title: Vac14 protein multimerization is a prerequisite step for Fab1 protein complex assembly and function.
abstract: Phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P2) helps control various endolysosome functions including organelle morphology, membrane recycling, and ion transport. Further highlighting its importance, PtdIns(3,5)P2 misregulation leads to the development of neurodegenerative diseases like Charcot-Marie-Tooth disease. The Fab1/PIKfyve lipid kinase phosphorylates PtdIns(3)P into PtdIns(3,5)P2 whereas the Fig4/Sac3 lipid phosphatase antagonizes this reaction. Interestingly, Fab1 and Fig4 form a common protein complex that coordinates synthesis and degradation of PtdIns(3,5)P2 by a poorly understood process. Assembly of the Fab1 complex requires Vac14/ArPIKfyve, a multimeric scaffolding adaptor protein that coordinates synthesis and turnover of PtdIns(3,5)P2. However, the properties and function of Vac14 multimerization remain mostly uncharacterized. Here we identify several conserved C-terminal motifs on Vac14 required for self-interaction and provide evidence that Vac14 likely forms a dimer. We also show that monomeric Vac14 mutants do not support interaction with Fab1 or Fig4, suggesting that Vac14 multimerization is likely the first molecular event in the assembly of the Fab1 complex. Finally, we show that cells expressing monomeric Vac14 mutants have enlarged vacuoles that do not fragment after hyperosmotic shock, which indicates that PtdIns(3,5)P2 levels are greatly abated. Therefore, our observations support an essential role for the Vac14 homocomplex in controlling PtdIns(3,5)P2 levels.
authors: Department of Chemistry and Biology and the Molecular Science Program, Ryerson University, Toronto, Ontario, Canada M5B 2K3.
authors: Tamadher A
authors: TA
authors: Alghamdi
authors: Cheuk Y
authors: CY
authors: Ho
authors: Amra
authors: A
authors: Mrakovic
authors: Danielle
authors: D
authors: Taylor
authors: Daniel
authors: D
authors: Mao
authors: Roberto J
authors: RJ
authors: Botelho
mesh_terms: D020816:Amino Acid Motifs; D002853:Chromatography, Liquid / Q000379:methods; D019281:Dimerization; D015966:Gene Expression Regulation, Fungal; D008565:Membrane Proteins / Q000378:metabolism*; D009154:Mutation; D019636:Neurodegenerative Diseases / Q000378:metabolism; D018129:Phosphatidylinositol Phosphates / Q000737:chemistry; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000378:metabolism*; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D055503:Protein Multimerization; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*; D014617:Vacuoles / Q000378:metabolism
chemical_list: D008565:Membrane Proteins; D018129:Phosphatidylinositol Phosphates; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; C452435:VAC14 protein, S cerevisiae; C106336:phosphatidylinositol 3,5-diphosphate; C095504:FAB1 protein, S cerevisiae; D017853:Phosphotransferases (Alcohol Group Acceptor)
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