Detergent interaction with band 3, a model polytopic membrane protein

Casey, Joseph R.; Reithmeier, Reinhart A.F.

doi:10.1021/bi00055a023

Cited by 55 publications

(33 citation statements)

References 41 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…Similarly, we demonstrated that six three-fragment combinations, two four-fragment combinations and, unexpectedly, a non-complementary pair of fragments lacking putative TM spans 6 and 7 generated DNDSsensitive chloride uptake [18]. By using non-denaturing detergents that are known to maintain the native structure of b3mem [19] followed by co-immunoprecipitation, we showed that at least five of these complementary pairs of fragments were able to coassemble specifically in oocyte and microsomal membranes [20]. The amino acid sequences of a large number of multispanning membrane proteins that act as transporters, channels or receptors have now been determined.…”

Section: Introductionmentioning

confidence: 59%

“…We have shown that contiguous pairs of band 3 fragments divided in the first, second, third or fourth exofacial loops or the fourth cytoplasmic loop co-associate in a specific and concentration-dependent manner [20]. This result was obtained by the solubilization of membranes containing these pairs of fragments in the non-ionic detergent Triton X-100, which does not denature band 3 [19], and then co-immunoprecipitation with the anti-(band 3) monoclonal antibodies BRIC155 (directed against the C-terminus of band 3) or BRIC170 (directed against the N-terminus of b3mem) and Protein A-agarose.…”

Section: Discussionmentioning

confidence: 96%

See 1 more Smart Citation

Structural model for the organization of the transmembrane spans of the human red-cell anion exchanger (band 3; AE1)

Groves

Tanner

1999

Biochemical Journal

View full text Add to dashboard Cite

We have examined the functional co-assembly of non-complementary pairs of N-and C-terminal polypeptide fragments of the anion transport domain (b3mem) of human red-cell band 3. cDNA clones encoding non-contiguous pairs of fragments with one transmembrane (TM) region omitted, or overlapping pairs of fragments with between one and ten TM regions duplicated, were co-expressed in Xenopus oocytes and a cell-free translation system. Stilbene disulphonate-sensitive chloride uptake assays in oocytes revealed that the omission of any single TM region of b3mem except spans 6 and 7 caused a complete loss of functional expression. In contrast, co-expressed pairs of fragments overlapping a single TM region 5, 6, 7, 8, 9-10 or 11-12 retained a high level of functionality, whereas fragments overlapping the clusters of TM regions 2-5, 4-5, 5-8 and 8-10 also mediated some stilbene disulphonate-sensitive uptake. The co-assembly of N-or C-terminal fragments with intact band 3, b3mem or other fragments was examined by co-immunoprecipitation in non-

show abstract

Section: Introductionmentioning

confidence: 59%

Section: Discussionmentioning

confidence: 96%

Structural model for the organization of the transmembrane spans of the human red-cell anion exchanger (band 3; AE1)

Groves

Tanner

1999

Biochemical Journal

View full text Add to dashboard Cite

show abstract

“…This is in contrast to the Band 3 protein from erythrocytes. This polytopic membrane protein (molecular mass of 95 kDa) eluted between thyroglobulin (667 kDa) and ferritin (440 kDa) protein markers during gel exclusion chromatography in the presence of C 12 E 8 detergent (23,24). Its large size was consistent with its being a dimer-detergent complex, and it was calculated that each monomer associated with one micelle of detergent.…”

Section: Figmentioning

confidence: 91%

The Minimum Functional Unit of Human P-glycoprotein Appears to be a Monomer

Loo

Clarke

1996

Journal of Biological Chemistry

View full text Add to dashboard Cite

Several studies have demonstrated the presence of oligomers of P-glycoprotein in multidrug-resistant cells. The minimum functional unit of P-glycoprotein, however, is not known. In order to determine whether the functional unit is an oligomer, we tested for associations between P-glycoproteins containing either a histidine tag or the epitope tag for monoclonal antibody A52 at the COOH-terminal end of the molecule. Both tagged molecules were active and had indistinguishable drug resistance profiles. The tagged P-glycoproteins were expressed contemporaneously in HEK 293 cells, purified by nickel-chelate chromatography followed by immunoblot analysis. We found that P-glycoprotein-A52 did not copurify with functionally active P-glycoprotein-(His) 10 , even when the former was overexpressed relative to the histidine-tagged protein. Similar results were obtained with phosphorylation-deficient mutants of P-glycoprotein. By contrast, we could purify and reconstitute drugstimulated ATPase activity when the half-molecules NH 2 -terminal half-(His) 10 /COOH-terminal half-A52 or NH 2 -terminal half-A52/COOH-terminal half-(His) 10 were coexpressed in HEK 293 cells. These results suggest that nickel-chelate chromatography may be a suitable method for studying protein-protein interactions in membrane proteins and that the minimal functional unit of P-glycoprotein is likely to be a monomer. P-glycoprotein is an ATP-dependent pump that transports a wide range of cytotoxic compounds out of the cell (1, 2). It may be one of several mechanisms responsible for multidrug resistance during chemotherapy. P-glycoprotein is expressed in a wide variety of tissues and cells. Its physiological role is unknown but it may act to protect the organism from endogenous and exogenous cytotoxic compounds. This is supported by studies on "knock-out" mice that were homozygous for a disruption of the mdr1a gene (3-5).Cloning and sequencing of human MDR1 (6) showed that P-glycoprotein contains 1280 amino acids organized into two tandem repeats of 610 amino acids joined by a linker region of 60 amino acids. Each repeat consists of an NH 2 -terminal hydrophobic domain containing six potential transmembranespanning helices followed by a hydrophilic domain containing a nucleotide-binding site. The arrangement is supported by the results of topology studies on the full-length molecule (7,8).The mechanism by which P-glycoprotein is able to couple ATP hydrolysis to the efflux of a broad range of lipophilic compounds is unknown. Although each half-molecule of Pglycoprotein possesses basal ATPase activity, drug-stimulated ATPase activity was only observed when both halves were expressed contemporaneously (9). Both nucleotide-binding sites are also essential for function since mutations in either site (10, 11) or chemical modification of either site (12, 13) inactivates any coupling of ATPase activity to drug binding. P-glycoprotein has been detected to be present in the membrane in the form of monomers, dimers, and higher oligomers (14 -17). In the membranes of...

show abstract

“…Since a properly arranged oligomeric complex appeared to be required for D2 dopamine receptor trafficking, we considered the possibility that monomers visualized following gel electrophoresis may have been part of a larger oligomeric complex formed prior to cell surface expression. Sodium dodecyl sulfate (SDS) has been shown to dissociate subunits of multimeric membrane proteins (Casey and Reithmeier, 1993). To investigate the possibility that oligomers were dissociated by SDS solubilization, we employed a native, non-denaturing gel electrophoresis technique in which the detergent used is perfluoro-octanoic acid (PFO) and not SDS (Ramjeesingh et al, 1999).…”

Section: Receptor Monomers In the Plasma Membranementioning

confidence: 99%