Newly synthesized canalicular ABC transporters are directly targeted from the Golgi to the hepatocyte apical domain in rat liver

Newly synthesized canalicular ectoenzymes and a cell adhesion molecule (cCA M105) have been shown to traffic from the Golgi to the basolateral plasma m embrane, from where they transcytose to the apical bile canalicular domain. It has been proposed that all canalicular proteins are targeted via this i ndirect route in hepatocytes, We studied the membrane targeting of rat cana licular proteins by in vivo [S-35]methionine metabolic labeling followed by preparation of highly purified Golgi membranes and canalicular (CMVs) and sinusoidal/basolateral (SMVs) membrane vesicles and subsequent immunoprecip itation, In particular, we compared membrane targeting of newly synthesized canalicular ABC (ATP-binding cassette) transporters MDR1, MDR2, and SPGP ( Sister of P-glycoprotein) with that of cCAM105. Significant differences wer e observed in metabolic pulse-chase labeling experiments with regard to mem brane targeting of these apical proteins. After a chase time of 15 min, cCA M105 appeared exclusively in SMVs, peaked at 1 h, and progressively decline d thereafter. In CMVs, cCAM105 was first detected after Ih and subsequently increased for 3 h, This findings confirm the transcytotic targeting of cCA M105 reported in earlier studies. In contrast, at no time point investigate d were MDR1, MDR2, and SPGP detected in SMVs. In CMVs, MDR1 and MDR2 appear ed after 30 min, whereas SPGP appeared after 2 h of labeling. In Golgi memb ranes, each of the ABC transporters peaked at 30 min and was virtually abse nt thereafter. These data suggest rapid, direct targeting of newly synthesi zed MDR1 and MDR2 from the Golgi to the bile canaliculus and transient sequ estering of SPGP in an intracellular pool en route from the Golgi to the ap ical plasma membrane. This study provides biochemical evidence for direct t argeting of newly synthesized apical ABC transporters from the Golgi to the bile canaliculus in vivo.