Two N-linked glycans are required to maintain the transport activity of the bile salt export pump (ABCB11) in MDCK II cells

2007 ◽  
Vol 292 (3) ◽  
pp. G818-G828 ◽  
Author(s):  
Kaori Mochizuki ◽  
Tatehiro Kagawa ◽  
Asano Numari ◽  
Matthew J. Harris ◽  
Johbu Itoh ◽  
...  
Keyword(s):  
Protein Stability ◽  
Bile Salt ◽  
Intracellular Trafficking ◽  
Double Mutant ◽  
Yellow Fluorescent Protein ◽  
Bile Salt Export Pump ◽  
Transport Activity ◽  
Wild Type ◽  
Mutant Proteins ◽  
Quadruple Mutant

The aim of this study was to determine the role of N-linked glycosylation in protein stability, intracellular trafficking, and bile acid transport activity of the bile salt export pump [Bsep (ATP-binding cassette B11)]. Rat Bsep was fused with yellow fluorescent protein, and the following mutants, in which Asn residues of putative glycosylation sites (Asn109, Asn116, Asn122, and Asn125) were sequentially replaced with Gln, were constructed by site-directed mutagenesis: single N109Q, double N109Q + N116Q, triple N109Q + N116Q + N122Q, and quadruple N109Q + N116Q + N122Q + N125Q. Immunoblot and glycosidase cleavage analysis demonstrated that each site was glycosylated. Removal of glycans decreased taurocholate transport activity as determined in polarized MDCK II cells. This decrease resulted from rapid decay of the mutant Bsep protein; biochemical half-lives were 3.76, 3.65, 3.24, 1.35, and 0.52 h in wild-type, single-mutant, double-mutant, triple-mutant, and quadruple-mutant cells, respectively. Wild-type and single- and double-mutant proteins were distributed exclusively along the apical membranes, whereas triple- and quadruple-mutant proteins remained intracellular. MG-132 but not bafilomycin A1 extended the half-life, suggesting a role for the proteasome in Bsep degradation. To determine whether a specific glycosylation site or the number of glycans was critical for protein stability, we studied the protein expression of combinations of N-glycan-deficient mutants and observed that Bsep with one glycan was considerably unstable compared with Bsep harboring two or more glycans. In conclusion, at least two N-linked glycans are required for Bsep protein stability, intracellular trafficking, and function in the apical membrane.

Phenotypic differences in PFIC2 and BRIC2 correlate with protein stability of mutant Bsep and impaired taurocholate secretion in MDCK II cells

2008 ◽  
Vol 294 (1) ◽  
pp. G58-G67 ◽  
Author(s):  
Tatehiro Kagawa ◽  
Norihito Watanabe ◽  
Kaori Mochizuki ◽  
Asano Numari ◽  
Yoshie Ikeno ◽  
...  
Keyword(s):  
Protein Expression ◽  
Apical Membrane ◽  
Bile Salt Export Pump ◽  
Transport Activity ◽  
Wild Type ◽  
Phenotypic Difference ◽  
Mutant Proteins ◽  
Phenotypic Differences ◽  
Benign Recurrent Intrahepatic Cholestasis ◽  
Taurocholate Transport

Progressive familial cholestasis (PFIC) 2 and benign recurrent intrahepatic cholestasis (BRIC) 2 are caused by mutations in the bile salt export pump (BSEP, ABCB11) gene; however, their prognosis differs. PFIC2 progresses to cirrhosis and requires liver transplantation, whereas BRIC2 is clinically benign. To identify the molecular mechanism(s) responsible for the phenotypic differences, eight PFIC2 and two BRIC2 mutations were introduced in rat Bsep, which was transfected in MDCK II cells. Taurocholate transport activity, protein expression, and subcellular distribution of these mutant proteins were studied in a polarized MDCK II monolayer. The taurocholate transport activity was approximately half of the wild-type (WT) in BRIC2 mutants (A570T and R1050C), was substantially less in two PFIC2 mutants (D482G and E297G), and was almost abolished in six other PFIC2 mutants (K461E, G982R, R1153C, R1268Q, 3767–3768insC, and R1057X). Bsep protein expression levels correlated closely with transport activity, except for R1057X. The half-life of the D482G mutant was shorter than that of the WT (1.35 h vs. 3.49 h in the mature form). BRIC2 mutants and three PFIC mutants (D482G, E297G, and R1057X) were predominantly distributed in the apical membrane. The other PFIC2 mutants remained intracellular. The R1057X mutant protein was stably expressed and trafficked to the apical membrane, suggesting that the COOH-terminal tail is required for transport activity but not for correct targeting. In conclusion, taurocholate transport function was impaired in proportion to rapid degradation of Bsep protein in the mutants, which were aligned in the following order: A570T and R1050C > D482G > E297G > K461E, G982R, R1153C, R1268Q, 3767–3768insC, and R1057X. These results may explain the phenotypic difference between BRIC2 and PFIC2.

