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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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Transport of vacuolar proteins and autophagy. TN125 (wild type), AKY13 (Δapg14), AKY15 (Δvps30), AKY114 (Δvps38), AKY109 (Δvps34), and AKY115 (Δvps15) 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 (Δypt7; lanes 1–3) and AKY131 (Δvps34; 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.
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Figure 3: Transport of vacuolar proteins and autophagy. TN125 (wild type), AKY13 (Δapg14), AKY15 (Δvps30), AKY114 (Δvps38), AKY109 (Δvps34), and AKY115 (Δvps15) 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 (Δypt7; lanes 1–3) and AKY131 (Δvps34; 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.

Mentions: We examined the effects of disruption of either VPS30, APG14, VPS38, VPS34, or VPS15 on several vacuolar trafficking pathways, including transport of CPY, proteinase A (PrA), proteinase B (PrB) and API, and autophagy. Transports of CPY, PrA, and PrB are initiated at the ER membrane, where they are translocated into lumen of the ER, and delivered by vesicular transport to the vacuole via the Golgi apparatus. The transport of API is completely different from that of CPY, PrA, and PrB. API is synthesized as a proform (pAPI) in the cytoplasm and targets directly to the vacuole via the Cvt pathway, where it is processed to the mature form (mAPI). The Cvt pathway uses a similar mechanism as the autophagic pathway, and most autophagy mutants are defective in the Cvt pathway (Harding et al. 1996; Scott et al. 1996; Baba et al. 1997). First, we examined transport of CPY by pulse–chase and immunoprecipitation experiments. Cells were pulse labeled with [35S]methionine/cysteine for 15 min and chased for 30 min at 28°C. The cells were then converted to spheroplasts and separated into intracellular (I) and extracellular (E) fractions, from which CPY was immunoprecipitated. In wild-type and Δapg14 cells, >95% of the newly synthesized CPY was present as a mature form (mCPY) in an intracellular fraction (Fig. 3 A, lanes 1 and 3). Δvps30, Δvps38, Δvps34, and Δvps15 cells missorted and secreted virtually all CPY as the Golgi-modified p2 form (Fig. 3 A, lanes 6, 8, 10, and 12). These results indicate that all Vps proteins examined (Vps15p, Vps34p, Vps38p, and Vps30p/Apg6p), but not Apg14p, are required for proper sorting of CPY, as reported previously (Robinson et al. 1988; Herman and Emr 1990; Herman et al. 1991a; Luo and Chang 1997; Seaman et al. 1997; Kametaka et al. 1998). Transport of PrA, PrB, and API was examined by immunoblotting. Δvps15 and Δvps34 cells accumulated Golgi forms of PrA (pPrA) and PrB (pPrB) (Fig. 3B and Fig. C, lanes 4 and 5), as shown previously (Robinson et al. 1988), indicating that sorting of PrA and PrB is severely impaired in these cells. Alternatively, in Δvps38 and Δvps30 cells, most PrA and PrB were found as mature forms (mPrA and mPrB), although very low levels of precursor forms were detected as well (Fig. 3B and Fig. C, lanes 2 and 6). Δapg14 cells exhibited normal sorting of PrA and PrB (Fig. 3B and Fig. C, lane 3). Transport of API was completely inhibited in Δvps30, Δapg14, Δvps34, and Δvps15 cells (Fig. 3 D, lanes 2–5), whereas Δvps38 cells showed normal targeting of API (Fig. 3 D, lane 6). We next examined autophagy using an ALP assay (Noda et al. 1995), which monitors autophagy-dependent processing of ALP Pho8Δ60 (Noda et al. 1995). In wild-type cells, the ALP activity increased in response to starvation, whereas in Δvps30, Δapg14, Δvps34, and Δvps15 cells its elevation was severely inhibited (Fig. 3 E). The ALP activity of Δvps38 cells was ∼70% of the activity of wild-type cells. These results indicate that Δvps30, Δapg14, Δvps34, and Δvps15 cells are defective both in the Cvt pathway and in the autophagy pathway, whereas in Δvps38 cells both pathways are nearly intact. Table summarizes these transport activities in the mutant cells. We can thus classify the mutants into 4 classes. Class I, which includes Δvps15 and Δvps34, display the most severe phenotype: all transport of vacuolar proteins examined is inhibited, and their growth is slow at 28°C and arrested at 37°C. Δvps30 (class II) is defective in both autophagy/Cvt and CPY sorting. However, Δapg14 (class III) and Δvps38 (class IV) are defective only in autophagy/Cvt or CPY sorting, respectively. These results suggest that Vps30p, Apg14p, and Vps38p may have regulatory roles, enabling the Vps34 PtdIns 3–kinase to perform specific functions.


