Fig. 6

ESCRTs affecting apical targeting of BSEP is upstream of Rab11. a VPS4-mediated the post-Golgi trafficking of BSEP was upstream of Rab11A. Hep G2 cells were co-expressed the indicated combination of FLAG-BSEP; EGFP-Rab11A or EGFP-Rab11A-S25N (EGFP-Rab11A-SN); mCherry-VPS4A or -VPS4A-E228Q (VPS4A-EQ); or mCherry-VPS4B or -VPS4B-E235Q (VPS4B-EQ) for temperature shift assay. A representative immunoblotting reveals the plasma-membrane (PM), organelle-membrane (OM) and cytosolic (Cyt) protein fractions detected by the indicated proteins and the fractionation controls. These experiments, described in (a), were independently triplicated. The values are the relative FLAG-BSEP signals normalized to the corresponding fractionation control signals. The independently triplicated results (n = 3, mean ± SD) were illustrated in (b). c Confocal microscopy demonstrates the BSEP-and-CHMP5 colocalized SACs are also positive with the recycling endosome marker Rab11 (arrowhead). Adult human liver samples were co-immunofluorescently stained for BSEP (green), CHMP5 (red), and Rab11 (blue pseudo-color). DAPI (white pseudo-color) stained the nuclei of hepatocytes. d After transcription and translation, BSEP undergoes core-and complete-glycosylation in the ER and Golgi, respectively, and subsequent post-Golgi trafficking. BSEP is poly-ubiquitinated via K63 linkages and bound with CHMP5 further targets to the sorting endosomes. The sorting endosome is an internal trafficking hub enriched with the small GTPase Rab5 proteins. Other ESCRT molecules, at least LIP5, may co-localize with BSEP and CHMP5 at the sorting endosome. VPS4 dissembles ESCRTs on the sorting endosomes, from which BSEP is transferred to the canalicular membrane via Rab11-mediated targeting and then cycles between the Rab11-recycling endosomes