Active PIKfyve Associates with and Promotes the Membrane Attachment of the Late Endosome-to-trans-Golgi Network Transport Factor Rab9 Effector p40

Abstract

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 membraneassociated 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.

Core Protein Machinery for Mammalian Phosphatidylinositol 3,5-Bisphosphate Synthesis and Turnover That Regulates the Progression of Endosomal Transport

“…In addition, in mammalian cells PIKfyve protein depletion was found to impair the early endosome-to-TGN traffic and proper endosome processing of GLUT4-containing vesicles in response to insulin (20,49,63). These data, together with the unconditional gross enlargement of mammalian early endosomes observed under these conditions, are consistent with a rate-limiting function of the PIKfyve-catalyzed PtdIns(3)P-to-PtdIns(3,5)P 2 conversion in the course of cargo transport and exit from early endosomes to later destinations, as we have suggested previously (21,22,48). In this study we provide compelling mechanistic evidence that deregulated PtdIns(3,5)P 2 homeostasis underlies the traffic arrest at early endosomes.…”

“…Total membrane fractions resuspended under HES ϩϩ buffer were mixed with iodixanol (OptiPrep; Sigma) in a Quick-Seal centrifuge tube to 30% iodixanol and 128 mM sucrose. A self-generating gradient was performed by centrifugation to equilibrium as specified previously (38,48). Fractions, collected from the bottom of the tube, were analyzed for protein concentration and immunoblotted with the indicated antibodies.…”

“…Stable HEK293 (TetOn) cell lines, inducibly expressing PIKfyve WT (clone 9) or PIKfyve K1831E (clone 5), were generated and maintained as described previously (44). HEK293, COS7, and PC12 cells were cultured under conditions described in our previous studies (38,43,44,48). BHK21 cells were maintained in Glasgow minimum essential medium, supplemented with 5% fetal bovine serum, 10% tryptose phosphate broth, and 1 mM glutamine as described (30).…”

“…Subcellular Fractionation and Equilibrium Centrifugation in Iodixanol Gradient-HEK293 stable cells induced to express PIKfyve WT were homogenized in HES ϩϩ buffer at 4°C and fractionated into total membranes and cytosols as described previously (38,48). Total membrane fractions resuspended under HES ϩϩ buffer were mixed with iodixanol (OptiPrep; Sigma) in a Quick-Seal centrifuge tube to 30% iodixanol and 128 mM sucrose.…”

Kinesin Adapter JLP Links PIKfyve to Microtubule-based Endosome-to-Trans-Golgi Network Traffic of Furin

“…Thus, PIKfyve has been found to interact with the late endosome-to-TGN transport factor Rab9 effector p40 (11). Furthermore, disruption of the PtdIns(3,5)P 2 homeostatic mechanism by means of expres-sion of dominant-negative kinase-deficient point mutants of PIKfyve, protein depletion, or pharmacological inhibition of PIKfyve activity was found to impair the exit of a subset of cargoes from early endosomes to the TGN and late endosomes or from the late endosomes (12)(13)(14)(15)(16).…”