The A1A0 ATPase from Methanosarcina mazei:: Cloning of the 5′ end of the aha operon encoding the membrane domain and expression of the proteolipid in a membrane-bound form in Escherichia coli

Three additional ATPase genes, clustered in the order ahaH, ahaI, and ahaK were found upstream of the previously characterized genes ahaECFABDG coding for the archaeal A(l)4(o) ATPase front Methanosarcina mazei. ahaH the first gene in the cluster, is preceded by a conserved promoter sequence. Northern blot analysis revealed that the clusters ahaHIK and akaECFABDG are transcribed as one message. AhaII is a hydrophilic polypeptide and is similar to peptides of previously unassigned function encoded by genes preceding postulated ATPase genes in Methanobacterium thermoautotrophicum and Methanococcus jannaschii..AhaI has a two-domain structure with a hydrophilic domain of 39 kDa and a hydrophobic domain with seven predicted transmembrane alpha helices, It is similar to the 100-kDa polypeptide of VlVo ATPases and is therefore suggested to participate in proton transport. AhaK is a hydrophobic polypeptide with two predicted transmembrane alpha helices and, on the basis of sequence comparisons and immunological studies, is identified as the proteolipid, a polypeptide which is essential for proton translocation. However, it is only one-half and one-third the size of the proteolipids from M. thermautotrophicum and M. jannaschii, respectively. ahaK is expressed in Escherichia coli, and it is incorporated into the cytoplasmic membrane despite the different chemical natures of lipids from archaea and bacteria, This is the first report on the expression and incorporation into E. coli lipids of a membrane integral enzyme from a methanogens, which will facilitate analysis of the structure and function of the membrane domain of the methanoarchaeal ATPase.