Functional role of cysteine residues in the Na+/H+ exchanger effects of mutation of cysteine residues on targeting and activity of the Na+/H+ exchanger.
We investigated the role of cysteine residues in activity and localization of the NHE1 isoform of the Na+/H+ exchanger. Each of the nine cysteine residues was mutated to serine or arginine. Mutation of the first serine (amino acid number 9) and serine number six (amino acid number 477) resulted in dramatic decreases in detectable activity of the Na+/H+ exchanger when transfected into AP-1 cells. Some other mutations resulted in minor decreases in activity of the protein. Confocal and light microscopy of mutant cells with decreased activity showed that the antiporter protein was mostly retained in an intracellular compartment which colocalized with the medial-Golgi cisternae. Smaller amounts of active protein still remained targeted to the plasma membrane in these mutants. Treatment of wild-type cells with DTT also caused the retention of the Na+/H+ exchanger to the same intracellular compartment. The results suggest that cysteines play an important role in intracellular folding and trafficking of the Na+/H+ exchanger.
Wang
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Department of Biochemistry, University of Alberta, 347 Medical Science Building, Edmonton, Alberta, T6G 2H7, Canada.
Singh
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Fliegel
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D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D001120:Arginine / Q000235:genetics; D002460:Cell Line; D006224:Cricetinae; D003545:Cysteine / Q000235:genetics / Q000502:physiology*; D006801:Humans; D007536:Isomerism; D051379:Mice; D016297:Mutagenesis, Site-Directed; D011817:Rabbits; D051381:Rats; D012694:Serine / Q000235:genetics; D017923:Sodium-Hydrogen Exchangers / Q000235:genetics / Q000502:physiology*; D013552:Swine
D017923:Sodium-Hydrogen Exchangers; C069139:growth factor-activatable Na-H exchanger NHE-1; D012694:Serine; D001120:Arginine; D003545:Cysteine