title: The Vac14-interaction network is linked to regulators of the endolysosomal and autophagic pathway.
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.
authors.affiliation: From the ‡Department of Internal Medicine D, Molecular Nephrology, University Hospital of Muenster, Albert-Schweitzer Campus 1, A14, D-48149 Muenster, Germany;
authors.forename: Ulf
authors.initials: U
authors.lastname: Schulze
authors.affiliation: From the ‡Department of Internal Medicine D, Molecular Nephrology, University Hospital of Muenster, Albert-Schweitzer Campus 1, A14, D-48149 Muenster, Germany;
authors.forename: Beate
authors.initials: B
authors.lastname: Vollenbröker
authors.affiliation: From the ‡Department of Internal Medicine D, Molecular Nephrology, University Hospital of Muenster, Albert-Schweitzer Campus 1, A14, D-48149 Muenster, Germany;
authors.forename: Daniela A
authors.initials: DA
authors.lastname: Braun
authors.affiliation: From the ‡Department of Internal Medicine D, Molecular Nephrology, University Hospital of Muenster, Albert-Schweitzer Campus 1, A14, D-48149 Muenster, Germany;
authors.forename: Truc
authors.initials: T
authors.lastname: Van Le
authors.affiliation: From the ‡Department of Internal Medicine D, Molecular Nephrology, University Hospital of Muenster, Albert-Schweitzer Campus 1, A14, D-48149 Muenster, Germany;
authors.forename: Daniel
authors.initials: D
authors.lastname: Granado
authors.affiliation: From the ‡Department of Internal Medicine D, Molecular Nephrology, University Hospital of Muenster, Albert-Schweitzer Campus 1, A14, D-48149 Muenster, Germany;
authors.forename: Joachim
authors.initials: J
authors.lastname: Kremerskothen
authors.affiliation: ‖Analytical Chemistry NC4/72, Biomolecular Mass Spectrometry/Proteincenter, Ruhr-University Bochum, Universitätsstr. 150, D-44801 Bochum, Germany;
authors.forename: Benjamin
authors.initials: B
authors.lastname: Fränzel
authors.affiliation: ‖Analytical Chemistry NC4/72, Biomolecular Mass Spectrometry/Proteincenter, Ruhr-University Bochum, Universitätsstr. 150, D-44801 Bochum, Germany;
authors.forename: Rafael
authors.initials: R
authors.lastname: Klosowski
authors.affiliation: ‡‡Institute of Plant Biology and Biotechnology, University of Muenster, Schlossplatz 8, D-48143 Muenster, Germany.
authors.forename: Johannes
authors.initials: J
authors.lastname: Barth
authors.affiliation: ‡‡Institute of Plant Biology and Biotechnology, University of Muenster, Schlossplatz 8, D-48143 Muenster, Germany.
authors.forename: Christian
authors.initials: C
authors.lastname: Fufezan
authors.affiliation: ‖Analytical Chemistry NC4/72, Biomolecular Mass Spectrometry/Proteincenter, Ruhr-University Bochum, Universitätsstr. 150, D-44801 Bochum, Germany;
authors.forename: Dirk A
authors.initials: DA
authors.lastname: Wolters
authors.affiliation: 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.
authors.forename: Hermann
authors.initials: H
authors.lastname: Pavenstädt
authors.affiliation: 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.
authors.forename: Thomas
authors.initials: T
authors.lastname: Weide
mesh_terms: D001343:Autophagy / Q000235:genetics*; D011992:Endosomes / Q000378:metabolism*; D005420:Flavoproteins / Q000378:metabolism; D005786:Gene Expression Regulation; D057809:HEK293 Cells; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D008247:Lysosomes / Q000378:metabolism*; D008565:Membrane Proteins / Q000096:biosynthesis* / Q000235:genetics; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism; D060066:Protein Interaction Maps / Q000235:genetics; D040901:Proteomics; D015398:Signal Transduction; D020691:rab GTP-Binding Proteins / Q000096:biosynthesis / Q000378:metabolism; D000090802:rab7 GTP-Binding Proteins
chemical_list: D005420:Flavoproteins; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D019869:Phosphatidylinositol 3-Kinases; D010744:Phosphoric Monoester Hydrolases; D020691:rab GTP-Binding Proteins; D000090802:rab7 GTP-Binding Proteins; C494631:VAC14 protein, human; C000716463:rab7 GTP-binding proteins, human; C492139:PIKFYVE protein, human; C522215:FIG4 protein, human; C079856:RAB9A protein, human