Cloning and Membrane Topology of a P type ATPase from Helicobacter pylori

Abstract

Southern blot screening of a genomic Helicobacter pylori library was employed to find a P type ATPase using a mixture of 16 DNA oligonucleotides coding for the DKTGT(I/L)T consensus sequence specific for the phosphorylation site of this family of ATPases. A positive clone, pRH439, was isolated and sequenced. The inserted 3.4-kb H. pylori DNA contained 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+ and Cu2+ ATPases. Genomic Southern blot analysis showed that this ATPase was present in all H. pylori strains examined, whereas it was not detectable in Campylobacter jejuni and other bacteria. The membrane topology of this ATPase was investigated using in vitro transcription/translation of fusion vectors to find signal anchor and/or stop transfer sequences. Eight regions of the H. pylori ATPase acted as signal anchor and/or stop transfer sequences and were ordered pairwise along the polypeptide chain placing the N and C-terminal amino acids in the cytoplasm. These transmembrane segments are contained between positions 73 and 92 (H1), 98 and 125 (H2), 128 and 148 (H3), 149 and 176 (H4), 309 and 327 (H5), 337 and 371 (H6), 637 and 658 (H7), and 659 and 685 (H8). The membrane domain of the ATPase, therefore, consists of at least four pairs of transmembrane segments with the phosphorylation site and ATP 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 several charged amino acids (3 Lys, 2 Glu, 2 Asp), predicted to be in the membrane 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 Lys, 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.

Characterization of a Thermophilic P-type Ag+/Cu+-ATPase from the ExtremophileArchaeoglobus fulgidus

“…1). This membrane topology has been experimentally confirmed only for CopA from H. pylori (23). A unique feature of these proteins is the presence of a putative metal binding sequence (CPC, CPH, or SPC) in their transmembrane region, specifically in the TM immediately upstream from the phosphorylation site ( Fig.…”

Membrane Topology and Insertion of Membrane Proteins: Search for Topogenic Signals

“…The membrane topology of the glutamate carrier GLT1 was studied by fusing a glycosylation consensus sequence behind a series of C-terminally truncated proteins (194). The in vitro translation-transcription system was also used to study the topology of the P-type ATPase from Helicobacter pylori (123) and the sodium ion-dependent citrate carrier of Kleb- FIG. 3.…”

“…Different types of systems have been developed for these studies. Insertion vehicles based on the H ϩ /K ϩ -ATPase (M0 and M1 vectors), the E. coli inner membrane protein leader peptidase (Lep), and the prolactin molecule have been used to analyze both eukaryotic (13,133) and bacterial (123,187) membrane proteins in the ER. Possible methods to perform similar studies in the bacterial membrane are discussed below (see "Similar studies in E. coli").…”

“…The M0 vector assays for the SA capacity of the inserted sequences, while the M1 vector identifies segments with ST activity. Membrane insertion sequences of the H ϩ /K ϩ -ATPase (13), the ER and sarcoplasmic Ca 2ϩ -ATPases (16), a P-type ATPase of Heliobacter pylori (123), and the rabbit gastric cholecystokinin A receptor (17) have been identified using the M0 and M1 system (15). Topogenic properties of putative transmembrane segments of the glycine transporter GLYT1 were analyzed in a similar system (132).…”

Co- and Posttranslational Translocation Mechanisms Direct Cystic Fibrosis Transmembrane Conductance Regulator N Terminus Transmembrane Assembly

“…Consistent with this view, any given polytopic orientation may be generated de novo from combinations of signal, stop transfer, and/or signal anchor sequences engineered into a single polypeptide (10 -12). Similarly, independent topogenic determinants have been identified from eukaryotic proteins such as bovine rhodopsin (13,14), acetylcholine receptor (15,16), P-glycoproteins (17)(18)(19), aquaporins (20,21), band 3 anion exchanger (22), and calcium and P-type ATPases (23,24). Whereas the molecular details of polytopic protein assembly into the ER membrane remain largely unknown, this process appears to require cytosolic and membrane bound components of the ER translocation machinery including signal recognition particle (25), the Sec61 complex, and TRAM (18,26,27).…”