Canalicular secretion of bile salts is a vital function of the vertebrate liver, yet the molecular identity of the involved ATP-dependent carrier protein has not been elucidated. We cloned the full-length cDNA of the sister of P-glycoprotein (spgp; M r ϳ160,000) of rat liver and demonstrated that it functions as an ATP-dependent bile salt transporter in cRNA injected Xenopus laevis oocytes and in vesicles isolated from transfected Sf9 cells. The latter demonstrated a 5-fold stimulation of ATP-dependent taurocholate transport as compared with controls. This spgp-mediated taurocholate transport was stimulated solely by ATP, was inhibited by vanadate, and exhibited saturability with increasing concentrations of taurocholate (K m Ӎ 5 M). Furthermore, spgp-mediated transport rates of various bile salts followed the same order of magnitude as ATP-dependent transport in canalicular rat liver plasma membrane vesicles, i.e. taurochenodeoxycholate > tauroursodeoxycholate ‫؍‬ taurocholate > glycocholate ‫؍‬ cholate. Tissue distribution assessed by Northern blotting revealed predominant, if not exclusive, expression of spgp in the liver, where it was further localized to the canalicular microvilli and to subcanalicular vesicles of the hepatocytes by in situ immunofluorescence and immunogold labeling studies. These results indicate that the sister of P-glycoprotein is the major canalicular bile salt export pump of mammalian liver.Bile formation is an important function of vertebrate liver (1). It is mediated by hepatocytes that generate bile flow within bile canaliculi by continuous vectorial secretion of bile salts and other solutes across their canalicular (apical) membrane (2). Studies in isolated membrane vesicles of rat and human livers have shown that canalicular bile salt transport is an ATP-dependent process (3-7). However, the molecular identity of the primary active canalicular bile salt transporter or bile salt export pump (BSEP) 1,2 has not yet been elucidated (8, 9). Although the canalicular ecto-ATPase has been proposed as a possible candidate (9, 10), other investigations have provided evidence that BSEP of mammalian liver is an ABC (ATP binding cassette)-type of membrane transporter (11,12). This assumption has recently been further supported by the cloning of an ATP-dependent bile salt transporter from Saccharomyces cerevisiae (13). This yeast bile salt transporter (BAT1) belongs to a subgroup of ABC-type proteins that includes also the canalicular multiorganic anion transporter or multidrug resistance protein MRP2 (human)/mrp2 (rat) (14 -16). Although MRP2/mrp2 mediates canalicular excretion of a broad range of divalent amphipathic anionic conjugates (1, 14, 17), it does not transport primary bile salts such as taurocholate or glycocholate (1, 18). Therefore, we designed degenerate oligonucleotide primers spanning the Walker A and B motifs of ABC proteins and performed reverse transcription-polymerase chain reactin with total rat liver mRNA. One of the amplified fragments revealed an 88% identity with the p...

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Poliovirus (PV) replicates its genome in association with membranous vesicles in the cytoplasm of infected cells. To elucidate the origin and mode of formation of PV vesicles, immunofluorescence labeling with antibodies against the viral vesicle marker proteins 2B and 2BC, as well as cellular markers of the endoplasmic reticulum (ER), anterograde transport vesicles, and the Golgi complex, was performed in BT7-H cells. Optical sections obtained by confocal laser scanning microscopy were subjected to a deconvolution process to enhance resolution and signal-to-noise ratio and to allow for a three-dimensional representation of labeled membrane structures. The mode of formation of the PV vesicles was, on morphological grounds, similar to the formation of anterograde membrane traffic vesicles in uninfected cells. ER-resident membrane markers were excluded from both types of vesicles, and the COPII components Sec13 and Sec31 were both found to be colocalized on the vesicular surface, indicating the presence of a functional COPII coat. PV vesicle formation during early time points of infection did not involve the Golgi complex. The expression of PV protein 2BC or the entire P2 and P3 genomic region led to the production of vesicles carrying a COPII coat and showing the same mode of formation as vesicles produced after PV infection. These results indicate that PV vesicles are formed at the ER by the cellular COPII budding mechanism and thus are homologous to the vesicles of the anterograde membrane transport pathway.Poliovirus (PV), the prototype member of the family Picornaviridae, contains an RNA genome of plus polarity with a single large open reading frame (ORF) coding for a polyprotein of 265 kDa. The translation product is cleaved by intrinsic viral proteases into about 20 proteins, of which intermediate as well as end products are functional (38, 83, 88; reviewed in reference 35). The proteins encoded by the P1 genomic region form the capsid, whereas most of the nonstructural proteins, encoded by the P2 and P3 genomic region, are involved in viral RNA replication (2,27,28,37,48,55,58,82). The viral genome replicates asymmetrically with an excess of plus strands produced in multistranded replicative intermediates (2,17,31,49). PV genome replication depends on the template RNA molecule carrying a cis-acting replicative element (see references 32 and 54), a primer (54), viral and cellular proteins and their proper interactions (2,29,30,53), and the presence of membranes (10,12,14,26,74).Genome replication of all plus-strand RNA viruses investigated so far takes place in membrane-bound replication complexes (see references 19 and 65 and references therein). However, the morphology of such replication complexes of different viruses is diverse, and different cellular compartments provide membranes for and are involved in viral replication (44,56,65). PV replication complexes are built up from individual membranous vesicles (PV vesicles) which are assembled into specific higher-order structures (11,12,15).The PV replicat...

The multidrug resistance-associated proteins (Mrps) constitute a family of cellular export pumps of the ATP-binding cassette transporter superfamily and play an important role in hepatobiliary excretion. We investigated the transport function and subcellular localization of mrp6, a novel member of the mrp family, in rat liver. Transport studies in vesicles isolated from mrp6 expressing Sf9 cells identified the anionic cyclopentapeptide and endothelin receptor antagonist BQ-123 as a substrate of mrp6 (K(m) approximately 17 microM). Besides BQ-123, which is also a substrate of mrp2 (K(m) approximately 124 microM), no other common substrates were found for mrp2, mrp6, and the canalicular bile salt export pump Bsep. The cyclic peptides endothelin I and Arg(8)-vasopressin were transported by mrp2 but not by mrp6. Using a polyclonal antiserum raised against a C-terminal peptide, mrp6 was found to be localized at the lateral and, to a lesser extent, at the canalicular plasma membrane of hepatocytes. The limited overlap of the substrate specificity with the canalicular export pumps mrp2 and Bsep indicates that mrp6 does not play a major role in canalicular organic anion excretion. However, its dual localization at the lateral and canalicular plasma membrane suggests that mrp6 might fulfill a "housekeeping" transport function involved in the regulation of paracellular and/or transcellular solute movement from blood into bile.