Two distinct Vps34 phosphatidylinositol 3-kinase complexes function in autophagy and carboxypeptidase Y sorting in Saccharomyces cerevisiae - PubMed (original) (raw)

Two distinct Vps34 phosphatidylinositol 3-kinase complexes function in autophagy and carboxypeptidase Y sorting in Saccharomyces cerevisiae

A Kihara et al. J Cell Biol. 2001.

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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Figures

Figure 1

Purification of Vps30p complexes. (A) AKY73 (wild type; lane 1) and AKY76 (Δ_vps30_; lane 2) cells grown in SC medium lacking methionine at 30°C were labeled with Express™ [35S]methionine/cysteine protein labeling mix (NEN Life Science Products) for 1 h. The labeled cells were converted to spheroplasts and lysed by extrusion through a polycarbonate filter with 3 μm pores. Thus, obtained lysates were solubilized with Triton X-100 and incubated with anti-Vps30p antibodies and protein A–Sepharose beads at 4°C for 2 h. Bound proteins were eluted, separated by SDS-PAGE, and detected by autoradiography using a PhosphorImager BAS2000 (Fuji Film). (B) Protein profiles during the purification of the Vps30p complexes. Total lysates prepared from AKY76 cells harboring pKHR25 (His6–Myc–VPS30; 2 μm) were solubilized with Triton X-100 and subjected to Ni-NTA agarose chromatography (load, lane 1; flow-through, lane 2). Proteins bound to Ni-NTA agarose were washed (lane 3) and eluted with 250 mM imidazole (lane 4). The eluates were then incubated with protein A–immobilized anti-Vps30p antibodies (flow-through, lane 5). The column was washed, and retained proteins were eluted with 100 mM glycine-HCl, pH 2.5 (lane 6; and C, lane 1). Proteins were separated by SDS-PAGE and visualized by Coomassie brilliant blue staining. (C) For control, a purification procedure as described in B was applied to lysates prepared from AKY76 cells harboring an empty pRS424 plasmid (lane 2). Proteins were analyzed by SDS-PAGE and visualized by silver staining. (D) Cells of AKY106 (wild type; lane 1) and AKY111 (Δ_vps30_; lane 2) 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 eluted, separated by SDS-PAGE, and detected by immunoblotting with anti-Vps30p, anti-Vps34p, anti-Vps38p, anti-Apg14p, and anti-Vps15p antibodies.

Figure 2

PtdIns 3–kinase activity in mutants lacking a component of Vps30p complexes. TN125 (wild type; lane 1), AKY15 (Δ_vps30_; lane 2), AKY13 (Δ_apg14_; lane 3), AKY114 (Δ_vps38_; lane 4), AKY109 (Δ_vps34_; lane 5), and AKY115 (Δ_vps15_; 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.

Figure 3

Transport of vacuolar proteins and autophagy. TN125 (wild type), AKY13 (Δ_apg14_), AKY15 (Δ_vps30_), AKY114 (Δ_vps38_), AKY109 (Δ_vps34_), and AKY115 (Δ_vps15_) cells were grown in SC medium lacking methionine (A) or in YPD (B–D) at 28°C. (A) Yeast cells were labeled with [35S]methionine/cysteine for 15 min and chased with unlabeled methionine and cysteine for 30 min. The labeled cells were converted to spheroplasts and separated into pellet (I, intracellular) and supernatant (E, extracellular) fractions. CPY was immunoprecipitated and visualized by autoradiography using BAS2000. (B–D) Total protein was separated by SDS-PAGE and detected by immunoblotting with anti-PrA (B), anti-Pr B (C), and anti-API (D). (E) Cells were grown in YPD (open bars) and shifted to SD(-N) medium for 6 h (filled bars) at 28°C. Lysates from each group of cells were subjected to the ALP assay (Noda and Ohsumi 1998) to measure autophagy activity. (F) KVY4 (Δ_ypt7_; lanes 1–3) and AKY131 (Δ_vps34_; lanes 4–6) cells grown in YPD to a log phase were transferred to SD(-N), and incubated for 4.5 h at 30°C. Total lysates were centrifuged at 13,000 g for 15 min. The pellets were treated with or without Triton X-100 and/or proteinase K as indicated on ice for 30 min. The samples were TCA-precipitated and subjected to immunoblotting with anti-API antibodies. pAPI*, digested pAPI fragment.

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.

Figure 5

Coimmunoprecipitaion with Vps30p. AKY112 (Δ_apg14_; lane 1), AKY113 (Δ_vps38_; lane 2), AKY110 (Δ_vps34_; lane 3), and AKY116 (Δ_vps15_; 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.

Figure 6

Cellular amount of components of Vps30p complexes. AKY106 (wild type; lane 1), AKY111 (Δ_vps30_; lane 2), AKY112 (Δ_apg14_; lane 3), AKY113 (Δ_vps38_; lane 4), AKY110 (Δ_vps34_; lane 5), and AKY126 (Δ_vps15_; lane 6) cells were grown at 28°C. Total cellular protein was separated by SDS-PAGE for the detection of Vps30p (A), Vps34p (B), and Vps15p (C) by immunoblotting. For detection of Apg14p and Vps38p, pKHR75 expressing His6–Myc–Apg14p or pKHR65 expressing Vps38-3xHA was introduced into the above respective strains. Total protein prepared from them was separated by SDS-PAGE and detected by immunoblotting using anti-Myc (9E10) (D) or anti-HA (16B12) (E) antibodies.

Figure 7

Kinase-defective Vps15p is unable to form Vps30p complexes. AKY109 (Δ_vps34_)/pKHR54 (VPS34) (lane 1), AKY109/pKHR60 (vps34-N736K) (lane 2), AKY115 (Δ_vps15_)/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.

Figure 8

Subcellular fractionation of Vps30p, Vps38p, and Vps34p. AKY106 (wild type), AKY110 (Δ_vps34_), AKY111 (Δ_vps30_), AKY112 (Δ_apg14_), and AKY126 (Δ_vps15_) cells, each bearing pKHR65 (_VPS38-3xHA), and AKY113 (Δ_vps38) cells bearing pRS313 (empty vector) were grown in SC without histidine at 28°C. Cell lysates were subjected to subcellular fractionation by differential centrifugation as described in Materials and Methods. Total cell lysate (lane 1), LSP (lane 2), HSP (lane 3), and HSS (lane 4) fractions were subjected to SDS-PAGE, followed by immunoblotting with anti-Vps30p (A), anti-HA (16B12) (B), anti-Vps34p (C), or anti-Pho8p (D) antibodies. Relative amounts of each fraction were indicated. The values in A and C were averages of three independent experiments.

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.

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Two distinct Vps34 phosphatidylinositol 3-kinase complexes function in autophagy and carboxypeptidase Y sorting in Saccharomyces cerevisiae - PubMed (original) (raw)