Cysteine-directed cross-linking localizes regions of the human erythrocyte anion-exchange protein (AE1) relative to the dimeric interface

Taylor, A. M.; Zhu, Qing; Casey, Joseph R.

doi:10.1042/bj3590661

Cited by 20 publications

(19 citation statements)

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“…Similar discrepancies have been observed for other proteins. In human erythrocyte anion exchange protein (Taylor et al, 2001), for example, only oxidation crosslinking or maleimide cross-linking was observed for some Cys pairs. The differences were interpreted in terms of the dynamics of the protein.…”

Section: Discussionmentioning

confidence: 99%

“…Cross-linking of Cys in either the signal anchor/linker or F-G loop regions resulted in covalently linked dimers of the 2C8 proteins. Considering the reported efficiency of cross-linking, which ranged from 25 to 80% for different Cys pairs in the human erythrocyte anion exchange protein (Taylor et al, 2001), a substantial fraction of CYP2C8 is likely to exist as a dimer.…”

Section: Discussionmentioning

confidence: 99%

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CYP2C8 Exists as a Dimer in Natural Membranes

Johnson

Kemper

2010

Drug Metab Dispos

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ABSTRACT:CYP2C8 with a modified N-terminal sequence (2C8H) crystallizes as a dimer, but it is not known whether native CYP2C8 exists as a dimer in natural membranes. We have examined the organization of 2C8H and CYP2C8 expressed in bacterial membranes and mammalian endoplasmic reticulum membranes, respectively, by cysteine scanning and cross-linking or oxidation of sulfhydryl groups. In both forms of CYP2C8, cross-linked dimers were observed that were eliminated by mutation of Cys-24 in the linker region. Introduction of individual cysteines in the N-terminal 21-amino acid membrane-spanning signal anchor resulted in a pattern of crosslinking consistent with an ␣-helical structure for the signal anchor. In the linker region, cross-linking was observed for cysteine substituted at residues 22, 23, or 24, just before three Arg residues, indicating close apposition of the two linker sequences despite the neighboring positive charges. Introduction into the F-G loop region of cysteine pairs optimally located for cross-linking based on the crystal structure resulted in cross-linked dimers in the Cys-24 mutant. Deletion of the signal anchor sequence eliminated crosslinking mediated by Cys-24 or by cysteines introduced in the F-G loop regions, indicating that the signal anchor interaction is required for stable dimer formation. These results indicate that the signal anchor sequence and the F-G loop region form interfaces for CYP2C8 intermolecular interactions in natural membranes.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

CYP2C8 Exists as a Dimer in Natural Membranes

Johnson

Kemper

2010

Drug Metab Dispos

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“…Cysteine-scanning mutagenesis also favored this orientation (37). However, in this domain, Lys 743 was shown to be accessible from the extracellular side or intracellular side depending on the kind of experiments (38,39). Kanki et al (40) have proposed an explanation to these contradictory observations, and they concluded that during synthesis this region is accessible from the extracellular side, and it is later occluded within the AE1 molecule.…”

Section: Discussionmentioning

confidence: 99%

Structural Model of the Anion Exchanger 1 (SLC4A1) and Identification of Transmembrane Segments Forming the Transport Site

