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.

The mammalian phosphatidylinositol (3,5)-bisphosphate (PtdIns(3,5)P 2 ) phosphatase Sac3 and ArPIKfyve, the associated regulator of the PtdIns3P-5 kinase PIKfyve, form a stable binary complex that associates with PIKfyve in a ternary complex to increase PtdIns(3,5)P 2 production. I41T rapid loss. Together, our data indentify a novel regulatory mechanism whereby ArPIKfyve enhances Sac3 abundance by attenuating Sac3 proteasome-dependent degradation and suggest that a failure of this mechanism could be the primary molecular defect in the pathogenesis of CMT4J.The phosphatase Sac3 and the kinase PIKfyve 2 are responsible for synthesis and turnover of phosphatidylinositol (PtdIns) (3,5)P 2 in mammalian cells (1). Intriguingly, both the endogenous and the ectopically expressed enzymes are found to reside in the same regulatory complex, called the PAS complex (for PIKfyve-ArPIKfyve-Sac3), organized by the PIKfyve regulator ArPIKfyve and its ability to homooligomerize (2, 3). Despite their antagonistic activities, PIKfyve and Sac3 appear to be enzymatically active in the ternary complex. Thus, in the case of PIKfyve, formation of the PAS regulatory core is critical for PIKfyve activation (3). Likewise, the Sac3 phosphatase retains its PtdIns(3,5)P 2 -hydrolyzing activity within the PAS ternary complex (4). These data reveal an unusual paradigm whereby a common complex relays two opposing activities, one for synthesis, another for degradation, the physiological meaning of which is yet to be understood (1, 5). Data from in vitro reconstitution studies indicate increased and decreased PtdIns(3,5)P 2 levels triggering mammalian endosome fission and fusion, respectively (2, 6). Thus, an association of the two active yet antagonistic enzymes in a common complex would be consistent with the critical requirement for a tight control of PtdIns(3,5)P 2 homeostasis related to dynamic endosome membrane remodeling through fission and fusion (1). Understanding the spatial and temporal regulation and the coordination of the individual enzyme activities within the PAS complex is essential in providing a better comprehension of the intricate PtdIns(3,5)P 2 homeostatic mechanism. Maintaining PtdIns(3,5)P 2 homeostasis is apparently indispensable for life as evidenced by the early lethality of Drosophila melanogaster or Caenorhabditis elegans PIKfyve-null mutants (7,8) and by the early death of ArPIKfyve and Sac3-deficient mouse models (9, 10). Concordantly, a defective Sac3 I41T allele in combination with a null allele is responsible for the pathogenesis of Charcot-Marie-Tooth type 4J (CMT4J) peripheral neuropathy, a recessively inherited disease with early onset manifested by progressive motor and sensory neuron degeneration (10, 11). The molecular and cellular mechanisms rendering this single I41T amino acid substitution pathogenic are currently unknown.ArPIKfyve associates with the Sac3 phosphatase independently of PIKfyve in a stable ArPIKfyve-Sac3 heterooligomer (3,4). This binary association is apparently a prerequisite for a produc...

In mammalian cells, the endosomal/endocytic system comprises an interconnected and morphologically complex network of membrane organelles that supports fundamental functions such as nutrient entry and delivery for degradation, removal and degradation of plasma membrane or Golgi proteins, regulation and integration of signaling pathways, and protein recycling to the cell surface or the TGN 2 (1-4). From the plasma membrane, the endocytosed cargo is first delivered to early endosomes/sorting endosomes. Cargoes destined for recycling to the cell surface then enter the endocytic recycling compartment, whereas others, intended for degradation, remain in early endosomes. Early endosomes undergo a series of changes, known as maturation, to give rise to maturing transport intermediates (herein ECV/MVBs; also Ref. 5) and to late endosomes that fuse with lysosomes to deliver cargo for degradation. Recycling or degradation is not the only outcome of the cell surface-originated cargoes. A set of internalized transmembrane proteins, including intracellular sorting receptors, enzymes, and toxins, are retrieved from the endosomal system and transported to the TGN. The endosome-to-TGN trafficking of the acid-hydrolase-sorting receptor, CI-MPR, the endopeptidase furin, and the putative cargo receptor TGN38 are the best studied examples. These cargoes are highly enriched in the TGN at steady state but arrive there from different compartments, utilizing distinct mechanisms. Thus, TGN38 enters the TGN from the endocytic recycling compartment by an iterative removal from the latter compartment, furin reaches the TGN by exiting the early/late endosomal system, and CI-MPR implements features of both pathways (4, 6 -9).Whereas the detailed molecular and cellular mechanisms underlying the membrane progression in the course of cargo transport through the endosomal system or retrieval from early/late endosomes to the TGN is still elusive, experimental evidence has been accumulating to implicate PIKfyve, the sole enzyme for PtdIns(3,5)P 2 synthesis (10). 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-