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Mol Cell Proteomics. 2014 Jun;13(6):1397-411. doi: 10.1074/mcp.M113.034108. Epub 2014 Feb 27.

The Vac14-interaction network is linked to regulators of the endolysosomal and autophagic pathway.

Author information

1
From the ‡Department of Internal Medicine D, Molecular Nephrology, University Hospital of Muenster, Albert-Schweitzer Campus 1, A14, D-48149 Muenster, Germany;
2
‖Analytical Chemistry NC4/72, Biomolecular Mass Spectrometry/Proteincenter, Ruhr-University Bochum, Universitätsstr. 150, D-44801 Bochum, Germany;
3
‡‡Institute of Plant Biology and Biotechnology, University of Muenster, Schlossplatz 8, D-48143 Muenster, Germany.
4
From the ‡Department of Internal Medicine D, Molecular Nephrology, University Hospital of Muenster, Albert-Schweitzer Campus 1, A14, D-48149 Muenster, Germany; Hermann.Pavenstaedt@ukmuenster.de weidet@uni-muenster.de.

Abstract

The scaffold protein Vac14 acts in a complex with the lipid kinase PIKfyve and its counteracting phosphatase FIG4, regulating the interconversion of phosphatidylinositol-3-phosphate to phosphatidylinositol-3,5-bisphosphate. Dysfunctional Vac14 mutants, a deficiency of one of the Vac14 complex components, or inhibition of PIKfyve enzymatic activity results in the formation of large vacuoles in cells. How these vacuoles are generated and which processes are involved are only poorly understood. Here we show that ectopic overexpression of wild-type Vac14 as well as of the PIKfyve-binding deficient Vac14 L156R mutant causes vacuoles. Vac14-dependent vacuoles and PIKfyve inhibitor-dependent vacuoles resulted in elevated levels of late endosomal, lysosomal, and autophagy-associated proteins. However, only late endosomal marker proteins were bound to the membranes of these enlarged vacuoles. In order to decipher the linkage between the Vac14 complex and regulators of the endolysosomal pathway, a protein affinity approach combined with multidimensional protein identification technology was conducted, and novel molecular links were unraveled. We found and verified the interaction of Rab9 and the Rab7 GAP TBC1D15 with Vac14. The identified Rab-related interaction partners support the theory that the regulation of vesicular transport processes and phosphatidylinositol-modifying enzymes are tightly interconnected.

PMID:
24578385
PMCID:
PMC4047462
DOI:
10.1074/mcp.M113.034108
[Indexed for MEDLINE]
Free PMC Article

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