title: The role of aspartic acid residues 405 and 416 of the kidney isotype of sodium-bicarbonate cotransporter 1 in its targeting to the plasma membrane.
abstract: The NH(2) terminus of the sodium-bicarbonate cotransporter 1 (NBCe1) plays an important role in its targeting to the plasma membrane. To identify the amino acid residues that contribute to the targeting of NBCe1 to the plasma membrane, polarized MDCK cells were transfected with expression constructs coding for green fluorescent protein (GFP)-tagged NBCe1 NH(2)-terminal deletion mutants, and the localization of GFP-tagged proteins was analyzed by confocal microscopy. Our results indicate that the amino acids between residues 399 and 424 of NBCe1A contain important sequences that contribute to its localization to the plasma membrane. Site-directed mutagenesis studies showed that GFP-NBCe1A mutants D405A and D416A are retained in the cytoplasm of the polarized MDCK epithelial cells. Examination of functional activities of D405A and D416A reveals that their activities are reduced compared with the wild-type NBCe1A. Similarly, aspartic acid residues 449 and 460 of pancreatic NBCe1 (NBCe1B), which correspond to residues 405 and 416 of NBCe1A, are also required for its full functional activity and accurate targeting to the plasma membrane. In addition, while replacement of D416 with glutamic acid did not affect the targeting or functional activity of NBCe1A, substitution of D405 with glutamic acid led to the retention of the mutated protein in the intracellular compartment and impaired functional activity. These studies demonstrate that aspartic acid residues 405 and 416 in the NH(2) terminus of NBCe1A are important in its accurate targeting to the plasma membrane.
authors.affiliation: Department of Medicine, University of Cincinnati, Ohio 45267-0585, USA. Hong.Li@uc.edu
authors.forename: Hong C
authors.initials: HC
authors.lastname: Li
authors.forename: Volodymyr
authors.initials: V
authors.lastname: Kucher
authors.forename: Emily Y
authors.initials: EY
authors.lastname: Li
authors.forename: Laura
authors.initials: L
authors.lastname: Conforti
authors.forename: Kamyar A
authors.initials: KA
authors.lastname: Zahedi
authors.forename: Manoocher
authors.initials: M
authors.lastname: Soleimani
mesh_terms: D000818:Animals; D001224:Aspartic Acid; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D016764:Cell Polarity; D004285:Dogs; D004847:Epithelial Cells / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000378:metabolism; D007668:Kidney / Q000166:cytology / Q000378:metabolism*; D008564:Membrane Potentials; D018613:Microscopy, Confocal; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009865:Oocytes / Q000378:metabolism; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D027982:Sodium-Bicarbonate Symporters / Q000235:genetics / Q000378:metabolism*; D013997:Time Factors; D014162:Transfection; D014981:Xenopus
chemical_list: D011993:Recombinant Fusion Proteins; D027982:Sodium-Bicarbonate Symporters; D049452:Green Fluorescent Proteins; D001224:Aspartic Acid