Reconstitution of bile acid transport in a heterologous cell by cotransfection of transporters for bile acid uptake and efflux.

The rat liver canalicular bile acid transporter/ecto-ATPase/cell CAM 105 (CBATP) is a 110-kDa transmembrane phosphoglycoprotein that is thought to have bile acid efflux, ecto-ATPase, and cell adhesion properties. Its extracellular amino-terminal domain is highly homologous to carcinoembryonic antigen (CEA), a glycophosphatidyl inositol-anchored membrane protein with cell adhesion properties and a marker for adenocarcinoma. In the current study, we examined the possibility of more clearly defining the role of CBATP in bile acid efflux by cotransfecting a heterologous cell, the COS cell, with cDNAs for a bile acid importer, the ileal bile acid transporter (IBAT), as well as for CBATP. The results show that when IBAT mediates uptake of [3H]taurocholate to a level 20-fold higher than that achieved previously by nonspecific pinocytosis, CBATP mediates time-, temperature- and concentration-dependent efflux. Efflux of [3H]taurocholate mediated by CBATP in the cotransfected COS cells is saturable and has curvilinear kinetic characteristics (Vmax = 400 pmol/mg protein/min, Km = 70 microM). It is inhibited by 4,4'-diisothiocyanostilbene-2,2-disulfonic acid and dependent on ATP but not dependent on membrane potential. Although CEA could not mediate bile acid efflux in COS cells cotransfected with IBAT and CEA, efflux of [3H]taurocholate was detected in COS cells cotransfected with IBAT and a chimeric molecule having the carboxyl-terminal tail and membrane spanning domain of CBATP and the amino-terminal extracellular tail of CEA. Taken together, these data provide further evidence that CBATP confers bile acid efflux properties on heterologous cells and that its cytoplasmic tail and membrane spanning segment are integral to this property. The data also establish a model system for more clearly defining the molecular determinants of bile acid transport mediated by this molecule.

Bile acid transport across the hepatocyte canalicular membrane.

Transport of bile acids across the canalicular membrane of the hepatocyte provides the primary motive force for generation of bile flow and is rate limiting in the vectorial movement of bile acids from blood to bile. Several distinct carriers for bile acids have been defined based on physiological studies in isolated hepatocytes, membrane vesicles, hepatocyte couples, and the perfused rat liver including membrane potential-driven and ATP-dependent mechanisms. Several groups have isolated and functionally reconstituted a canalicular bile acid transport protein of M(r) approximately 110 kDa. The ATP-dependent mechanism for secretion of monovalent bile acids appears to be mediated by a yet to be identified protein of the ATP binding cassette family of transporters. However, it remains conjectural whether the ATP-dependent and membrane potential-driven components of canalicular bile acid transport are mediated by one or more transport proteins. Bile acid sulfates and glucuronides are substrates for the canalicular multispecific organic anion transporter whose activity has recently been associated with the multidrug resistance-associated protein.

Site-directed mutagenesis within an ectoplasmic ATPase consensus sequence abrogates the cell aggregating properties of the rat liver canalicular bile acid transporter/ecto-ATPase/cell CAM 105 and carcinoembryonic antigen.

Recent studies of the rat liver canalicular bile acid transporter/ecto-ATPase/cell CAM 105 (CBATP), a member of the carcinoembryonic antigen (CEA) supergene family, indicate that it is a multifunctional protein possessing bile acid efflux, ecto-ATPase, and intercellular aggregating properties. Cheung et al. (Cheung, P. H., Luo, W., Qiu, Y., Zhang, K. E., Millron, P., Lin, S. H. (1993) J. Biol. Chem. 268, 24303-24310) have shown that the amino-terminal Ig V-like domain of this protein is required for its aggregating properties, much like the homologous amino-terminal domain of CEA is required for its aggregating properties. The amino-terminal domains of both CBATP and CEA include a consensus ATPase sequence. Site-directed mutagenesis within this ATPase consensus sequence completely eliminates the ecto-ATPase activity of CBATP (Sippel, C. J., McCollum, M., Perlmutter, D. H. (1994) J. Biol. Chem. 269, 2820-2826). In this study we examined the possibility that it is this ATPase consensus sequence which is required for the cell aggregating properties of CBATP and CEA and whether there is a relationship between ATPase, aggregating, and bile acid efflux activities. For this we used a baculovirus vector to express in Sf9 cells wild type as well as mutant and chimeric CBATP and CEA molecules. The results indicate that Arg-98 in the ATPase consensus sequence of CBATP and the corresponding residue of CEA are essential for the aggregating properties of these molecules. Moreover Arg-98 is essential for CBATP to interact with itself, CEA to interact with itself, and CBATP to interact with CEA. However, the role of Arg-98 in aggregation is distinct from its role in ecto-ATPase activity and the aggregating properties cannot be attributed to a change in ATP metabolism in the pericellular milieu.