scholarly journals Intracellular Trafficking of Bile Salt Export Pump (ABCB11) in Polarized Hepatic Cells: Constitutive Cycling between the Canalicular Membrane and rab11-positive Endosomes

2004 ◽  
Vol 15 (7) ◽  
pp. 3485-3496 ◽  
Author(s):  
Yoshiyuki Wakabayashi ◽  
Jennifer Lippincott-Schwartz ◽  
Irwin M. Arias
Keyword(s):  
Bile Salt ◽  
Intracellular Trafficking ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Confocal Imaging ◽  
Bile Salt Export Pump ◽  
Microtubule Organizing Center ◽  
Globular Structure ◽  
Canalicular Membrane ◽  
Organizing Center

The bile salt export pump (BSEP, ABCB11) couples ATP hydrolysis with transport of bile acids into the bile canaliculus of hepatocytes. Its localization in the apical canalicular membrane is physiologically regulated by the demand to secrete biliary components. To gain insight into how such localization is regulated, we studied the intracellular trafficking of BSEP tagged with yellow fluorescent protein (YFP) in polarized WIF-B9 cells. Confocal imaging revealed that BSEP-YFP was localized at the canalicular membrane and in tubulo-vesicular structures either adjacent to the microtubule-organizing center or widely distributed in the cytoplasm. In the latter two locations, BSEP-YFP colocalized with rab11, an endosomal marker. Selective photobleaching experiments revealed that single BSEP-YFP molecules resided in canalicular membranes only transiently before exchanging with intracellular BSEP-YFP pools. Such exchange was inhibited by microtubule and actin inhibitors and was unaffected by brefeldin A, dibutyryl cyclic AMP, taurocholate, or PI 3-kinase inhibitors. Intracellular carriers enriched in BSEP-YFP elongated and dissociated as tubular elements from a globular structure adjacent to the microtubule-organizing center. They displayed oscillatory movement toward either canalicular or basolateral membranes, but only fused with the canalicular membrane. The pathway between canalicular and intracellular membranes that BSEP constitutively cycles within could serve to regulate apical pools of BSEP as well as other apical membrane transporters.

Bile salt export pump is highly conserved during vertebrate evolution and its expression is inhibited by PFIC type II mutations

2001 ◽  
Vol 281 (2) ◽  
pp. G316-G322 ◽  
Author(s):  
Shi-Ying Cai ◽  
Lin Wang ◽  
Nazzareno Ballatori ◽  
James L. Boyer
Keyword(s):  
Bile Salt ◽  
Bile Secretion ◽  
Sf9 Cells ◽  
Bile Salt Export Pump ◽  
Raja Erinacea ◽  
Mutant Proteins ◽  
Liver Cdna Library ◽  
Function Relationship ◽  
Dependent Transport ◽  
Relationship Of

Bile secretion is a fundamental function of the liver of all vertebrates and is generated by ATP-dependent transport proteins at the canalicular membrane of hepatocytes, particularly by the bile salt export pump BSEP. To determine the evolutionary origin and structure-function relationship of this transport mechanism, a liver cDNA library from the marine skate Raja erinacea, a 200 million-year-old vertebrate, was screened for BSEP orthologues. A full-length clone was isolated that encodes for 1,348 amino acids and shares 68.5% identity to human BSEP. Northern blot analysis revealed a 5-kb transcript only in skate liver. Expression of skate Bsep in Sf9 cells demonstrated a sixfold stimulation of ATP-dependent taurocholate transport compared with controls, with a Michaelis-Menten constant of 15 μM, which is comparable to rat Bsep. Sequences at the site of published mutations in human BSEP are also conserved in skate Bsep. When two of these mutations were introduced into the skate Bsep cDNA, this resulted in defective expression of the mutant proteins in Sf9 cells. These studies demonstrate that Bsep is a liver-specific ATP-dependent export pump that is highly conserved throughout evolution and provide insights into critical determinants for the function of this transporter in higher vertebrates.

scholarly journals Functional and mechanistic roles of the human proton-coupled folate transporter transmembrane domain 6–7 linker

2016 ◽  
Vol 473 (20) ◽  
pp. 3545-3562 ◽  
Author(s):  
Mike R. Wilson ◽  
Zhanjun Hou ◽  
Lucas J. Wilson ◽  
Jun Ye ◽  
Larry H. Matherly
Keyword(s):  
Secondary Structure ◽  
Targeted Delivery ◽  
Transmembrane Domain ◽  
Surface Expression ◽  
Chimeric Proteins ◽  
Transport Activity ◽  
Wild Type ◽  
Intracellular Loop ◽  
Mutant Proteins ◽  
Sequence Elements

