(Received for publication, August 15, 1995; and in revised form, September 12, 1995)
From the
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 Cd and
Cu
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.
P type ion motive ATPases are widely distributed in nature,
occurring in bacteria, fungi, plants, and
animals(1, 2, 3, 4) . They are
polytopic integral membrane proteins, with usually one but occasionally
more subunits(2) . An aspartate residue, which forms the
aspartyl phosphate intermediate during the catalytic cycle, resides in
a conserved DKTGT(I/T)T consensus sequence identified in all known P
type sequences(1, 2, 3) . In contrast to this
phosphorylation signature sequence and another sequence within the ATP
binding domain, P type ATPases diverge widely in their amino acid
sequences, in part reflecting their functional diversity in ion
transport. Interpretation of hydropathy suggests that perhaps they can
be divided into two groups, those containing eight and those containing
10 transmembrane helices.
Helicobacter pylori is a human
gastric pathogen associated with peptic ulcer disease as well as
chronic gastritis, which may predispose to gastric
cancer(5, 6, 7, 8, 9, 10) .
Several virulence factors are known to play a role in pathogenicity of H. pylori infections(6, 8, 9, 11) , but
the means whereby H. pylori colonizes and survives in the
human stomach is poorly understood. One possibility is the expression
of selected P type ATPases. P type ATPases are involved in many
bacterial functions such as maintenance of pH,
turgor pressure, and intracellular ion
composition(4, 12, 13) . The ions transported
by P type ATPases of bacteria vary due to need for selective adaptation
to a varying environment.
The first bacterial P type ATPase
described was the three-subunit Kdp ATPase, a high affinity uptake
transporter for K in Escherichia
coli(14) . The ion-translocating activity of another
putative K
ATPase (15, 16) found in Enterococcus hirae (formerly Streptococcus faecalis)
is now thought to have a role in H
export(17) . This enzyme is composed of a single subunit
of 78-kDa based on purification and reconstitution. Another E.
hirae P type ATPase, CopB, was one of the earliest single subunit
P type ATPases cloned(18) . Additional bacterial P type ATPases
are now being cloned and expressed. The copper pumps, CopA and
CopB(18, 19, 20) , and the
Cd
-ATPase (21) have significant homology to
Menkes gene and other putative copper-transporting ATPases in mammalian
cells(22, 23, 24, 25, 26, 27, 28) .
One or more of these ATPases might be essential for the survival of H. pylori in the human stomach.
We used the homology in the
phosphorylation consensus sequence for isolation of an H. pylori gene encoding a P type ATPase. The DNA predicts a 75-kDA protein
including the conserved sites of phosphorylation and ATP binding. It
has significant homology to the bacterial Cu and
Cd
transporting ion pumps (18, 19, 20, 21) as well as to the
eukaryotic Cu
pumps(26, 27, 28) . The membrane domain
of the protein contains the ion transport pathway. The mammalian pumps,
such as the SERCA family of Ca
-ATPases(29) ,
the Na
,K
-ATPases(30) , and
the gastric H
,K
-ATPase (31) are often expressed at sufficient levels to allow
biochemical methods to be used for finding transmembrane segments.
Bacterial P type ATPases are not expressed in large quantity, and a
fusion protein approach was taken to define the 10 membrane segments of
the Mg
-ATPase of Salmonella typhimurium(32) . A fusion protein vector system in pGEM7sz+ has
been developed that allows scanning of the membrane insertion
properties of putative transmembrane segments of any protein by in
vitro translation(33) .
In this work, we have cloned an H. pylori P type ATPase using a DNA oligonucleotide probe
targeted to the phosphorylation consensus sequence of these pumps.
Southern blot analysis indicated that the encoded ion pump may be
unique to H. pylori. A variety of hydropathy algorithms
predict six to eight transmembrane helices (H1 to H6 or H8) ()for this enzyme. In vitro transcription/translation, using a fusion vector containing
putative transmembrane segments, showed the presence of at least eight
transmembrane segments in this protein. Protein sequence suggests that
this ATPase contains divalent cation binding sites and is a
cation-transporting ATPase.
To insert amplified DNA fragments of the H. pylori P type ATPase, the DNA linker fragment of the M0 or M1 vector was first removed by BglII/HindIII restriction digestion. This was followed by in-frame insertion of selected DNA fragments carrying BglII/HindIII recognition sites at their 5`- and 3`-ends, obtained by PCR amplification of the H. pylori DNA with appropriate primers. These vectors are suitable for transcription-coupled in vitro translation of fusion proteins under control of the pGEM7zf+ T7 promoter.
