Topology and sites in the H,K-ATPase

Understanding the membrane topology of the EP-type pumps has been approached largely by analysis of hydrophobicity plots, which are confusing in the COOH-terminal third of the proteins. Each pair of predicted membrane-spanning segments with the extracytoplasmic loop contains at least one cysteine, allowing fluorescent labeling of these regions of the enzymes by cysteine reagents once the cytoplasmic domain has been removed. The membrane segment arrangement of the H,K and sr Ca ATPases was investigated by tryptic cleavage of intact cytoplasmic face-out vesicles. This was followed by fluorescein or coumarin maleimide labeling of the SDS solubilized residual membrane fragments, tricine gradient gel separation, and sequencing. The presence of four membrane-spanning pairs was demonstrated for the alpha subunit of the H,K-ATPase, with no membrane retention of H9 and H10, although H9 has four cysteines based on cDNA sequencing. A similar observation was made for the Ca pump, except that fluorescein-labeled H9 was detected in the membrane with a molecular weight of 4 kD, showing that cleavage had occurred at lys958 predicted to be extracytoplasmic in a 10 membrane segment model. It seems likely that for both these enzymes the membrane domain contains only 8 alpha helical spanning segments. Cleavage at ala236 in the beta subunit was found only in leaky, not in ion-tight vesicles, arguing for a single membrane segment in this subunit. In the H,K-ATPase additional evidence for the presence and arrangement of the first, third, and fourth pair of segments was obtained by labeling the intact enzyme with extracytoplasmic inhibitory reagents. The K competitive reagent, an imidazopyridine, MeDAZIP+, labeled the first pair of membrane segments. The acid-activated SH reagent class, the pyridinyl methyl sulfinyl benzimidazoles, labeled cysteines 813 and 822 in the M5/M6 region as well as cysteine 892 in the extracytoplasmic loop between M7 and M8. No labeling of the beta subunit was found, indicating the presence of three disulfide bonds in the extracytoplasmic domain of this subunit. Both sets of extracytoplasmic reagents are predicted to bind close to the fatty acid/phospholipid head group interface. Inhibition by these reagents shows that conformational changes are transmitted between cytoplasmic and extracytoplasmic domains.