The proton-coupled folate transporter (PCFT; SLC46A1) is a folate–proton symporter expressed in solid tumors and is used for tumor-targeted delivery of cytotoxic antifolates. Topology modeling suggests that the PCFT secondary structure includes 12 transmembrane domains (TMDs) with TMDs 6 and 7 linked by an intracellular loop (positions 236–265) including His247, implicated as functionally important. Single-cysteine (Cys) mutants were inserted from positions 241 to 251 in Cys-less PCFT and mutant proteins were expressed in PCFT-null (R1-11) HeLa cells; none were reactive with 2-aminoethyl methanethiosulfonate biotin, suggesting that the TMD6–7 loop is intracellular. Twenty-nine single alanine mutants spanning the entire TMD6–7 loop were expressed in R1-11 cells; activity was generally preserved, with the exception of the 247, 250, and 251 mutants, partly due to decreased surface expression. Coexpression of PCFT TMD1–6 and TMD7–12 half-molecules in R1-11 cells partially restored transport activity, although removal of residues 252–265 from TMD7–12 abolished transport. Chimeric proteins, including a nonhomologous sequence from a thiamine transporter (ThTr1) inserted into the PCFT TMD6–7 loop (positions 236–250 or 251–265), were active, although replacement of the entire loop with the ThTr1 sequence resulted in substantial loss of activity. Amino acid replacements (Ala, Arg, His, Gln, and Glu) or deletions at position 247 in wild-type and PCFT–ThTr1 chimeras resulted in differential effects on transport. Collectively, our findings suggest that the PCFT TMD6–7 connecting loop confers protein stability and may serve a unique functional role that depends on secondary structure rather than particular sequence elements.

scholarly journals Enhancing ubiquitin crystallization through surface-entropy reduction

2014 ◽  
Vol 70 (10) ◽  
pp. 1434-1442 ◽  
Author(s):  
Patrick J. Loll ◽  
Peining Xu ◽  
John T. Schmidt ◽  
Scott L. Melideo
Keyword(s):  
Surface Density ◽  
Double Mutant ◽  
High Surface ◽  
Wild Type ◽  
Mutant Proteins ◽  
X Ray ◽  
Surface Entropy ◽  
Lysine Residues ◽  
Entropy Reduction ◽  
Packing Interactions

Ubiquitin has many attributes suitable for a crystallization chaperone, including high stability and ease of expression. However, ubiquitin contains a high surface density of lysine residues and the doctrine of surface-entropy reduction suggests that these lysines will resist participating in packing interactions and thereby impede crystallization. To assess the contributions of these residues to crystallization behavior, each of the seven lysines of ubiquitin was mutated to serine and the corresponding single-site mutant proteins were expressed and purified. The behavior of these seven mutants was then compared with that of the wild-type protein in a 384-condition crystallization screen. The likelihood of obtaining crystals varied by two orders of magnitude within this set of eight proteins. Some mutants crystallized much more readily than the wild type, while others crystallized less readily. X-ray crystal structures were determined for three readily crystallized variants: K11S, K33S and the K11S/K63S double mutant. These structures revealed that the mutant serine residues can directly promote crystallization by participating in favorable packing interactions; the mutations can also exert permissive effects, wherein crystallization appears to be driven by removal of the lysine rather than by addition of a serine. Presumably, such permissive effects reflect the elimination of steric and electrostatic barriers to crystallization.

Downregulated in adenoma gene encodes a chloride transporter defective in congenital chloride diarrhea

1999 ◽  
Vol 276 (1) ◽  
pp. G185-G192 ◽  
Author(s):  
Richard H. Moseley ◽  
Pia Höglund ◽  
Gary D. Wu ◽  
Debra G. Silberg ◽  
Siru Haila ◽  
...  
Keyword(s):  
Double Mutant ◽  
Xenopus Oocytes ◽  
Ph Gradient ◽  
Transport Activity ◽  
Wild Type ◽  
Sulfate Transporter ◽  
Functional Mutation ◽  
Congenital Chloride Diarrhea

Congenital chloride diarrhea (CLD) is a recessively inherited disorder characterized by massive loss of chloride in stool. We previously identified mutations in the downregulated in adenoma ( DRA) gene in patients with CLD and demonstrated that DRA encodes an intestine-specific sulfate transporter. To determine whether DRA is an intestinal chloride transporter and how mutations affect transport, Xenopus oocytes were injected with wild-type and mutagenized DRA cRNA and uptake of Cl− and[Formula: see text] was assayed. Both Cl− and[Formula: see text] were transported by wild-type DRA and an outwardly directed pH gradient stimulated Cl− uptake, consistent with Cl−/OH−exchange. Among three mutants, C307W transported both anions as effectively as wild-type, whereas transport activity was lost in V317del and the double mutant identified in 32 of 32 Finnish CLD patients. We conclude that DRA is a chloride transporter defective in CLD and that V317del is a functional mutation and C307W a silent polymorphism.