Gels were dried and placed into cassettes. X-ray films (Hyperfilm
MP, Amersham) were exposed for 6-24 h at -80 °C. The
presence of glycosylation due to a signal anchor sequence was seen by
the increase in M of the product when the M0
vector with insert was translated in the presence of membranes. The
presence of a stop transfer sequence was shown by the inhibition of
normal glycosylation of the M1 vector in the presence of membranes.
Figure 1: DNA sequence and predicted amino acid sequence of pRH439 isolated from H. pylori DNA library. The figure shows the 3,399-bp H. pylori DNA sequence of the Sau3A insertion of pRH439 as determined by DNA sequencing. There is one complete open reading frame beginning with an ATG translational start codon at positions 1,214-1,216 predicting a putative P type ATPase 686 amino acids in length. Amino acids are shown by single letter code. The 5` adjacent sequence of the 74,937-kDa open reading frame contains an AGGGA ribosome binding site (underlined). Potential transcriptional termination sequences are contained in the 3`-flanking DNA sequence (underlined). The putative P type ATPase gene is preceded by an additional open reading frame, which is disrupted by the BglII-Sau3A restriction site (not translated).
Figure 2: Comparison of P type ATPases: regions of significant sequence identity of prominent members of the bacterial P type ATPase family. P type ATPases analyzed were the novel H. pylori P type ATPase (hpATPase), S. aureus CadA ATPase, the hpCopA/P ATPase of H. pylori (hpCopA, hpCopP), CopA and CopB of E. hirae (ehCopA, ehCopB), FixI ATPase of R. meliloti (rmFixI), and the histidine-rich P type ATPase Hra1 of E. coli (ecHra1 ATPase). All amino acid sequences were originally compared for homologies using the Genetics Computer Group programs PILEUP and PRETTY. P Type ATPases were aligned manually to show similar domains along the proteins. This revealed a block of very strong homology around the phosphorylation site (P, dark vertical bar) and the ATP binding region (ATP, dark vertical bar). This region was used as an anchor region for the manual alignment of all sequences. Indicated in the sequences are positions of histidine residues (light vertical bars) and cysteine residues (circles). The eight putative transmembrane helical regions of the novel H. pylori ATPase are shown as open boxes as determined using the in vitro translation mapping technique as described in this paper.
Significant homology was seen to portions of other P
type ATPases, such as the E. coli KdpB gene product (46) and in particular to the human copper-transporting pumps
associated with Menkes disease (26, 27, 28) ,
and the gastric H,K
-ATPase (47) (data not shown).
Figure 3: Fluorogram of Southern blot analysis using DNA isolated from various H. pylori strains and related and unrelated bacteria. Genomic DNAs were prepared from H. pylori ATCC 49503 (lane 5), ATCC 43526 (lane 6), ATCC 43629 (lane 7) ATCC 51111 (lane 8), ATCC 51110 (lane 9), ATCC 43504 (lane 10) and clinical isolate H. pylori 69A (lane 11). Other DNAs were isolated from H. felis ATCC 49179 (lane 1), P. vulgaris ATCC 13315 (lane 2), E. coli MM294 ATCC 33625 (lane 3), and C. jejuni ATCC 33560 (lane 4). DNAs were digested with HindIII and separated by agarose gel electrophoresis. Lanes containing the different DNAs are given in parentheses. For detection of the cloned P type ATPase gene, a digoxigenin-labeled 2.1-kilobase pair EcoRI-HindIII restriction fragment of pRH439 was used for hybridization. Under stringent hybridization conditions used here, pRH439-derived P type ATPase sequences were detectable in all H. pylori strains analyzed but not in genomic DNA of H. felis, P. vulgaris, C. jejuni, and E. coli. Using less stringent hybridization conditions, a DNA fragment was detected in H. felis DNA (data not shown).
Figure 4:
Kyte/Doolittle hydropathy profile of the H. pylori P type ATPase and the primary amino acid sequence.
The predicted Kyte/Doolittle hydropathy plot of the P type ATPase using
a moving average of 11 amino acids is shown. The numbers at the top indicate the eight segments of the pump that were shown to act as
transmembrane segments by using in vitro transcription and
translation of insertion detection vectors (this study). The
corresponding peaks of the plot are shaded.
