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© The Rockefeller University Press,
0021-9525/2001//519 $5.00
The Journal of Cell Biology, Volume 152, Number 3,
, 2001 519-530
Original Article |
Two Distinct Vps34 Phosphatidylinositol 3–Kinase Complexes Function in Autophagy and Carboxypeptidase Y Sorting inSaccharomyces cerevisiae
yohsumi{at}nibb.ac.jp
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.
Key Words: Phosphatidylinositol 3–kinase • autophagy • Vps34p • Vps30p/Apg6p • CPY sorting
© 2001 The Rockefeller University Press
N. Ishihara's present address is Department of Molecular Biology, Graduate School of Medical Science, Kyushu University, Maidashi Fukuoka, 812-8582, Japan.Abbreviations used in this paper: 2-ME, 2-mercaptoethanol; ALP, alkaline phosphatase; API, aminopeptidase I; CPY, carboxypeptidase Y; Cvt, cytoplasm-to-vacuole targeting; HSP, high speed pellet; HSS, high speed supernatant; LSP, low speed pellet; PrA, proteinase A; PrB, proteinase B; PtdIns, phosphatidylinositol; PtdIns(3)P, PtdIns 3–phosphate; PVC, prevacuolar compartment; SC, synthetic complete.
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