A Protein Kinase/Lipid Kinase Complex Required for Yeast Vacuolar Protein Sorting

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.

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Vps34p required for yeast vacuolar protein sorting is a multiple specificity kinase that exhibits both protein kinase and phosphatidylinositol-specific PI 3-kinase activities.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(18)31729-0 ◽

1994 ◽

Vol 269 (50) ◽

pp. 31552-31562

Author(s):

J H Stack ◽

S D Emr

Keyword(s):

Protein Kinase ◽

Protein Sorting ◽

Vacuolar Protein Sorting ◽

Vacuolar Protein

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Faculty Opinions recommendation of Mycobacterium tuberculosis virulence is mediated by PtpA dephosphorylation of human vacuolar protein sorting 33B.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1108938.567055 ◽

2008 ◽

Author(s):

Vojo Deretic

Keyword(s):

Mycobacterium Tuberculosis ◽

Protein Sorting ◽

Vacuolar Protein Sorting ◽

Vacuolar Protein

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Faculty Opinions recommendation of Mycobacterium tuberculosis virulence is mediated by PtpA dephosphorylation of human vacuolar protein sorting 33B.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1108938.566090 ◽

2008 ◽

Author(s):

Lalita Ramakrishnan

Keyword(s):

Mycobacterium Tuberculosis ◽

Protein Sorting ◽

Vacuolar Protein Sorting ◽

Vacuolar Protein

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An Interorganellar Bidding War: Vps13 Localization by Competitive Organelle-Specific Adaptors

Contact ◽

10.1177/2515256418814621 ◽

2018 ◽

Vol 1 ◽

pp. 251525641881462

Author(s):

Samantha K. Dziurdzik ◽

Björn D.M. Bean ◽

Elizabeth Conibear

Keyword(s):

Endoplasmic Reticulum ◽

Recent Work ◽

Protein Sorting ◽

Repeat Region ◽

Vacuolar Protein Sorting ◽

Membrane Contact Sites ◽

Contact Sites ◽

Prospore Membrane ◽

Membrane Contact ◽

Vacuolar Protein

Membrane contact sites are regulated through the controlled recruitment of constituent proteins. Yeast vacuolar protein sorting 13 (Vps13) dynamically localizes to membrane contact sites at endosomes, vacuoles, mitochondria, and the endoplasmic reticulum under different cellular conditions and is recruited to the prospore membrane during meiosis. Prior to our recent work, the mechanism for localization at contact sites was largely unknown. We identified Ypt35 as a novel Vps13 adaptor for endosomes and the nucleus-vacuole junction. Furthermore, we discovered a conserved recruitment motif in Ypt35 and found related motifs in the prospore membrane and mitochondrial adaptors, Spo71 and Mcp1, respectively. All three adaptors compete for binding to a six-repeat region of Vps13, suggesting adaptor competition regulates Vps13 localization. Here, we summarize and discuss the implications of our work, highlighting key outstanding questions.

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