Active PIKfyve associates with and promotes the membrane attachment of the late endosome-to-trans-Golgi network transport factor Rab9 effector p40

摘要: PIKfyve, a kinase that displays specificity for phosphatidylinositol (PtdIns), PtdIns 3-phosphate (3-P), and proteins, is important in multivesicular body/late endocytic function. Enzymatically inactive PIKfyve mutants elicit enormous dilation of late structures, suggesting role endosome-to-trans-Golgi network (TGN) membrane retrieval. Here we report p40, Rab9 effector reported previously to bind Rab9-GTP stimulate endosome-to-TGN transport, interacts with as determined by yeast two-hybrid assays, glutathione S-transferase (GST) pull-down co-immunoprecipitation doubly transfected HEK293 cells. The interaction engages the chaperonin domain four out six C-terminally positioned kelch repeats p40. Differential centrifugation cell line, stably expressing PIKfyveWT, showed membrane-associated immunoreactive p40 co-sedimenting high speed pellet (HSP) fraction. Remarkably, similar analysis line dominant-negative kinase-deficient PIKfyveK1831E demonstrated marked depletion from HSP GST-p40 failed specifically associate lipid products 5-P 3,5-P2 liposome binding assay but was found be an vitro substrate serine activity. A band electrophoretic mobility react phosphoserine-specific antibody mainly PIKfyveWT-containing fractions obtained density gradient sedimentation total membranes PIKfyveWT-expressing Together these results identify partner suggest p40-PIKfyve subsequent PIKfyve-catalyzed phosphorylation anchor discrete facilitating transport.