Also marked is the site of phosphorylation (P) at position
Asp. The primary amino acid sequence consists of 686
residues, as predicted from the isolated DNA clone pRH439. The putative
membrane-spanning segments are shaded differentially for
sequences that behaved as both signal anchor and stop transfer
sequences and for those that behaved only as stop transfer
sequences.
In Fig. 5A, the M0 and M1 fusion protein products are shown as they were obtained by in vitro translation of the M0 and M1 vectors without inserts from the H. pylori P type ATPase. The M1 fusion protein was glycosylated in the presence of membranes, while the M0 protein was not, as shown previously(33) .
Figure 5:
In vitro transcription/translation of M0 and M1 vectors without and with
insertions corresponding to H1 (amino acids 73-92),
H2(98-125), and H1+H2(73-125). S-Methionine-labeled protein products were visualized by
autoradiography. Protein was obtained by in vitro transcription/translation of DNA vectors in the presence of
S-methionine in the absence(-) or presence of
microsomal membranes. The protein products were separated by SDS-PAGE
followed by autoradiography. Panel A shows the result when the
basic M0 vector and M1 vector, without carrying any DNA insertions,
were subjected to in vitro translation as control. These M0/M1
control reactions were carried out in every experiment. Whereas the M
of the M0
-
fusion protein product is
unaffected by the presence of membranes, it is shown that the presence
of the first signal anchor transmembrane segment of the gastric
H
,K
-ATPase resulted in a significant
shift in molecular weight. Panel B shows the autoradiogram
obtained by translation of the putative TM1 region (amino acids
Leu
-Leu
) of the H. pylori P
type ATPase inserted into M0 (PY-36) and M1 vector (PY-89). C,
the result obtained by translation of H2 (amino acids
Pro
-Arg
) in M0 (PY-37) and M1 vector
(PY-51) is shown. D, translation of H1+H2 (amino acids
Leu
-Arg
) in M0 (PY-63) and M1 vector
(PY-102). Panel E shows the result obtained when the modified
H1+H2 sequence element, Leu
-Val
,
was used in M0 (PY-40).
In the following we describe a series of experiments using various arrangements of putative transmembrane sequences as defined by hydrophobicity, singly, in pairs, or in larger combinations, to determine their ability to membrane-insert during translation.
Figure 6:
In vitro transcription/translation of the H3 (amino acids 128-148),
H4(149-176), and H3+H4(128-177) segments of the H.
pylori pump using the M0/M1 vector system. The autoradiograms shown were obtained by in vitro transcription/translation
of vectors in the absence (-) and presence (+) of membranes
followed by SDS-PAGE to separate the S-methionine-labeled
translation products. Panel A shows the translation products
when the H3 segment of the H. pylori P type ATPase,
Phe
-Cys
, was present in the M0
(PY-94) and the M1 vector (PY-95), respectively. Panel B shows
the autoradiogram obtained with the extended H3 segment consisting of
amino acids from position Arg
to Glu
. Using
PY-90, the sequence was expressed as insertion of the M0 protein,
whereas in PY-91 it was expressed as internal part of the M1 protein. C, in these lanes the products from translation of the H4
sequence are shown, starting with a valine residue in position 149 and
ending with the serine residue of position 176. Translation of PY-83
resulted in the expression of the Val
-Ser
containing M0 fusion protein. Using PY-84, the same insert was
expressed adjacent to the first transmembrane segment of the gastric
ATPase cloned in the M1 vector. D, translation products
obtained using the C-terminal truncated
Glu
-Lys
region when expressed in M0
(PY-92) and M1 vector (PY-93). Panel E shows the products that
were expressed with the vector PY-100 containing the putative second
pair of transmembrane sequences, Arg
-Lys
,
cloned into M0. Products containing the same sequence inserted into the
M1 protein are obtained by using vector
PY-101.
