Functional consequences of mutations in the transmembrane domain and the carboxy-terminus of the murine AE1 anion exchanger

M. N. Chernova, B. D. Humphreys, D. H. Robinson, A. K. Stuart-Tilley, A. M. Garcia, F. C. Brosius, S. L. Alper

Research output: Contribution to journalArticle

26 Scopus citations

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/M730I. 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/M730I 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 Δ(c)890 and the internal deletion Δ(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.

Original languageEnglish
Pages (from-to)111-123
Number of pages13
JournalBiochimica et Biophysica Acta - Biomembranes
Volume1329
Issue number1
DOIs
StatePublished - Oct 2 1997
Externally publishedYes

Keywords

  • Autoradiography
  • Chloride bicarbonate exchange
  • Chymotrypsin
  • Immunoprecipitation
  • In vitro translation
  • Isotopic flux
  • Metabolic labeling
  • Mutagenesis
  • Plasma membrane
  • Proteolysis
  • Xenopus oocyte

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