Bile acid efflux mediated by the rat liver canalicular bile acid transport/ecto-ATPase protein requires serine 503 phosphorylation and is regulated by tyrosine 488 phosphorylation.

Transfection of cDNA for a hepatocyte canalicular phosphoprotein, the rat liver canalicular bile acid transporter/ecto-ATPase/cell CAM 105, confers bile acid efflux and ecto-ATPase activities on heterologous cells (Sippel, C. J., Suchy, F. J., Ananthanarayanan, M., and Perlmutter D. H. (1993) J. Biol. Chem. 268, 2083-2091). Our previous studies have also indicated that there is a positive correlation between the degree of phosphorylation of this transporter and its bile acid efflux activity. In this study, we introduced site-specific mutations of amino acid residues within a protein kinase C-dependent (T502A, S503A) and a tyrosine kinase-dependent (Y488F) phosphorylation consensus sequence in the cytoplasmic tail of this transporter in order to map the sites that are phosphorylated in vivo and to examine the functional significance of each. COS cells were transfected with mutant and wild type constructs using the pCDM8 expression vector. Metabolic labeling and cell surface labeling showed that the mutant proteins were synthesized and delivered to the cell surface as efficiently as the wild type. Phosphoamino acid analysis using lysates of transfected cells showed that the T502A, S503A mutant contained [32P]phosphotyrosine, the Y488F mutant contained [32P]phosphoserine, and the wild type contained both 32P-labeled amino acids, proving that Ser503 and Tyr488 are the only amino acids phosphorylated in this system under control conditions. Bile acid transport activity was completely abrogated in cells transfected with the T502A, S503A mutant cDNA and was retained but altered in kinetic characteristics in cells transfected with the Y488F mutant cDNA, even though both of these constructs conferred ecto-ATPase activity to the same extent as the wild type cDNA. Taken together, these data show that the bile acid efflux activity of this transporter requires site-specific phosphorylation of Ser503 and is regulated by site-specific phosphorylation of Tyr488.

Bile acid transport by the rat liver canalicular bile acid transport/ecto-ATPase protein is dependent on ATP but not on its own ecto-ATPase activity.

We have shown that bile acid efflux and ecto-ATPase activities are two distinct properties of a single rat liver hepatocyte canalicular membrane protein (Sippel, C. J., Suchy, F. J., Ananthanarayanan, M., and Perlmutter, D. H. (1993) J. Biol. Chem. 268, 2083-2091). Bile acid efflux in COS cells transfected with this rat hepatocyte canalicular bile acid transport/ectoATPase cDNA is stimulated by ATP and inhibited by nonhydrolyzable ATP analogs. In this study, we depleted transfected COS cells of ATP to examine whether bile acid efflux mediated by this transporter was dependent on ATP or just stimulated by ATP. We also used mutagenesis of an ATPase consensus sequence in the ectoplasmic domain to examine the relationship of ATPase activity to bile acid efflux mediated by the same polypeptide. The results indicate that bile acid transport is abrogated by ATP depletion and reconstituted by exogenous ATP in a concentration-dependent and saturable manner. Introduction of mutations at amino acids Gly97 and Arg98 in the ATPase consensus sequence abrogated ATPase activity but did not affect synthesis or cell surface delivery of the transporter and did not affect its bile acid transport activity. Taken together, the data indicate that bile acid efflux mediated by the rat hepatocyte canalicular bile acid transport/ecto-ATPase protein is dependent on ATP but not on its own ATPase activity. The data, therefore, imply that 1) ATP affects its bile acid transport activity through an entirely distinct mechanism; and 2) if there is any functional relationship between the ecto-ATPase and bile acid transport properties, it is mediated indirectly through regulation of net ATP concentrations in the canalicular space by the ecto-ATPase.

