PMCCPMCCPMCC

Search tips
Search criteria 

Advanced

 
Logo of jcellbiolHomeThe Rockefeller University PressEditorsContactInstructions for AuthorsThis issue
 
J Cell Biol. 1995 April 2; 129(2): 321–334.
PMCID: PMC2199917

Vesicle-mediated protein transport: regulatory interactions between the Vps15 protein kinase and the Vps34 PtdIns 3-kinase essential for protein sorting to the vacuole in yeast

Abstract

A membrane-associated complex composed of the Vps15 protein kinase and the Vps34 phosphatidylinositol 3-kinase (PtdIns 3-kinase) is essential for the delivery of proteins to the yeast vacuole. An active Vps15p is required for the recruitment of Vps34p to the membrane and subsequent stimulation of Vps34p PtdIns 3-kinase activity. Consistent with this, mutations altering highly conserved residues in the lipid kinase domain of Vps34p lead to a dominant-negative phenotype resulting from titration of activating Vps15 proteins. In contrast, catalytically inactive Vps15p mutants do not produce a dominant mutant phenotype because they are unable to associate with Vps34p in a wild-type manner. These data indicate that an intact Vps15p protein kinase domain is necessary for the association with and activation of Vps34p, and they demonstrate that a functional Vps15p-Vps34p complex is absolutely required for the efficient delivery of proteins to the vacuole. Analysis of a temperature-conditional allele of VPS15, in which a shift to the nonpermissive temperature leads to a decrease in cellular PtdIns(3)P levels, indicates that the loss of Vps15p function leads to a defect in activation of Vps34p. In addition, characterization of a temperature-sensitive allele of VPS34 demonstrates that inactivation of Vps34p leads to the immediate missorting of soluble vacuolar proteins (e.g., carboxypeptidase Y) without an apparent defect in the sorting of the vacuolar membrane protein alkaline phosphatase. This rapid block in vacuolar protein sorting appears to be the result of loss of PtdIns 3- kinase activity since cellular PtdIns(3)P levels decrease dramatically in vps34 temperature-sensitive mutant cells that have been incubated at the nonpermissive temperature. Finally, analysis of the defects in cellular PtdIns(3)P levels in various vps15 and vsp34 mutant strains has led to additional insights into the importance of PtdIns(3)P intracellular localization, as well as the roles of Vps15p and Vps34p in vacuolar protein sorting.

