Abstract
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.
Highlights
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 trans-Golgi network (TGN) is still elusive, experimental evidence has been accumulating to implicate PIKfyve, the sole enzyme for PtdIns[3,5]P2 synthesis [10]
PIKfyve-JLP Association Required in Endosome-to-TGN Transport 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]
Consistent with previous studies (6 – 8), we found that under these conditions Tac-furin reaches steady-state distribution at the TGN in control cells, which is manifested by the accumulation of the anti-Tac immunofluorescence signals as a cluster at the perinuclear region (Fig. 5A, panel a). siRNAmediated PIKfyve protein depletion (Ͼ85%; Ref. 15 and this study, not shown) in the TRVb1/TTF cells profoundly inhibited the endosome-to-TGN delivery of Tac-furin
Summary
Yeast Two-hybrid Screening and Cloning of Full-length Mouse JLPL cDNA—The interaction mating version of two-hybrid screening of the human HeLa cell cDNA library with the PIKfyve 252-aa peptide fragment of the conserved cpn60_TCP1 domain (pNLexNLS-PIKfyve-(616 – 868)) was described previously [11]. 48 h following transfection with the siRNA duplexes, the TRVb1/ TTF cells were transfected (Lipofectamine, Invitrogen) with the cDNAs of human JLP-S or mouse/yeast HA-PIKfyve-cFab chimera. Anti-Tac Uptake and Tac Chimera Steady-state Localization— The uptake of Alexa555-labeled anti-Tac IgG in TRVb1/TTF or TRVb1/Tac-TGN38 cells was performed as described previously (6 – 8). To reveal whether steady-state localization of the Tacchimeric protein was affected by the treatments indicated, fixed cells were permeabilized (0.5% Triton X-100 in phosphate-buffered saline) and incubated with anti-Tac IgG, which was detected by Alexa568-conjugated anti-mouse IgG. Statistical analysis was performed by Student’s t test, with p Ͻ 0.05 considered as significant
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