The rat liver ecto-ATPase is also a canalicular bile acid transport protein.

A approximately 110-kDa glycoprotein purified from canalicular vesicles by bile acid affinity chromatography has been identified as the canalicular bile acid transport protein. Internal amino acid sequence and chemical and immunochemical characteristics of this protein were found to be identical to a rat liver canalicular ecto-ATPase. In order to definitively determine whether these were two activities of a single polypeptide, we examined the possibility that transfection of cDNA for the ecto-ATPase would confer bile acid transport characteristics, as well as ecto-ATPase activity, on heterologous cells. The results show that transfection of the ecto-ATPase cDNA conferred on COS cells de novo synthesis of a approximately 110-kDa polypeptide, as immunoprecipitated by antibody to the purified canalicular bile acid transport protein and conferred on COS cells the capacity to pump out [3H]taurocholate with efflux characteristics comparable with those previously determined in canalicular membrane vesicles (Km = 100 microM; Vmax = 200 pmol/mg of protein/20 s). A truncated ecto-ATPase cDNA, missing the cytoplasmic tail, was targeted correctly to the cell surface but did not confer bile acid transport activity on COS cells. The results of this study also show that the canalicular ecto-ATPase/bile acid transport protein is phosphorylated on its cytoplasmic tail and that its phosphorylation is stimulated by activation of protein kinase C and inhibited by inhibitors of protein kinase C activation. Moreover, inhibition of protein kinase C activation by staurosporine completely abrogates bile acid transport but does not affect ATPase activity. This study, therefore, demonstrates that the rat liver canalicular ecto-ATPase is also a bile acid transport protein, that the capacity to pump out bile acid can be conferred on a heterologous cell by DNA-mediated gene transfer, and that phosphorylation within the cytoplasmic tail of the transporter is essential for bile acid efflux activity but not for ATPase activity.

An ontogenically regulated 48-kDa protein is a component of the Na(+)-bile acid cotransporter of rat liver.

Recent evidence suggests that the Na(+)-coupled carrier mechanism for bile acids on the hepatocyte basolateral plasma membrane is a polypeptide in the molecular weight range of 48,000-50,000. In this study we used a strategy for the identification and isolation of this transport protein based on the observation that Na(+)-dependent transport activity is abruptly expressed in fetal rat liver just before birth [Suchy et al. Am. J. Physiol. 251 (Gastrointest. Liver Physiol. 14): G665-G673, 1986]. Analysis of basolateral plasma membranes by SDS-PAGE revealed that a protein of apparent molecular weight 48,000 was absent from fetal rat liver on day 19 of gestation, barely detectable on day 20, and thereafter increased progressively with postnatal development. Monospecific, polyclonal antibodies raised against the 48-kDa protein but not preimmune antibodies significantly inhibited the initial rate of Na(+)-dependent taurocholate uptake by isolated rat hepatocytes. In contrast, Na(+)-independent taurocholate transport and uptake of another anion, 35SO4(2-), were not affected by antibody treatment. When an extract containing the total complement of basolateral proteins was incorporated into asolectin liposomes, Na+ gradient-dependent uptake of taurocholate was observed, including a 2- to 2.5-fold accumulation of substrate above its equilibrium concentration (overshoot). However, if the membrane extract was first selectively depleted of the 48-kDa protein by immunoprecipitation with the anti-48-kDa antibody before reconstitution, Na(+)-dependent stimulation of taurocholate transport was completely abolished. These studies indicate that an ontogenically regulated 48-kDa protein is a component of the basolateral Na(+)-dependent transport system for bile acids.

Postnatal expression of the canalicular bile acid transport system of rat liver.