Figure 7:
In vitro transcription/translation of vectors with insertions corresponding
to the H5 (amino acids 309-327), H6(337-371), and
H5+H6(309-371) sequences of the H. pylori P type
ATPase. In vitro reactions were performed in the
absence(-) and presence (+) of microsomal membranes to assay
for insertions with membrane-spanning properties. Panel A shows the autoradiogram obtained from the S-methionine-labeled products expressed from vectors
carrying the H5 insertion, Ser
-Gly
,
in the M0 (PY-43) and M1 vectors (PY-54). B, in the lanes shown, the products using the M0 (PY-77) and the M1 vector
(PY-78), which contained the Leu
-Lys
H6
region, are depicted. Panel C shows the products obtained when
the shorter H6 sequence ending at Lys
was translated as
part of the M0 (PY-45) and of the M1
-
fusion protein
(PY-56). D, the autoradiogram is shown found with the
Leu
-Ser
sequence inserted into M0
(PY-44) and M1 (PY-55). Panel E shows the results obtained by in vitro transcription/translation when the sequence from
Ser
-Lys
corresponding to the third
pair of transmembrane sequences, H5+H6, was expressed in both the
M0 vector (PY-79) and the M1 vector
(PY-80).
Figure 8:
In vitro transcription/translation of vectors containing the H7 (amino
acids 637-658), H8(659-685), and H7+H8(637-685)
sequences of the H. pylori P type ATPase.
Transcription/translation was carried out in the absence (-) and
presence (+) of microsomal membranes. A, products shown
here were expressed from the M0 (PY-49) and the M1 vector (PY-60)
carrying insertions that represent the seventh transmembrane segment
(Ile-Gly
). B, also shown is
the result obtained by insertion of the last stop transfer sequence,
Val
-Arg
, into both M0 (PY-50) and M1
vector (PY-61). C, the right two lanes show the autoradiograms that were obtained by using in vitro transcription/translation of vectors including H7 through H8
(Ile
-Arg
) inserted in M0 (PY-70) and
M1 (PY-76).
Figure 9:
In vitro transcription/translation of M0/M1 vector constructs containing
longer C-terminal sequences (amino acids 578-658 and
578-685). Panel A shows the autoradiogram obtained when
the M0 and M1 vectors with the Ala-Gly
region insertion sequence, including H7, were translated in the
absence(-) and presence (+) of microsomal membranes (PY-68
and PY-74). B, products obtained by expression of the
Ala
-Arg
sequence, which includes
H7+H8, inserted into M0 vector PY-69 and into M1 vector
PY-75.
For example, when Pro-Gly
(H2-H5, PY-73) was translated in the M1 vector, the product
was glycosylated as would be predicted from the signal anchor
activities of H5 (Fig. 10A). Extension of the insert to
Lys
(PY-72) and, therefore, addition of the next
putative membrane-spanning segment
(Lys
-Lys
), which had been shown to act as
an efficient stop transfer sequence did indeed inhibit glycosylation,
as predicted for this construct ending with the stop transfer sequence
H6 (Fig. 10B). No glycosylation was found when the
Leu
-Lys
vector (PY-67) containing the
H1-H6 segment was translated in the M0 vector (Fig. 10C) indicating that the three N-terminal pairs
of transmembrane segments co-insert well during translation. In
combination with the data for translation/insertion of PY-69, these
constructs provide data consistent with the individual insertion
results discussed above.
Figure 10:
In vitro transcription/translation of the M1 vectors containing H2-H5
and H2-H6 and the M0 vector with H1-H6. S-Methionine-labeled protein products were detected by
SDS-PAGE followed by autoradiography. Protein was synthesized in the
absence(-) and presence (+) of microsomal membranes. A, the left two lanes show the translation product of
the sequence corresponding to H2-H5,
Pro
-Gly
, expressed in the M1 vector
(PY-73). B, the protein products shown in the middle two lanes
were obtained when the region representing H2-H6,
Pro
-Lys
, was expressed as part of the
M1
-
fusion protein (PY-72). Panel C shows the
protein product when the M0 vector contained the sequence representing
H1-H6 of the H. pylori P type ATPase
(PY-67).
Figure 11:
In vitro transcription/translation of M0/M1 vectors carrying insertions of
other possible TM segments (amino acids 493-513, 541-563,
and 578-601). Reactions were performed in the absence(-)
and presence (+) of membranes. A, the left two lanes show the results of translation of the M0 vector PY-46 and the M1
vector PY-57, both carrying the sequence starting at amino acid
Thr and ending at position Ile
. B,
the autoradiogram shown was obtained by using the M0 vector
PY-47 and the M1 vector PY-58, both containing the insertion
corresponding to amino acids Ser
-Lys
. Panel C shows the protein products obtained when the sequence
Ala
-Gly
was translated as part of the
M0 (PY-48) and the M1 fusion protein
(PY-59).
