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JIPs (c-Jun N-terminal kinase interacting proteins), which scaffold JNK/p38 MAP kinase signaling modules, also bind conventional kinesins and are implicated in microtubule-based membrane trafficking in neuronal cells. Here we have identified a novel splice variant of the Jip4 gene product JLP(L) (JNK-interacting leucine zipper protein) in yeast-two hybrid screens with the phosphoinositide kinase PIKfyve. The interaction was confirmed by pulldown and coimmunoprecipitation assays in native cells. It engages the PIKfyve cpn60_TCP1 consensus sequence and the last 75 residues of the JLP C terminus. Subpopulations of both proteins cofractionated and populated similar structures at the cell perinuclear region. Because PIKfyve is essential in endosome-to-trans-Golgi network (TGN) cargo transport, we tested whether JLP is a PIKfyve functional partner in this trafficking pathway. Short interfering RNA (siRNA)-mediated depletion of endogenous JLP or PIKfyve profoundly delayed the microtubule-based transport of chimeric furin (Tac-furin) from endosomes to the TGN in a CHO cell line, which was rescued upon ectopic expression of siRNA-resistant JLP or PIKfyve constructs. Peptides from the contact sites in PIKfyve and JLP, or a dominant-negative PIKfyve mutant introduced into cells by ectopic expression or microinjection, induced a similar defect. Because Tac-TGN38 delivery from endosomes to the TGN, unlike that of Tac-furin, does not require intact microtubules, we monitored the effect of JLP and PIKfyve depletion or the interacting peptides administration on Tac-TGN38 trafficking. Remarkably, neither maneuver altered the Tac-TGN38 delivery to the TGN. Our data indicate that JLP interacts with PIKfyve and that both proteins and their association are required in microtubule-based, but not in microtubule-independent, endosome-to-TGN cargo transport.