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

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 endocytic structures, suggesting a role for PIKfyve in endosome- to- trans- Golgi network ( TGN) membrane retrieval. Here we report that p40, a Rab9 effector reported previously to bind Rab9- GTP and stimulate endosome- to- TGN transport, interacts with PIKfyve as determined by yeast two- hybrid assays, glutathione S- transferase ( GST) pull- down assays, and co- immunoprecipitation in doubly transfected HEK293 cells. The interaction engages the PIKfyve chaperonin domain and four out of the six C- terminally positioned kelch repeats in p40. Differential centrifugation in a HEK293 cell line, stably expressing PIKfyve(WT), showed the membrane-associated immunoreactive p40 co- sedimenting with PIKfyve in the high speed pellet ( HSP) fraction. Remarkably, similar analysis in a HEK293 cell line stably expressing dominant- negative kinase- deficient PIKfyve(K1831E) demonstrated a marked depletion of p40 from the HSP fraction. GST- p40 failed to specifically associate with the PIKfyve lipid products PtdIns 5- P and PtdIns 3,5- P-2 in a liposome binding assay but was found to be an in vitro substrate of the PIKfyve serine kinase activity. A band with the p40 electrophoretic mobility was found to react with a phosphoserine- specific antibody mainly in the PIKfyve(WT)- containing fractions obtained by density gradient sedimentation of total membranes from PIKfyve(WT) -expressing HEK293 cells. Together these results identify the Rab9 effector p40 as a PIKfyve partner and suggest that p40- PIKfyve interaction and the subsequent PIKfyve-catalyzed p40 phosphorylation anchor p40 to discrete membranes facilitating late endosome- to- TGN transport.