Canalicular plasma membrane (CPM) vesicles prepared by a Ca2+ precipitation method from developing (7 and 14 days old) and adult rat liver were used to directly examine the postnatal ontogenesis of taurocholate (TC) transport. The initial rate of 50 microM TC uptake by vesicles derived from 14-day-old and adult but not 7-day-old animals was markedly inhibited by the anion transport inhibitor 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS). DIDS-sensitive TC uptake was 21.6 +/- 5.6 (SE) at 14 days compared with 58.1 +/- 8.1 pmol.mg protein-1.5 s-1 in adults (P less than or equal to 0.01). Kinetic studies were performed by preloading these predominantly "right-side out" vesicles with TC (25-800 microM) and measuring the initial rate (5 s) of efflux into bile salt-free medium. Computer analysis of the DIDS-sensitive portion of efflux revealed saturable kinetics with a similar Vmax (2.72 +/- 0.36 vs. 1.97 +/- 0.17 nmol.mg protein-1.min-1; P = NS) but a threefold higher Km (0.35 +/- 0.09 vs. 0.11 +/- 0.02 mM; P less than or equal to 0.05) in 14 day vs. adult CPM vesicles. In contrast, efflux from 7 day CPM vesicles increased linearly with increasing concentrations of TC and was not inhibited by DIDS. Immunoblots of canalicular membranes, probed with an antibody against the 100-kDa bile acid transport protein, showed that the amount of immunoreactive carrier protein in the membranes of 14-day-old and adult rats was similar but was only 37% of the adult level at 7 days of age.(ABSTRACT TRUNCATED AT 250 WORDS)

Role of influenza B virus in hepatic steatosis and mitochondrial abnormalities in a mouse model of Reye syndrome.

The hepatic steatosis observed in the influenza B virus mouse model of Reye syndrome has been attributed to infectious virus or, alternately, to decreased food intake in the virus-treated mice or impurities in the virus preparation. To resolve this issue, 4- to 6-wk-old male Balb C mice were given, by intravenous injection, 12,800 hemagglutination units of influenza B Lee/40 virus in phosphate buffered saline/1% bovine serum albumin using virus prepared by ultra-centrifugation from infected allantoic fluid, by sucrose density-gradient purification of virus prepared by ultracentrifugation from infected allantoic fluid or by irradiation of virus prepared by ultracentrifugation from infected allantoic fluid to inactivate virus. The infectivity titer of virus prepared by ultracentrifugation from infected allantoic fluid was much higher than that of sucrose density-gradient purified virus prepared from infected allantoic fluid: 50% egg infectious dose for virus prepared by ultracentrifugation from infected allantoic fluid was 3.9 x 10(4)/hemagglutination unit vs. 8.7 50% egg infectious dose/hemagglutination unit for sucrose density-gradient purified virus prepared from infected allantoic fluid. Control mice received phosphate-buffered saline/1% bovine serum albumin or uninfected allantoic fluid diluted in phosphate-buffered saline/1% bovine serum albumin. Mice were fasted to eliminate dietary variation, and livers were obtained 36 hr after virus administration. Of the above treatments, only virus prepared by ultracentrifugation from infected allantoic fluid caused clinical illness and increased hepatic triglycerides (p less than 0.02) compared with controls. Hepatic triglycerides in virus prepared by ultracentrifugation from infected allantoic fluid correlated with histopathological vacuolization scores (r = 0.5773; p less than 0.03).(ABSTRACT TRUNCATED AT 250 WORDS)

Isolation and characterization of the canalicular membrane bile acid transport protein of rat liver.

The aim of this study was to isolate the Na(+)-independent bile acid transporter from rat canalicular plasma membranes by affinity chromatography. The affinity matrix used consisted of lysylcholic acid covalently linked to CH-Sepharose 4B, resulting in an anionic ligand essentially identical to glycocholic acid. The protein fraction, adsorbed and eluted from the affinity column, was markedly enriched in a 100-kDa band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) compared with the total membrane and membrane extract. The 100-kDa band, further purified by preparative SDS-PAGE, was electroeluted from excised gel fragments and used as an immunogen for antibody production in rabbits. The immune serum, but not preimmune serum, specifically recognized a single, 100-kDa polypeptide on one- and two-dimensional immunoblots of canalicular membranes. In contrast, no reactivity was observed with proteins in liver basolateral or ileal brush-border membranes. The 125I-labeled protein was immunoprecipitated from membrane extracts solubilized in NP-40 and was found to migrate with a pI of 5.3 on two-dimensional electrophoresis. The apparent molecular weight of the protein was reduced by 50% after deglycosylation with N-glycanase. The 100-kDa protein was localized specifically and exclusively by immunocytochemical methods to the bile canalicular domain of the hepatocyte plasma membrane. Moreover, the immunoglobin G fraction prepared from the antiserum significantly inhibited taurocholate transport by canalicular membrane vesicles and decreased the covalent labeling of the 100-kDa protein by the anion transport inhibitor 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid. Thus the isolation of a single 100-kDa protein by bile acid-affinity chromatography, as well as the inhibitory effects of antibodies directed against this polypeptide, provide further support for its role in the canalicular transport of bile acids.

