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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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Apg14p and Vps38p exist in distinct complexes. (A and B) AKY73 cells were grown in YPD at 30°C. Total lysates were solubilized with Triton X-100 and incubated with protein A–immobilized anti-Apg14p antibodies (lane 1) or anti-Vps38p antibodies (lane 2). The beads were then separated into two fractions and were subjected to immunoblotting (A) or PtdIns 3–kinase assays (B). In A, bound proteins were eluted from the beads, separated by SDS-PAGE, and detected by immunoblotting with anti-Vps30p, anti-Vps34p, anti-Vps15p, anti-Apg14p, and anti-Vps38p antibodies. In B, labeled lipids were extracted, separated by TLC, and visualized by autoradiography using BAS2000. (C) AKY112/pAUR112 (vector; lane 1) and AKY112/pKHR69 (His6–Myc–APG14; lane 2) cell lysates were solubilized with Triton X-100 and loaded on a Ni-NTA agarose column. Bound proteins were eluted with 250 mM imidazole and were subjected to immunoblotting with anti-Vps30p, anti-Myc (9E10), anti-Vps34p, and anti-Vps38p antibodies.
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Figure 4: Apg14p and Vps38p exist in distinct complexes. (A and B) AKY73 cells were grown in YPD at 30°C. Total lysates were solubilized with Triton X-100 and incubated with protein A–immobilized anti-Apg14p antibodies (lane 1) or anti-Vps38p antibodies (lane 2). The beads were then separated into two fractions and were subjected to immunoblotting (A) or PtdIns 3–kinase assays (B). In A, bound proteins were eluted from the beads, separated by SDS-PAGE, and detected by immunoblotting with anti-Vps30p, anti-Vps34p, anti-Vps15p, anti-Apg14p, and anti-Vps38p antibodies. In B, labeled lipids were extracted, separated by TLC, and visualized by autoradiography using BAS2000. (C) AKY112/pAUR112 (vector; lane 1) and AKY112/pKHR69 (His6–Myc–APG14; lane 2) cell lysates were solubilized with Triton X-100 and loaded on a Ni-NTA agarose column. Bound proteins were eluted with 250 mM imidazole and were subjected to immunoblotting with anti-Vps30p, anti-Myc (9E10), anti-Vps34p, and anti-Vps38p antibodies.

Mentions: As shown above, Apg14p and Vps38p are required for different membrane trafficking pathways. To discriminate whether Apg14p and Vps38p compose a single complex that functions both in autophagy/Cvt and in CPY sorting, or Apg14p and Vps38p compose distinct complexes that function separately, detergent extracts prepared from wild-type cells were subjected to coimmunoprecipitation experiments using anti-Apg14p or anti-Vps38p antibodies. Immunoprecipitates were then visualized by immunoblotting using anti-Vps30p, anti-Vps34p, anti-Vps15p, anti-Apg14p, and anti-Vps38p antibodies (Fig. 4 A). Both Vps30p and Vps34p were coimmunoprecipitated with their respective specific antibodies (Fig. 4 A, lanes 1 and 2). A substantial level of Vps15p was detected in the immunoprecipitates of anti-Vps38p antibodies (Fig. 4 A, lane 2). Anti-Apg14p antibodies precipitated a very low but detectable amount of Vps15p (Fig. 4 A, lane 1). The low level of Vps15p was probably due to instability of Vps15p in cell lysates and low abundance of the Apg14p-containing complex (see below). However, Vps38p could not be detected in the immunoprecipitates obtained using anti-Apg14p antibodies at all (Fig. 4 A, lane 1). Immunoprecipitates obtained using anti-Vps38p antibodies did not contain Apg14p either (Fig. 4 A, lane 2). These results suggest that Vps38p and Apg14p are not present in the same complex. Both complexes possess PtdIns 3–kinase activity, although immunoprecipitates obtained using anti-Apg14p antibodies showed ∼10-fold less activity than anti-Vps38p antibody immunoprecipitates (Fig. 4 B).


