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Topology of the C‐terminal Transmembrane Region of NBCe1‐A Differs from AE1
Author(s) -
Zhu Quansheng,
Kao Liyo,
Liu Weixin,
Newman Debra,
Orozco Nicholas,
Azimov Rustam,
Pushkin Alexander,
Abuladze Natalia,
Kurtz Ira
Publication year - 2009
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.23.1_supplement.800.8
Subject(s) - membrane topology , transmembrane domain , transmembrane protein , cysteine , topology (electrical circuits) , chemistry , biochemistry , biophysics , amino acid , biology , enzyme , mathematics , receptor , combinatorics
Sodium‐dependent bicarbonate cotransporter 1 (NBCe1) and sodium‐independent chloride/bicarbonate exchanger 1 (AE1) belong to the SLC4 transport family of membrane proteins that mediate bicarbonate transport. The extensively analyzed topology of AE1 highlights the structural and functional importance of the C‐terminal transmembrane region, which is considered to form the anion transport site. Residues in the C‐terminal transmembrane region of NBCe1‐A and AE1 are 40% homologous, however, no information is available on the topology of this region in NBCe1‐A. In this study, we performed introduced cysteine scanning mutagenesis coupled with sulfhydryl specific chemistry in the C‐terminal transmembrane region of NBCe1‐A. One hundred sixty cysteine mutations were individually introduced between Ala800 and Asp960 on a NBCe1‐A construct in which five cytoplasmic endogenous cysteines were changed to serine. These mutations completely covered the C‐terminal transmembrane region of NBCe1‐A, which includes the two proposed re‐entrant loops in AE1. The location of introduced cysteine residues were determined by whole cell labeling with two cysteine specific reagents: membrane permeant biotin maleimide (BM) and membrane impermeant MTS‐TAMRA. Of 160 introduced cysteine mutants, 31 were labeled with BM and 10 were labeled with MTS‐TAMRA. Our data demonstrate that topology of the C‐terminal transmembrane region of NBCe1‐A differs from AE1 in the following aspects: 1) NBCe1‐A lacks the two AE1 re‐entrant loops; 2) NBCe1‐A has 5 rather than 4 transmembrane helices (TM) in this region; 3) the average size of NBCe1‐A C‐terminal TMs is greater than AE1. On the basis of results, we propose a new topology model for the C‐terminal transmembrane region of NBCe1‐A. Supported by NIH