Rapid microprocedure for extraction of plasmid DNA from Staphylococcus aureus.

A rapid micromethod for extraction of plasmid DNA from Staphylococcus aureus in quantities sufficient for restriction endonuclease analysis was developed. The method of "fingerprinting" is useful for identification of epidemic strains in the clinical setting and is more reliable than frequently used determinations of plasmid molecular weight, particularly when small numbers of plasmids are involved.

The regulation of ubiquinone-6 biosynthesis by Saccharomyces cerevisiae.

Increasing concentrations of glucose (1-5%) in the growth medium depressed ubiquinone-6 biosynthesis in continuously cultured wild type Saccharomyces cerevisiae. In addition, an early intermediate in the pathway of ubiquinone-6 biosynthesis, i.e. 3,4-dihydroxy-5-hexaprenylbenzoate (3,4-DHHB), was found to accumulate. The increase in 3,4-DHHB levels varied inversely with the diminished levels of ubiquinone-6, suggesting that O-methylation of 3,4-DHHB is a regulated step in catabolite repression. Experiments using protoplasts demonstrated that the effect of catabolite repression on this pathway was reversible by 1.2 mM cAMP but not by other nucleotides and cyclic nucleotides. This response to cAMP was unaltered by the protein synthesis inhibitor cycloheximide, indicating that the regulatory control for this reaction must occur at the enzymatic level. Additional experiments demonstrated the presence of a heat-labile component of the cytoplasm, which was essential for this effect of cAMP. This observation suggests that this cytosolic effector may be translocated to the inner membrane of the mitochondria, the intracellular site for ubiquinone-6 biosynthesis.

Identification of 3-methoxy-4-hydroxy-5-hexaprenylbenzoic acid as a new intermediate in ubiquinone biosynthesis by Saccharomyces cerevisiae.

A ubiquinone-deficient mutant strain of Saccharomyces cerevisiae, 26H, was found to accumulate a previously unidentified intermediate in ubiquinone biosynthesis when grown in the presence of p-hydroxy[7-14C]- or -[u-14C]benzoic acid. This intermediate was isolated from the lipid extracts of a 100-L culture of 26H and purified by various chromatographic procedures to yield 20 mg of product. Analysis by means of NMR, IR, UV, and mass spectrometry revealed the structure of this new intermediate to be 3-methoxy-4-hydroxy-5-hexaprenylbenzoic acid (3-MHHB). In vitro experiments with isolated yeast and rat mitochondria showed that 3-MHHB could be converted to ubiquinone-6. These findings indicate that 3-O-methylation precedes decarboxylation of the prenylated protocatechuic acid intermediate in the biosynthesis of ubiquinone in eukaryotes.

Identification of 3,4-dihydroxy-5-hexaprenylbenzoic acid as an intermediate in the biosynthesis of ubiquinone-6 by Saccharomyces cerevisiae.

The mutant strain of Saccharomyces cerevisiae E3-24 is unable to synthesize ubiquinone-6. When this mutant is grown in the presence of p-hydroxy[U-14C]benzoate or p-hydroxy[carboxy-14C]benzoate, a radioactive compound accumulates. This new metabolite has been isolated and identified as 3,4-dihydroxy-5-hexaprenylbenzoate (3,4-DHHB). Aerobically grown prototrophic strains of S. cerevisiae were found to contain only low levels of this compound. When strain X963-18C, blocked at homoserine O-transacetylase (in methionine biosynthesis), was deprived of methionine, ubiquinone biosynthesis ceased, and 3,4-DHHB was observed to accumulate. This suggested that S-adenosylmethionine (SAM) could be the methyl donor for 3,4-DHHB. Restoration of methionine to the cultures released this block and resulted in the conversion of 3,4-DHHB to ubiquinone-6, demonstrating a precursor--product relationship. The identification of 3,4-DHHB as an intermediate in ubiquinone biosynthesis in yeast establishes an alternate pathway for ubiquinone biosynthesis in eukaryotes.