Full Text

The Full Text of this article is available as a PDF (2.1M).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Auger KR, Carpenter CL, Cantley LC, Varticovski L. Phosphatidylinositol 3-kinase and its novel product, phosphatidylinositol 3-phosphate, are present in Saccharomyces cerevisiae. J Biol Chem. 1989 Dec 5;264(34):20181–20184. [PubMed]
  • Auger KR, Serunian LA, Soltoff SP, Libby P, Cantley LC. PDGF-dependent tyrosine phosphorylation stimulates production of novel polyphosphoinositides in intact cells. Cell. 1989 Apr 7;57(1):167–175. [PubMed]
  • Bankaitis VA, Johnson LM, Emr SD. Isolation of yeast mutants defective in protein targeting to the vacuole. Proc Natl Acad Sci U S A. 1986 Dec;83(23):9075–9079. [PubMed]
  • Cantley LC, Auger KR, Carpenter C, Duckworth B, Graziani A, Kapeller R, Soltoff S. Oncogenes and signal transduction. Cell. 1991 Jan 25;64(2):281–302. [PubMed]
  • Carlberg K, Tapley P, Haystead C, Rohrschneider L. The role of kinase activity and the kinase insert region in ligand-induced internalization and degradation of the c-fms protein. EMBO J. 1991 Apr;10(4):877–883. [PubMed]
  • Carpenter CL, Auger KR, Chanudhuri M, Yoakim M, Schaffhausen B, Shoelson S, Cantley LC. Phosphoinositide 3-kinase is activated by phosphopeptides that bind to the SH2 domains of the 85-kDa subunit. J Biol Chem. 1993 May 5;268(13):9478–9483. [PubMed]
  • Carpenter CL, Cantley LC. Phosphoinositide kinases. Biochemistry. 1990 Dec 25;29(51):11147–11156. [PubMed]
  • Cowles CR, Emr SD, Horazdovsky BF. Mutations in the VPS45 gene, a SEC1 homologue, result in vacuolar protein sorting defects and accumulation of membrane vesicles. J Cell Sci. 1994 Dec;107(Pt 12):3449–3459. [PubMed]
  • Downing JR, Roussel MF, Sherr CJ. Ligand and protein kinase C downmodulate the colony-stimulating factor 1 receptor by independent mechanisms. Mol Cell Biol. 1989 Jul;9(7):2890–2896. [PMC free article] [PubMed]
  • Fantl WJ, Escobedo JA, Martin GA, Turck CW, del Rosario M, McCormick F, Williams LT. Distinct phosphotyrosines on a growth factor receptor bind to specific molecules that mediate different signaling pathways. Cell. 1992 May 1;69(3):413–423. [PubMed]
  • Giorgetti S, Ballotti R, Kowalski-Chauvel A, Tartare S, Van Obberghen E. The insulin and insulin-like growth factor-I receptor substrate IRS-1 associates with and activates phosphatidylinositol 3-kinase in vitro. J Biol Chem. 1993 Apr 5;268(10):7358–7364. [PubMed]
  • Hawkins PT, Stephens LR, Piggott JR. Analysis of inositol metabolites produced by Saccharomyces cerevisiae in response to glucose stimulation. J Biol Chem. 1993 Feb 15;268(5):3374–3383. [PubMed]
  • Herman PK, Emr SD. Characterization of VPS34, a gene required for vacuolar protein sorting and vacuole segregation in Saccharomyces cerevisiae. Mol Cell Biol. 1990 Dec;10(12):6742–6754. [PMC free article] [PubMed]
  • Herman PK, Stack JH, DeModena JA, Emr SD. A novel protein kinase homolog essential for protein sorting to the yeast lysosome-like vacuole. Cell. 1991 Jan 25;64(2):425–437. [PubMed]
  • Herskowitz I. Functional inactivation of genes by dominant negative mutations. Nature. 1987 Sep 17;329(6136):219–222. [PubMed]
  • Hiles ID, Otsu M, Volinia S, Fry MJ, Gout I, Dhand R, Panayotou G, Ruiz-Larrea F, Thompson A, Totty NF, et al. Phosphatidylinositol 3-kinase: structure and expression of the 110 kd catalytic subunit. Cell. 1992 Aug 7;70(3):419–429. [PubMed]
  • Horazdovsky BF, Busch GR, Emr SD. VPS21 encodes a rab5-like GTP binding protein that is required for the sorting of yeast vacuolar proteins. EMBO J. 1994 Mar 15;13(6):1297–1309. [PubMed]
  • Ito H, Fukuda Y, Murata K, Kimura A. Transformation of intact yeast cells treated with alkali cations. J Bacteriol. 1983 Jan;153(1):163–168. [PMC free article] [PubMed]
  • Johnson LM, Bankaitis VA, Emr SD. Distinct sequence determinants direct intracellular sorting and modification of a yeast vacuolar protease. Cell. 1987 Mar 13;48(5):875–885. [PubMed]
  • Joly M, Kazlauskas A, Fay FS, Corvera S. Disruption of PDGF receptor trafficking by mutation of its PI-3 kinase binding sites. Science. 1994 Feb 4;263(5147):684–687. [PubMed]
  • Kapeller R, Chakrabarti R, Cantley L, Fay F, Corvera S. Internalization of activated platelet-derived growth factor receptor-phosphatidylinositol-3' kinase complexes: potential interactions with the microtubule cytoskeleton. Mol Cell Biol. 1993 Oct;13(10):6052–6063. [PMC free article] [PubMed]
