CLONING AND MEMBRANE TOPOLOGY OF A P-TYPE ATPASE FROM HELICOBACTER-PYLORI

Southern blot screening of a genomic Helicobacter pylori library was e mployed to find a P type ATPase using a mixture of 16 DNA oligonucleot ides coding for the DKTGT(I/L)T consensus sequence specific for the ph osphorylation site of this family of ATPases, A positive clone, pRH439 , was isolated and sequenced, The inserted 3.4-kb H. pylori DNA contai ned an intact open reading frame encoding a protein of 686 amino acids carrying the consensus sites for phosphorylation and ATP binding, The amino acid sequence exhibits a 25-30% identity with bacterial Cd2+ an d Cu2+ ATPases, Genomic Southern blot analysis showed that this ATPase was present in all H. pylori strains examined, whereas it was not det ectable in Campylobacter jejuni and other bacteria, The membrane topol ogy of this ATPase was investigated using in vitro transcription/trans lation of fusion vectors to find signal anchor and/or stop transfer se quences. Eight regions of the II, pylori ATPase acted as signal anchor and/or stop transfer sequences and were ordered pairwise along the po lypeptide chain placing the N and C-terminal amino acids in the cytopl asm, These transmembrane segments are contained between positions 73 a nd 92 (H1), 98 and 125 (H2), 128 and 148 (H3), 149 and 176 (H4), 309 a nd 327 (H5), 337 and 371 (H6), 637 and 658 (H7), and 659 and 685 (HS), The membrane domain of the ATPase, therefore, consists of at least fo ur pairs of transmembrane segments with the phosphorylation site and A TP binding domain located in the large cytoplasmic loop between H6 and H7. The cytoplasmic domain contains several histidines and cysteines, perhaps indicative of divalent cation binding sites. There are severa l charged amino acids (3 Lys, 2 Glu, 2 Asp) predicted to be in the mem brane domain mainly in H2, H3, and H4 and a Cys-Pro-Cys putative metal ion site in H6. The extracytoplasmic domain also has several charged amino acids (5 Glu, 1 Asp, 1 Lye, 1 Arg), It is likely that this novel protein is a heavy metal cation transporting ATPase and belongs to a family of P type ATPases containing eight transmembrane segments.