title: Functional consequences of mutations in the transmembrane domain and the carboxy-terminus of the murine AE1 anion exchanger.
abstract: We have characterized mouse AE1-mediated 36Cl- influx and surface AE1 polypeptide expression in Xenopus oocytes injected with cRNA encoding two classes of loss-of-function mutants. The first arose spontaneously. Chimeric mutants constructed with a functional AE1 cDNA localized the site of spontaneous mutation to the transmembrane domain, and DNA sequencing revealed two missense mutations encoding the double-mutant polypeptide V728F/M7301. Each mutation individually produced only partial loss of AE1 transport activity, and coexpression of the individual mutants did not restore full activity. The functional changes produced by the mutations correlated with reduced fractional accumulation of polypeptides at the oocyte surface. The V728F/M7301 polypeptide expressed in mammalian cells displayed complete endoH resistance and rapid degradation. We also examined the effect on AE1 function of engineered removal of its hydrophilic carboxy-terminus. Both delta(c)890 and the internal deletion delta(c)890-917 were functionally inactive in Xenopus oocytes. Lack of transport activity correlated with lack of detectable polypeptide accumulation at the oocyte surface. Coexpression with wt AE1 of some, but not all, of these AE1 mutants partially suppressed wt AE1-mediated 36Cl- uptake. In contrast, coexpression with wt AE1 of soluble N-terminal AE1 fragments was not inhibitory.
authors.affiliation: Beth Israel Hospital, Department of Medicine, Harvard Medical School, Boston, MA 02215, USA.
authors.forename: M N
authors.initials: MN
authors.lastname: Chernova
authors.forename: B D
authors.initials: BD
authors.lastname: Humphreys
authors.forename: D H
authors.initials: DH
authors.lastname: Robinson
authors.forename: A K
authors.initials: AK
authors.lastname: Stuart-Tilley
authors.forename: A M
authors.initials: AM
authors.lastname: Garcia
authors.forename: F C
authors.initials: FC
authors.lastname: Brosius
authors.forename: S L
authors.initials: SL
authors.lastname: Alper
mesh_terms: D000818:Animals; D001457:Anion Exchange Protein 1, Erythrocyte / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002712:Chlorides / Q000378:metabolism* / Q000493:pharmacokinetics; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D005786:Gene Expression Regulation; D051379:Mice; D008845:Microinjections; D016297:Mutagenesis, Site-Directed; D009865:Oocytes / Q000378:metabolism; D014176:Protein Biosynthesis / Q000235:genetics; D018075:RNA, Complementary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017422:Sequence Analysis, DNA; D014162:Transfection / Q000235:genetics; D014981:Xenopus
chemical_list: D001457:Anion Exchange Protein 1, Erythrocyte; D002712:Chlorides; D018075:RNA, Complementary; D011993:Recombinant Fusion Proteins