  • Klionsky DJ, Emr SD. Membrane protein sorting: biosynthesis, transport and processing of yeast vacuolar alkaline phosphatase. EMBO J. 1989 Aug;8(8):2241–2250. [PubMed]
  • Klionsky DJ, Herman PK, Emr SD. The fungal vacuole: composition, function, and biogenesis. Microbiol Rev. 1990 Sep;54(3):266–292. [PMC free article] [PubMed]
  • Kornfeld S. Structure and function of the mannose 6-phosphate/insulinlike growth factor II receptors. Annu Rev Biochem. 1992;61:307–330. [PubMed]
  • Kornfeld S, Mellman I. The biogenesis of lysosomes. Annu Rev Cell Biol. 1989;5:483–525. [PubMed]
  • Marcusson EG, Horazdovsky BF, Cereghino JL, Gharakhanian E, Emr SD. The sorting receptor for yeast vacuolar carboxypeptidase Y is encoded by the VPS10 gene. Cell. 1994 May 20;77(4):579–586. [PubMed]
  • Muhlrad D, Hunter R, Parker R. A rapid method for localized mutagenesis of yeast genes. Yeast. 1992 Feb;8(2):79–82. [PubMed]
  • Paravicini G, Horazdovsky BF, Emr SD. Alternative pathways for the sorting of soluble vacuolar proteins in yeast: a vps35 null mutant missorts and secretes only a subset of vacuolar hydrolases. Mol Biol Cell. 1992 Apr;3(4):415–427. [PMC free article] [PubMed]
  • Pleiman CM, Hertz WM, Cambier JC. Activation of phosphatidylinositol-3' kinase by Src-family kinase SH3 binding to the p85 subunit. Science. 1994 Mar 18;263(5153):1609–1612. [PubMed]
  • Robinson JS, Klionsky DJ, Banta LM, Emr SD. Protein sorting in Saccharomyces cerevisiae: isolation of mutants defective in the delivery and processing of multiple vacuolar hydrolases. Mol Cell Biol. 1988 Nov;8(11):4936–4948. [PMC free article] [PubMed]
  • Rothman JH, Howald I, Stevens TH. Characterization of genes required for protein sorting and vacuolar function in the yeast Saccharomyces cerevisiae. EMBO J. 1989 Jul;8(7):2057–2065. [PubMed]
  • Rothman JH, Stevens TH. Protein sorting in yeast: mutants defective in vacuole biogenesis mislocalize vacuolar proteins into the late secretory pathway. Cell. 1986 Dec 26;47(6):1041–1051. [PubMed]
  • Schu PV, Takegawa K, Fry MJ, Stack JH, Waterfield MD, Emr SD. Phosphatidylinositol 3-kinase encoded by yeast VPS34 gene essential for protein sorting. Science. 1993 Apr 2;260(5104):88–91. [PubMed]
  • Serunian LA, Auger KR, Cantley LC. Identification and quantification of polyphosphoinositides produced in response to platelet-derived growth factor stimulation. Methods Enzymol. 1991;198:78–87. [PubMed]
  • Soltoff SP, Carpenter CL, Auger KR, Kapeller R, Schaffhausen B, Cantley LC. Phosphatidylinositol-3 kinase and growth regulation. Cold Spring Harb Symp Quant Biol. 1992;57:75–80. [PubMed]
  • Stack JH, Emr SD. Vps34p required for yeast vacuolar protein sorting is a multiple specificity kinase that exhibits both protein kinase and phosphatidylinositol-specific PI 3-kinase activities. J Biol Chem. 1994 Dec 16;269(50):31552–31562. [PubMed]
  • Stack JH, Herman PK, Schu PV, Emr SD. A membrane-associated complex containing the Vps15 protein kinase and the Vps34 PI 3-kinase is essential for protein sorting to the yeast lysosome-like vacuole. EMBO J. 1993 May;12(5):2195–2204. [PubMed]
  • Stephens L, Cooke FT, Walters R, Jackson T, Volinia S, Gout I, Waterfield MD, Hawkins PT. Characterization of a phosphatidylinositol-specific phosphoinositide 3-kinase from mammalian cells. Curr Biol. 1994 Mar 1;4(3):203–214. [PubMed]
  • Valls LA, Hunter CP, Rothman JH, Stevens TH. Protein sorting in yeast: the localization determinant of yeast vacuolar carboxypeptidase Y resides in the propeptide. Cell. 1987 Mar 13;48(5):887–897. [PubMed]
  • Valls LA, Winther JR, Stevens TH. Yeast carboxypeptidase Y vacuolar targeting signal is defined by four propeptide amino acids. J Cell Biol. 1990 Aug;111(2):361–368. [PMC free article] [PubMed]
  • Walsh JP, Caldwell KK, Majerus PW. Formation of phosphatidylinositol 3-phosphate by isomerization from phosphatidylinositol 4-phosphate. Proc Natl Acad Sci U S A. 1991 Oct 15;88(20):9184–9187. [PubMed]
  • Whitman M, Downes CP, Keeler M, Keller T, Cantley L. Type I phosphatidylinositol kinase makes a novel inositol phospholipid, phosphatidylinositol-3-phosphate. Nature. 1988 Apr 14;332(6165):644–646. [PubMed]

Articles from The Journal of Cell Biology are provided here courtesy of The Rockefeller University Press