FIGURE 5.

Tac-furin but not Tac-TGN38 trafficking to the TGN is arrested by PIKfyve or JLP protein depletion or by PIKfyve^K1831E expression. TRVb1/TTF or TRVb1/Tac-TGN38 cell lines stably expressing Tac-furin or Tac-TGN38 chimera were seeded on coverslips placed onto 35-mm plates. Cells were transfected with siRNA duplexes (100 nm) targeting specific regions in mouse JLP, PIKfyve, or cyclophilin B (Control; A and B) and after 48 h were cotransfected with pcDNA3-hJLP^WT-S or the pCMV5-HA-PIKfyve-cFab1 chimera as indicated (B). Cells were transfected with pEGFP-HA-PIKfyve^K1831E cDNA (C). At 72 h (A and B) or 12 h post-transfection (C), cells were incubated with Alexa555-anti-Tac monoclonal antibody (10 min, 37 °C), chased (40 min), fixed, and then processed for fluorescence microscopy as detailed under “Experimental Procedures.” Tac chimera trafficking was monitored by Alexa555 fluorescence (A, panels a–f; B, panels a and c; C, panels a and c). Expression of HA-PIKfyve-cFab1 (B, panel b), hJLP-S (B, panel d), and eGFP-PIKfyve^K1831E (C, panels b and d) was detected by polyclonal anti-HA antibodies, anti-JLP antibodies (the dilution detects only the overexpressed protein), and GFP fluorescence signals, respectively. Cells were observed in a Nikon Eclipse TE 200 inverted fluorescence microscope. Note the Tac-furin distribution to cytoplasmic punctae in cells with JLP or PIKfyve depletion (A, panels b and c) or PIKfyve^K1831E expression (C, panel a) and no apparent perinuclear TGN clusters. Expression of HA-PIKfyve-cFab1 and hJLP^WT-S restores the steady-state TGN localization of Tac-furin in the PIKfyve- and JLP-depleted cells, respectively (B, arrowheads in panels a and c) in >85% of the expressing cells. Bar, 10 μm.