Newly Synthesized Canalicular ABC Transporters Are Directly Targeted from the Golgi to the Hepatocyte Apical Domain in Rat Liver

Abstract

Newly synthesized canalicular ectoenzymes and a cell adhesion molecule (cCAM105) have been shown to traffic from the Golgi to the basolateral plasma membrane, from where they transcytose to the apical bile canalicular domain. It has been proposed that all canalicular proteins are targeted via this indirect route in hepatocytes. We studied the membrane targeting of rat canalicular proteins by in vivo [35 S]methionine metabolic labeling followed by preparation of highly purified Golgi membranes and canalicular (CMVs) and sinusoidal/basolateral (SMVs) membrane vesicles and subsequent immunoprecipitation. 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 were observed in metabolic pulse-chase labeling experiments with regard to membrane targeting of these apical proteins. After a chase time of 15 min, cCAM105 appeared exclusively in SMVs, peaked at 1 h, and progressively declined thereafter. In CMVs, cCAM105 was first detected after 1 h and subsequently increased for 3 h. This findings confirm the transcytotic targeting of cCAM105 reported in earlier studies. In contrast, at no time point investigated were MDR1, MDR2, and SPGP detected in SMVs. In CMVs, MDR1 and MDR2 appeared after 30 min, whereas SPGP appeared after 2 h of labeling. In Golgi membranes, each of the ABC transporters peaked at 30 min and was virtually absent thereafter. These data suggest rapid, direct targeting of newly synthesized MDR1 and MDR2 from the Golgi to the bile canaliculus and transient sequestering of SPGP in an intracellular pool en route from the Golgi to the apical plasma membrane. This study provides biochemical evidence for direct targeting of newly synthesized apical ABC transporters from the Golgi to the bile canaliculus in vivo.The bile canalicular membrane of the mammalian hepatocyte contains several primary active transporters that couple ATP hydrolysis to the transport of specific substrates into the bile canaliculus (1-4). These transporters are members of the superfamily of ABC (ATP-binding cassette) membrane transport proteins (5) and currently include P-glycoprotein or MDR1 (multidrug resistance protein; organic cations) (6), MDR2 (phosphatidylcholine) (7,8), SPGP (sister of P-glycoprotein; bile acids) (9), and MRP2 (multidrug resistance-associated protein; non-bile acid organic anions) (10).The amount of each ABC transporter in the canalicular membrane is regulated by the physiological demand to excrete bile acids. Intravenous administration of rats with taurocholate or dibutyryl cAMP rapidly and selectively increased the functional activity and amount of ABC transporters in the canalicular membrane. This increase was inhibited by prior administration of colchicine, which disrupts microtubules (11), and wortmannin, which inhibits phosphatidylinositol 3-kinase (12). These observations indicate that an intracellular microtubule-dependent transport mechanism that is...

Raft-mediated Trafficking of Apical Resident Proteins Occurs in Both Direct and Transcytotic Pathways in Polarized Hepatic Cells: Role of Distinct Lipid Microdomains

“…This would suggest that a direct vesicular transport pathway between TGN and BC membrane does exist in hepatocytes, raising the question whether and if so which proteins might participate in this transport event. Very recently, such a direct pathway for the polytopic apical plasma membrane ATP-binding cassette (ABC) transporter proteins, multidrug resistance protein 1 (MDR1), and sister of p-glycoprotein (SPGP) has been proposed, based upon in vitro and in vivo experiments in liver cells (Sai et al, 1999;Kipp and Arias, 2000). Most importantly, these data question the long-standing dogma of an exclusive indirect transfer of resident apical proteins in liver cells.…”

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