Conformational changes in the First Transmembrane Helix of the H+-ATPase, AHA2, during H+ Transport

Abstract

Conformational changes in the first transmembrane alpha helix of the Arabidopsis H+ pump, AHA2, were studied with a cysteine scanning mutagenesis technique when the enzyme was expressed in Saccharomyces. Cysteine-reactive compounds, including N-ethylmaleimide (NEM), fluorescein maleimide (FM), positively (MTSET) and negatively charged methanethiosulfonate (MTSES) reagents, were applied to intact yeast that were actively respiring (non-starved) or cultured overnight at 4°C in glucose-free media (starved). When yeast were exposed to FM, extracellular accessibility of substituted cysteines was determined by measuring fluorescent protein on PAGE gels. In respiring yeast, FM bound only to residues predicted to be in the extracellular 1-2 loop of AHA2; however, in starved yeast, FM binding was observed at residues predicted to be in the extracellular 1-2 loop and deep within the first transmembrane alpha helix, down to residue 73. Pre-incubation with other cysteine-reactive agents showed that MTSET only bound to residues in the extracellular 1-2 loop and MTSES did not bind at all in this area of the enzyme. These results suggest that the conformation of the first transmembrane helix, or adjacent alpha helices, depend on enzyme transport activity, with the adjoining extracellular loop favoring binding of positively-charged ions. One interpretation of these data is that conformational changes involving the first transmembrane alpha helix affect the disposition of H2O molecules in the membrane domain of the enzyme. These data, together with crystallographic data for other P-type ATPases, suggest that this region in the enzyme's transmembrane domain contains an extracellularly-facing cavity. We speculate that this cavity could contribute to a release pathway for H+ during the ion transport cycle.