Fig. 3
From: The ESCRT-III molecules regulate the apical targeting of bile salt export pump
Aberrant interaction with CHMP5 affected the polarized trafficking of BSEP-R487H and BSEP-N490D mutants. a Representative immunostaining demonstrates the impaired canalicular targeting of BSEP-R487H and BSEP-N490D mutants at the steady state in vivo. FLAG-BSEP wild type and mutants were expressed in Bsep knockout mouse livers for 7 days via hydrodynamic injection. b Confocal images reveal the impaired post-Golgi trafficking and membrane targeting of BSEP-R487H and BSEP-N490D mutants. Temperature shift assay was performed with Hep G2 cells co-expressing the membrane marker Mem-DsRed plus FLAG-tagged BSEPs (stained with anti-mCherry and anti-FLAG antibodies, respectively). Arrows indicate the plasma membrane. c The bar graph (n = 3, mean ± SD) illustrates the subcellular distribution index of FLAG-BSEP wild-type and the two mutations after temperature shift assays. Total 98 to 131 cells were counted. (*P ≤ 0.05). d Immunoblotting reveals the decreased plasma-membrane targeting of BSEP-R487H and BSEP-N490D. Hep G2 cells expressing FLAG-BSEP wild-type or two mutants were subjected to temperature shift assay followed by subcellular fractionation. The plasma-membrane (PM), organelle-membrane (OM) and cytosolic (Cyt) fractions were analyzed to detect the indicated proteins and the fractionation control. Numbers are the relative ratio of the signal density of FLAG-BSEPs in the PM normalized to that of the PM control. e BSEP polypeptides (amino acids [a.a.] 484-558) harboring either R487H or N490D mutations could interact with CHMP5 in yeast two-hybrid assays. The BSEP-484-55 and BSEP-351-454 fragments were used as the positive and negative CHMP5-interaction control, respectively. Lamin was negative control. f Immunoblotting demonstrates aberrant association between the two BSEP mutants and ESCRT-III molecules. Hep G2 cells were co-expressed CHMP5-3HA and FLAG-BSEP-WT or the BSEP mutants and then fractionated. The pCMV6-AC-3HA was the vector control (Vector CTL). FLAG-tagged BSEP proteins in the total membrane (tM) fractions were immunoprecipitated and probed the indicated proteins. The bar graph illustrates the relative signal density normalized to FLAG-BSEPs of the “tM fraction + IP” panel