Functional analysis of polar amino-acid residues in membrane associated regions of the NHE1 isoform of the mammalian Na+/H+ exchanger.

The NHE1 isoform of the Na+/H+ exchanger is a ubiquitous plasma membrane protein that regulates intracellular pH in mammalian cells. Site-specific mutagenesis was used to examine the functional role of conserved, polar amino-acid residues occurring in segments of the protein associated with the membrane. Seventeen mutant proteins were assessed by characterization of intracellular pH changes in stably transfected cells that lacked an endogenous Na+/H+ exchanger. All of the mutant proteins were targeted correctly to the plasma membrane and were expressed at similar levels. Amino-acid residues Glu262 and Asp267 were critical to Na+/H+ exchanger activity while mutation of Glu391 resulted in only a partial reduction in activity. The Glu262-->Gln mutant was expressed partially as a deglycosylated protein with increased sensitivity to trypsin treatment in presence of Na+. Substitution of mutated Glu262, Asp267 and Glu391 with alternative acidic residues restored Na+/H+ exchanger activity. The Glu262-->Asp mutant had a decreased affinity for Li+, but its activity for Na+ and H+ ions was unaffected. The results support the hypothesis that side-chain oxygen atoms in a few, critically placed amino acids are important in Na+/H+ exchanger activity and the acidic amino-acid residues at positions 262, 267 and 391 are good candidates for being involved in Na+ coordination by the protein.

Isolation and characterization of the Saccharomyces cerevisiae SDH4 gene encoding a membrane anchor subunit of succinate dehydrogenase.

Succinate dehydrogenase (EC 1.3.99.1) is an intrinsic bacterial or inner mitochondrial membrane protein that catalyses the oxidation of succinate and donates electrons to the respiratory chain via quinone acceptors. It is a heterotetramer composed of a flavoprotein, an iron-sulfur, and two hydrophobic subunits. We purified succinate dehydrogenase by blue native gel electrophoresis, determined the amino-terminal sequence of the Sdh4p subunit and used this information to clone the SDH4 gene. It encodes a precursor protein of 181 amino acids that is converted to the 150-amino acid mature Sdh4p protein with a mass of 16,638 Da. Hydrophobicity analysis predicts that Sdh4p forms three transmembrane alpha-helices. We have constructed an SDH4 mutant by targeted gene disruption; it retains the ability to grow on rich glycerol medium. Western blot analysis of SDH4 disruption mutant membrane fractions indicates that membrane attachment of the flavoprotein and iron-sulfur subunits is impaired but not abolished. This membrane-bound enzyme is able to reduce ubiquinone, although less efficiently than the wild-type enzyme. These findings indicate that Sdh4p contributes both to the membrane attachment of the catalytic flavoprotein and iron-sulfur subunits and to electron transfer to ubiquinone.