title: Identification of membrane topography of the electrogenic sodium bicarbonate cotransporter pNBC1 by in vitro transcription/translation.
abstract: The transmembrane topography of the human pancreatic electrogenic sodium bicarbonate cotransporter pNBC1 was investigated using in vitro transcription/translation of HK-M0 and HK-M1 fusion vectors designed to test membrane insertion properties of pNBC1 hydrophobic sequences (H). These vectors encode N-terminal 101 (HK-M0) or 139 (HK-M1) amino acids of the H,K-ATPase alpha-subunit, a linker region and the C-terminal 177 amino acids of the H,K-ATPase beta-subunit that contain five N-linked glycosylation consensus sites (Bamberg, K., and Sachs, G. (1994) J. Biol. Chem. 269, 16909-16919). The glycosylation status of the beta-region was used as a reporter to determine whether a given hydrophobic sequence possesses signal anchor and/or stop transfer properties in the HK-M0 and HK-M1 vectors. The linker region of each vector was replaced either with individual hydrophobic sequences or combinations thereof. The transcription/translation products of these fusion vectors in reticulocyte lysate system +/- microsomal membranes were identified by [(35)S]-autoradiography following separation using SDS-PAGE. The results of the in vitro transcription/translation analysis indicated that 10 (H1, H2N, H3, H5, H6, H7, H8, H9, H11, and H12) out of 12 hydrophobic sequences were able to insert into the plasma membrane. Two hydrophobic sequences, H4 and H10, had no membrane insertion activity even when upstream and downstream sequences were present. These data and immunocytochemical studies indicate that pNBC1 contains 10 transmembrane domains with N- and C-termini oriented intracellularly. This is the first characterization of the membrane topography of a sodium bicarbonate cotransporter.
authors.affiliation: Division of Nephrology, David Geffen School of Medicine at UCLA, Los Angeles, California 90095, USA.
authors.forename: Sergei
authors.initials: S
authors.lastname: Tatishchev
authors.forename: Natalia
authors.initials: N
authors.lastname: Abuladze
authors.forename: Alexander
authors.initials: A
authors.lastname: Pushkin
authors.forename: Debra
authors.initials: D
authors.lastname: Newman
authors.forename: Weixin
authors.initials: W
authors.lastname: Liu
authors.forename: David
authors.initials: D
authors.lastname: Weeks
authors.forename: George
authors.initials: G
authors.lastname: Sachs
authors.forename: Ira
authors.initials: I
authors.lastname: Kurtz
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001693:Biological Transport, Active / Q000235:genetics; D002460:Cell Line; D003593:Cytoplasm / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004285:Dogs; D005822:Genetic Vectors; D006031:Glycosylation; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D007425:Intracellular Membranes / Q000737:chemistry* / Q000378:metabolism; D017136:Ion Transport / Q000235:genetics; D008861:Microsomes / Q000737:chemistry / Q000378:metabolism; D008969:Molecular Sequence Data; D010179:Pancreas / Q000737:chemistry / Q000378:metabolism*; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014176:Protein Biosynthesis; D017434:Protein Structure, Tertiary / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D027982:Sodium-Bicarbonate Symporters / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D014158:Transcription, Genetic
chemical_list: D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D027982:Sodium-Bicarbonate Symporters