Barneaud-Rocca

Etchebest

Guizouarn

2013

Journal of Biological Chemistry

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Background: There is still no high resolution three-dimensional structure available for the membrane-spanning domain of anion exchanger 1 (AE1). Results: A three-dimensional model of AE1 membrane-spanning domain has been generated in silico and experimentally assessed. Conclusion: Transmembrane segments forming AE1 transport site have been identified. Significance: This is the first three-dimensional model of AE1 membrane-spanning domain based on a cation symporter.The anion exchanger 1 (AE1), a member of bicarbonate transporter family SLC4, mediates an electroneutral chloride/bicarbonate exchange in physiological conditions. However, some point mutations in AE1 membrane-spanning domain convert the electroneutral anion exchanger into a Na ؉ and K ؉ conductance or induce a cation leak in a still functional anion exchanger. The molecular determinants that govern ion movement through this transporter are still unknown. The present study was intended to identify the ion translocation pathway within AE1. In the absence of a resolutive three-dimensional structure of AE1 membrane-spanning domain, in silico modeling combined with site-directed mutagenesis experiments was done. A structural model of AE1 membrane-spanning domain is proposed, and this model is based on the structure of a uracilproton symporter. This model was used to design cysteine-scanning mutagenesis on transmembrane (TM) segments 3 and 5. By measuring AE1 anion exchange activity or cation leak, it is proposed that there is a unique transport site comprising TM3-5 and TM8 that should function as an anion exchanger and a cation leak.The anion exchanger 1 (AE1, 2 SLC4A1, or band 3) is the main membrane protein in vertebrate red cells. It fulfills different tasks in these cells: a structural role by linking plasma membrane to cytoskeleton, a respiratory role by improving CO 2 transport capacity of red cells, and an antigenic function (Diego blood group and senescence), and it is also involved in cytokinesis and red cell volume regulation (1). At the molecular level, this protein is divided into two main structural domains: a cytoplasmic N-terminal domain (about 400 amino acids) and a membrane-spanning domain (about 450 amino acids) with a short C-terminal tail in the cytoplasm. These two entities seem to function independently: the large cytoplasmic domain is involved in interactions with enzymes, hemoglobin, and structural proteins, whereas the membrane-spanning domain is responsible for the transport activity of the protein (2, 3). In addition to red cells, AE1 is also expressed in kidney ␣-intercalated cells and cardiac myocytes (4). In physiological conditions, AE1 exchanges one chloride for one bicarbonate by an electroneutral transport mechanism with the driving force for ion movement provided by their electrochemical gradient. A few years ago, some specific point mutations were characterized and proposed to convert the electroneutral anion exchanger into a cation-conductive pathway (5, 6). These mutations are associated with human pathologies (hered...

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“…In either case, the conformational alterations within the AE1 SAO/wild-type heterodimer, detectable in ovalocytes as increased protease sensitivity and increased access to surface biotinylation (46), do not reduce detectably the efficiency of AE1 SAO-mediated rescue of Cl Ϫ /HCO 3 Ϫ exchange by AE1 LDAAA in Xenopus oocytes. Taylor et al (58) have documented inter-protomeric cross-linking between identical introduced cysteine residues in the Nterminal-most four exofacial loops connecting AE1 transmembrane spans, as well as less extensive cross-linking at the fifth (possibly re-entrant) exofacial loop. However, aa 821 in another candidate re-entrant loop, and aa 855 in the C-terminal-most external loop, were not subject to cross-linking, consistent with an earlier model of the AE1 homodimer (59) and with chemical cross-linking of the related AE2 anion exchanger (60).…”

Section: Interprotomeric Rescue Of Ae1-mediated Hcomentioning

confidence: 99%

Deficient HCO3- Transport in an AE1 Mutant with Normal Cl- Transport Can be Rescued by Carbonic Anhydrase II Presented on an Adjacent AE1 Protomer

Dahl¹,

Jiang

Chernova

et al. 2003

Journal of Biological Chemistry

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؊ exchange by an AE1 missense mutant devoid of CA2 binding, with activity further enhanced by CA2 co-expression. The same transport-incompetent AE1 mutants failed to rescue Cl ؊ /HCO 3 ؊ exchange by the AE1 truncation mutant 896X, despite preservation of the latter's core CA2 binding site. These data increase the minimal extent of a functionally defined CA2 binding site in AE1. The inter-protomeric rescue of HCO 3 ؊ transport within the AE1 dimer shows functional proximity of the C-terminal cytoplasmic tail of one protomer to the anion translocation pathway in the adjacent protomer within the AE1 heterodimer. The data strongly support the hypothesis that an intact transbilayer anion translocation pathway is completely contained within an AE1 monomer.

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Cysteine-directed cross-linking localizes regions of the human erythrocyte anion-exchange protein (AE1) relative to the dimeric interface

Cited by 20 publications

References 29 publications

CYP2C8 Exists as a Dimer in Natural Membranes

CYP2C8 Exists as a Dimer in Natural Membranes

Structural Model of the Anion Exchanger 1 (SLC4A1) and Identification of Transmembrane Segments Forming the Transport Site

Deficient HCO3- Transport in an AE1 Mutant with Normal Cl- Transport Can be Rescued by Carbonic Anhydrase II Presented on an Adjacent AE1 Protomer

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