scholarly journals Phosphorylation of UT-A1 urea transporter at serines 486 and 499 is important for vasopressin-regulated activity and membrane accumulation

2008 ◽  
Vol 295 (1) ◽  
pp. F295-F299 ◽  
Author(s):  
Mitsi A. Blount ◽  
Abinash C. Mistry ◽  
Otto Fröhlich ◽  
S. Russ Price ◽  
Guangping Chen ◽  
...  
Keyword(s):  
Double Mutant ◽  
Transport Activity ◽  
Wild Type ◽  
Urea Transporter ◽  
Camp Production ◽  
Loop Region ◽  
Collecting Ducts ◽  
Phosphopeptide Analysis ◽  
Central Loop ◽  
Urine Concentrating Mechanism

The UT-A1 urea transporter plays an important role in the urine concentrating mechanism. Vasopressin (or cAMP) increases urea permeability in perfused terminal inner medullary collecting ducts and increases the abundance of phosphorylated UT-A1, suggesting regulation by phosphorylation. We performed a phosphopeptide analysis that strongly suggested that a PKA consensus site(s) in the central loop region of UT-A1 was/were phosphorylated. Serine 486 was most strongly identified, with other potential sites at serine 499 and threonine 524. Phosphomutation constructs of each residue were made and transiently transfected into LLC-PK1 cells to assay for UT-A1 phosphorylation. The basal level of UT-A1 phosphorylation was unaltered by mutation of these sites. We injected oocytes, assayed [14C]urea flux, and determined that mutation of these sites did not alter basal urea transport activity. Next, we tested the effect of stimulating cAMP production with forskolin. Forskolin increased wild-type UT-A1 and T524A phosphorylation in LLC-PK1 cells and increased urea flux in oocytes. In contrast, the S486A and S499A mutants demonstrated loss of forskolin-stimulated UT-A1 phosphorylation and reduced urea flux. In LLC-PK1 cells, we assessed biotinylated UT-A1. Wild-type UT-A1, S486A, and S499A accumulated in the membrane in response to forskolin. However, in the S486A/S499A double mutant, forskolin-stimulated UT-A1 membrane accumulation and urea flux were totally blocked. We conclude that the phosphorylation of UT-A1 on both serines 486 and 499 is important for activity and that this phosphorylation may be involved in UT-A1 membrane accumulation.

Mutagenesis of theN-glycosylation site of hNaSi-1 reduces transport activity

2003 ◽  
Vol 285 (5) ◽  
pp. C1188-C1196 ◽  
Author(s):  
Hongyan Li ◽  
Ana M. Pajor
Keyword(s):  
Xenopus Oocytes ◽  
Polyclonal Antibodies ◽  
Membrane Vesicles ◽  
Glycosylation Site ◽  
Transport Activity ◽  
Wild Type ◽  
Glutathione S Transferase ◽  
Mutant Proteins ◽  
Amino Acid Peptide

The human Na+-sulfate cotransporter (hNaSi-1) belongs to the SLC13 gene family, which also includes the high-affinity Na+-sulfate cotransporter (hSUT-1) and the Na+-dicarboxylate cotransporters (NaDC). In this study, the location and functional role of the N-glycosylation site of hNaSi-1 were studied using antifusion protein antibodies. Polyclonal antibodies against a glutathione S-transferase fusion protein containing a 65-amino acid peptide of hNaSi-1 (GST-Si65) were raised in rabbits, purified, and then used in Western blotting and immunofluorescence experiments. The antibodies recognized native NaSi-1 proteins in pig and rat brush-border membrane vesicles as well as the recombinant proteins expressed in Xenopus oocytes. Wild-type hNaSi-1 and two N-glycosylation site mutant proteins, N591Y and N591A, were functionally expressed and studied in Xenopus oocytes. The apparent mass of N591Y was not affected by treatment with peptide- N-glycosylase F, in contrast to the mass of wild-type hNaSi-1, which was reduced by up to 15 kDa, indicating that Asn591is the N-glycosylation site. Although the cell surface abundance of the two glycosylation site mutants, N591Y and N591A, was greater than that of wild-type hNaSi-1, both mutants had greatly reduced Vmax, with no change in Km. These results suggest that Asn591and/or N-glycosylation is critical for transport activity in NaSi-1.

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