38p (E) antibodies.
Mentions: Previous studies suggested that Vps15p directly phosphorylates Vps34p (Stack et al. 1993). These proteins appear to form the cores of complexes I and II. To address the organization of complexes I and II, the coimmunoprecipitation experiments using anti-Vps30p antibodies, as shown in Fig. 1 D, were performed on Δapg14, Δvps38, Δvps34, and Δvps15 cells. Consistent with the results that Apg14p and Vps38p compose separate complexes, the interaction between Vps30p and Apg14p was not affected by deletion of VPS38 (Fig. 5 D, lane 2), and deletion of APG14 did not affect the Vps30p–Vps38p interaction (Fig. 5 E, lane 1). We also investigated their cellular amounts in the respective deletion strains by immunoblotting. The amounts of Apg14p and Vps38p were not changed compared with wild-type cells in Δvps38 (Fig. 6 D, lane 4) or Δapg14 (Fig. 6 E, lane 3) cells, respectively. The Vps30p–Vps38p interaction and the cellular amount of Vps38p were unchanged in Δvps34 and Δvps15 cells (Fig. 5 E, lanes 3 and 4; Fig. 6 E, lanes 5 and 6). However, Vps38p was not detected in Δvps30 cells (Fig. 6 E, lane 2). These results suggest that Vps30p directly binds to Vps38p and protects Vps38p from degradation. In contrast, although both Vps15p and Vps34p were present in Δvps38 cells (Fig. 6 B, lane 4; Fig. 6 C, lane 4), they could not interact with Vps30p in the absence of Vps38p (Fig. 5 B, lane 2; Fig. 5 C, lane 2). These results imply that the interaction between Vps30p and the Vps34p–Vps15p core complex is indirect and is mediated by Vps38p. Apg14p was barely detected in Δvps30, Δvps34, and Δvps15 cells (Fig. 6 D, lanes 2, 5, and 6). Accordingly, a reduced amount of Apg14p was coimmunoprecipitated with Vps30p in Δvps34 cells (Fig. 5 D, lane 3), and Apg14p was not detected in the immunoprecipitates from Δvps15 cells (Fig. 5 D, lane 4). These results suggest that direct binding of Vps30p and Vps34p–Vps15p to Apg14p may protect Apg14p from proteolysis by hindering recognition sites against proteases (proteolytic systems) or by inducing Apg14p to assume a tight conformation. Deletion of APG14 appears not to affect the interaction between Vps30p and Vps34p–Vps15p (Fig. 5 B, lane 1; Fig. 5 C, lane 1), although deletion of VPS38 leads to dramatic effects on it (Fig. 5 B, lane 2; Fig. 5 C, lane 2). These results suggest that complex I is much less abundant than complex II in yeast cells. In fact, the total amount of Apg14p is very low (data not shown).

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