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)

Apg14p and Vps38p exist in distinct complexes. (A and B) AKY73 cells were grown in YPD at 30°C. Total lysates were solubilized with Triton X-100 and incubated with protein A–immobilized anti-Apg14p antibodies (lane 1) or anti-Vps38p antibodies (lane 2). The beads were then separated into two fractions and were subjected to immunoblotting (A) or PtdIns 3–kinase assays (B). In A, bound proteins were eluted from the beads, separated by SDS-PAGE, and detected by immunoblotting with anti-Vps30p, anti-Vps34p, anti-Vps15p, anti-Apg14p, and anti-Vps38p antibodies. In B, labeled lipids were extracted, separated by TLC, and visualized by autoradiography using BAS2000. (C) AKY112/pAUR112 (vector; lane 1) and AKY112/pKHR69 (His6–Myc–APG14; lane 2) cell lysates were solubilized with Triton X-100 and loaded on a Ni-NTA agarose column. Bound proteins were eluted with 250 mM imidazole and were subjected to immunoblotting with anti-Vps30p, anti-Myc (9E10), anti-Vps34p, and anti-Vps38p antibodies.
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Figure 4: Apg14p and Vps38p exist in distinct complexes. (A and B) AKY73 cells were grown in YPD at 30°C. Total lysates were solubilized with Triton X-100 and incubated with protein A–immobilized anti-Apg14p antibodies (lane 1) or anti-Vps38p antibodies (lane 2). The beads were then separated into two fractions and were subjected to immunoblotting (A) or PtdIns 3–kinase assays (B). In A, bound proteins were eluted from the beads, separated by SDS-PAGE, and detected by immunoblotting with anti-Vps30p, anti-Vps34p, anti-Vps15p, anti-Apg14p, and anti-Vps38p antibodies. In B, labeled lipids were extracted, separated by TLC, and visualized by autoradiography using BAS2000. (C) AKY112/pAUR112 (vector; lane 1) and AKY112/pKHR69 (His6–Myc–APG14; lane 2) cell lysates were solubilized with Triton X-100 and loaded on a Ni-NTA agarose column. Bound proteins were eluted with 250 mM imidazole and were subjected to immunoblotting with anti-Vps30p, anti-Myc (9E10), anti-Vps34p, and anti-Vps38p antibodies.
Mentions: As shown above, Apg14p and Vps38p are required for different membrane trafficking pathways. To discriminate whether Apg14p and Vps38p compose a single complex that functions both in autophagy/Cvt and in CPY sorting, or Apg14p and Vps38p compose distinct complexes that function separately, detergent extracts prepared from wild-type cells were subjected to coimmunoprecipitation experiments using anti-Apg14p or anti-Vps38p antibodies. Immunoprecipitates were then visualized by immunoblotting using anti-Vps30p, anti-Vps34p, anti-Vps15p, anti-Apg14p, and anti-Vps38p antibodies (Fig. 4 A). Both Vps30p and Vps34p were coimmunoprecipitated with their respective specific antibodies (Fig. 4 A, lanes 1 and 2). A substantial level of Vps15p was detected in the immunoprecipitates of anti-Vps38p antibodies (Fig. 4 A, lane 2). Anti-Apg14p antibodies precipitated a very low but detectable amount of Vps15p (Fig. 4 A, lane 1). The low level of Vps15p was probably due to instability of Vps15p in cell lysates and low abundance of the Apg14p-containing complex (see below). However, Vps38p could not be detected in the immunoprecipitates obtained using anti-Apg14p antibodies at all (Fig. 4 A, lane 1). Immunoprecipitates obtained using anti-Vps38p antibodies did not contain Apg14p either (Fig. 4 A, lane 2). These results suggest that Vps38p and Apg14p are not present in the same complex. Both complexes possess PtdIns 3–kinase activity, although immunoprecipitates obtained using anti-Apg14p antibodies showed ∼10-fold less activity than anti-Vps38p antibody immunoprecipitates (Fig. 4 B).

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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