ArPIKfyve Homomeric and Heteromeric Interactions Scaffold PIKfyve and Sac3 in a Complex to Promote PIKfyve Activity and Functionality

摘要: PtdIns(3,5)P(2) (with PtdIns indicating phosphatidylinositol) is vital in the differentiation and development of multicellular organisms because knockout PtdIns(3,5)P(2)-synthesizing enzyme PIKfyve (phosphoinositide kinase for position 5 containing a FYVE finger domain) or its associated regulator ArPIKfyve lethal. In previous work with endogenous proteins, we identified that Sac3, phosphatase turns over PtdIns(3,5)P(2), associates PIKfyve-ArPIKfyve biosynthetic complex. However, whether three proteins suffice organization/maintenance this complex [referred to as PAS (PIKfyve-ArPIKfyve-Sac3) complex], how they interact one another, what functional relevance ternary association would be remained unresolved. Using co-immunoprecipitation analyses transfected mammalian cells increased decreased levels singly double versus triple combinations, herein report triad sufficient form maintain principal organizer interacting both Sac3 PIKfyve, whereas permissive maximal We further scaffolds through homomeric interactions, mediated via conserved C-terminal domain. Introduction peptide fragment ArPIKfyve-ArPIKfyve contact sites effectively disassembled reduced vitro lipid activity. Exploring insulin-regulated GLUT4 translocation 3T3L1 adipocytes readout, process positively regulated by activity levels, determined ectopic expression inhibits surface accumulation. Our data indicate organized provide optimal functionality maintained heteromeric interactions.