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)

Transport of vacuolar proteins and autophagy. TN125 (wild type), AKY13 (Δapg14), AKY15 (Δvps30), AKY114 (Δvps38), AKY109 (Δvps34), and AKY115 (Δvps15) 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 (Δypt7; lanes 1–3) and AKY131 (Δvps34; 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.
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Figure 3: Transport of vacuolar proteins and autophagy. TN125 (wild type), AKY13 (Δapg14), AKY15 (Δvps30), AKY114 (Δvps38), AKY109 (Δvps34), and AKY115 (Δvps15) 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 (Δypt7; lanes 1–3) and AKY131 (Δvps34; 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.
Mentions: We examined the effects of disruption of either VPS30, APG14, VPS38, VPS34, or VPS15 on several vacuolar trafficking pathways, including transport of CPY, proteinase A (PrA), proteinase B (PrB) and API, and autophagy. Transports of CPY, PrA, and PrB are initiated at the ER membrane, where they are translocated into lumen of the ER, and delivered by vesicular transport to the vacuole via the Golgi apparatus. The transport of API is completely different from that of CPY, PrA, and PrB. API is synthesized as a proform (pAPI) in the cytoplasm and targets directly to the vacuole via the Cvt pathway, where it is processed to the mature form (mAPI). The Cvt pathway uses a similar mechanism as the autophagic pathway, and most autophagy mutants are defective in the Cvt pathway (Harding et al. 1996; Scott et al. 1996; Baba et al. 1997). First, we examined transport of CPY by pulse–chase and immunoprecipitation experiments. Cells were pulse labeled with [35S]methionine/cysteine for 15 min and chased for 30 min at 28°C. The cells were then converted to spheroplasts and separated into intracellular (I) and extracellular (E) fractions, from which CPY was immunoprecipitated. In wild-type and Δapg14 cells, >95% of the newly synthesized CPY was present as a mature form (mCPY) in an intracellular fraction (Fig. 3 A, lanes 1 and 3). Δvps30, Δvps38, Δvps34, and Δvps15 cells missorted and secreted virtually all CPY as the Golgi-modified p2 form (Fig. 3 A, lanes 6, 8, 10, and 12). These results indicate that all Vps proteins examined (Vps15p, Vps34p, Vps38p, and Vps30p/Apg6p), but not Apg14p, are required for proper sorting of CPY, as reported previously (Robinson et al. 1988; Herman and Emr 1990; Herman et al. 1991a; Luo and Chang 1997; Seaman et al. 1997; Kametaka et al. 1998). Transport of PrA, PrB, and API was examined by immunoblotting. Δvps15 and Δvps34 cells accumulated Golgi forms of PrA (pPrA) and PrB (pPrB) (Fig. 3B and Fig. C, lanes 4 and 5), as shown previously (Robinson et al. 1988), indicating that sorting of PrA and PrB is severely impaired in these cells. Alternatively, in Δvps38 and Δvps30 cells, most PrA and PrB were found as mature forms (mPrA and mPrB), although very low levels of precursor forms were detected as well (Fig. 3B and Fig. C, lanes 2 and 6). Δapg14 cells exhibited normal sorting of PrA and PrB (Fig. 3B and Fig. C, lane 3). Transport of API was completely inhibited in Δvps30, Δapg14, Δvps34, and Δvps15 cells (Fig. 3 D, lanes 2–5), whereas Δvps38 cells showed normal targeting of API (Fig. 3 D, lane 6). We next examined autophagy using an ALP assay (Noda et al. 1995), which monitors autophagy-dependent processing of ALP Pho8Δ60 (Noda et al. 1995). In wild-type cells, the ALP activity increased in response to starvation, whereas in Δvps30, Δapg14, Δvps34, and Δvps15 cells its elevation was severely inhibited (Fig. 3 E). The ALP activity of Δvps38 cells was ∼70% of the activity of wild-type cells. These results indicate that Δvps30, Δapg14, Δvps34, and Δvps15 cells are defective both in the Cvt pathway and in the autophagy pathway, whereas in Δvps38 cells both pathways are nearly intact. Table summarizes these transport activities in the mutant cells. We can thus classify the mutants into 4 classes. Class I, which includes Δvps15 and Δvps34, display the most severe phenotype: all transport of vacuolar proteins examined is inhibited, and their growth is slow at 28°C and arrested at 37°C. Δvps30 (class II) is defective in both autophagy/Cvt and CPY sorting. However, Δapg14 (class III) and Δvps38 (class IV) are defective only in autophagy/Cvt or CPY sorting, respectively. These results suggest that Vps30p, Apg14p, and Vps38p may have regulatory roles, enabling the Vps34 PtdIns 3–kinase to perform specific functions.

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