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Vac14 multimerization is required for Fab1 complex assembly and function
Author(s) -
Ho Shan Cheuk Ying,
Alghamdi Tamadher,
Botelho Roberto J
Publication year - 2013
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.27.1_supplement.1019.5
Subject(s) - immunoprecipitation , function (biology) , phosphatase , yeast , biology , mutant , plasma protein binding , mutagenesis , microbiology and biotechnology , phosphorylation , biochemistry , gene
Phosphatidylinositol‐3,5‐bisphosphate (PtdIns3,5P 2 ) is a poorly understood regulator of endolysosome function. In yeast, PtdIns3,5P 2 is synthesized by the Fab1 PtdIns3P 5‐kinase and degraded by the antagonistic Fig4 phosphatase. Interestingly, Fab1 and Fig4 exist in a common protein complex containing Vac14. Vac14 is a self‐interacting protein necessary for Fab1 complex assembly. However, the exact function of Vac14 self‐interaction is not known, which we explore here. Size‐exclusion chromatography and velocity ultracentrifugation suggest that the Vac14 multimer is non‐globular and exists as a homodimer and/or homotrimer. Moreover, using sequential Vac14 truncations, site‐directed mutagenesis and co‐immunoprecipitation studies with both yeast and recombinant proteins, we identified several motifs in the C‐terminal half of Vac14 that are necessary for self‐interaction. Monomeric Vac14 mutants do not support interaction with Fab1 and Fig4, and consequently, did not rescue phenotypic defects in vac14Δ . Together, these results suggest that Vac14 multimerization is necessary and is the first step in the formation of the Fab1 complex and the synthesis of PtdIns3,5P 2 . Funded by NSERC.