American Society for Biochemistry and Molecular Biology, Molecular and Cellular Proteomics, 6(13), p. 1397-1411
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 (PI3P) to phosphatidylinositol-3,5-bisphosphate (PI(3,5)P2). Dysfunctional Vac14 mutants, deficiency of one of the Vac14 complex components or inhibition of PIKfyve enzymatic activity result in the formation of large vacuoles in cells. How these vacuoles are generated and which processes are involved is only poorly understood. Here we show that ectopic overexpression of wildtype 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 are bound to the membrane of these enlarged vacuoles. To decipher the linkage between the Vac14 complex and regulators of the endolysosomal pathway, a protein affinity approach combined with the multidimensional protein identification technology (MudPIT) was conducted and unraveled novel molecular links. 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 regulation of vesicular transport processes as well as phosphatidylinositol modifying enzymes are tightly interconnected.