Table 1summarizes the sequences that were analyzed in the two vectors and the effects of the inserts on induction of glycosylation in M0 and prevention of glycosylation in M1 along with the figures in which the data are presented. This method, therefore, reveals the presence of eight regions of the protein with membrane insertion properties.
Using the conserved DKTGT(L/I)T signature sequence of phosphorylation of bacterial P type ATPases we have isolated a clone from an H. pylori gene library predicting a 75-kDa protein. In terms of homology, hydrophobicity, and conserved sequence elements, there are several features that show it to be a member of the family of cation-pumping P type ATPases. The predicted amino acid sequence, 686 residues in length, contains an aspartate residue at position 388 as part of the DKTGTLT phosphorylation signature sequence, which is homologous with the phosphorylation sequence of another P type ATPase, hpCopA, recently cloned from H. pylori(20) . A GDGINDAP sequence is found in the cytoplasmic loop at positions 582-589 of the novel protein. This sequence matches the GDGXNDXP sequence element of other P type ATPases postulated to be within the region of ATP binding(12) .
This
protein of H. pylori shows a strong overall sequence homology
to the bacterial efflux protein CadA (21) and the bacterial and
eukaryotic copper-transporting
ATPases(19, 20, 24, 25, 26, 27, 28) .
Two Cu P type ATPases have been cloned from E.
hirae, CopA and CopB. While the CopA gene product was postulated
to be a copper-importing protein, the CopB protein is responsible for
copper export. The main sequence difference between these two related
pumps resides in their first 100 N-terminal amino acids(19) .
Compared with these pumps, the N-terminal amino acid region of the H. pylori P type ATPase analyzed here exhibits more homology
to the N terminus of the E. hirae CopA ion pump as compared
with the N terminus of the CopB protein. This might suggest an uptake
function for this new ATPase. The N-terminal region of the cloned H. pylori pump, which contains a CXXC ion-binding
motif, as found in staphylococcal CadA and enterococcal CopA, is also
related to a cation binding protein, hpCopP, operon-associated with the
hpCopA P type ATPase gene of H. pylori(20) . This gene
is predicted to have only six transmembrane segments, but it should be
pointed out that the original sequence for the CopA pump of E.
hirae was later found to be truncated at the N-terminal end, and
the full-length cDNA contained two additional predicted transmembrane
segments(19) .
The N-terminal His/Cys-containing sequence
element, HIHNLDCPDC, found at positions 5-13 in this P type
ATPase, contains the CXXC sequence found in the N-terminal
region in members of the P type ATPase family, which is thought to be
involved in cation binding(12) . Strikingly, the CPDC sequence
of the cloned enzyme is preceded by a HIHNLD sequence revealing a
His/Cys-rich motif. Such motifs are thought to be part of an
ion-binding motif for divalent ions, in particular Zn and Ni
(50) . The N-terminal
CXXC sequence element is also present in both the
Cd
-ATPase of S. aureus(21) and the
CopA ATPase of E. hirae(19) and is missing in the
611-amino acid CopA gene product of H. pylori, but, as
discussed above, its absence in this cDNA might reflect a cloning
artifact. As part of the bicistronic CopA/P operon the CopA message of H. pylori is co-expressed with the CopP gene product, the
latter containing a CXXC element(20) . In contrast to
the published sequence of H. pylori CopA/P ATPase, the H.
pylori pump cloned in our laboratory is a single polypeptide chain
as was found for the E. hirae ATPase, which has been purified
and reconstituted(17) .
A further motif of the H. pylori enzyme, as found in other pumps of this class is the membrane-associated CPC sequence, as predicted from the hydrophobicity plot, located in positions 344-346. It was postulated that this sequence motif, a proline residue flanked by two cysteines, is conserved in heavy metal transporting pumps(12) . However, the Fix1 gene product, which represents a P type ATPase of unidentified function of the nitrogen-fixing genus R. meliloti, also contains an intramembranous CPC sequence (44) perhaps indicative, with respect to the nitrogen assimilation machinery of R. meliloti, of a more extended substrate specificity of these pumps.
Additionally, the cloned P type ATPase contains several
histidine and cysteine residues in the region around the conserved
sites of phosphorylation and ATP binding, which may reflect the
presence of Cu or Ni
binding sites
in this cytoplasmic region(45, 50) . The presence of
putative cation binding sites in this domain could reflect regulation
by divalent cations or transport of the cation itself. However,
expression of the protein in E. coli did not alter
Ni
uptake, whereas synthetic peptides containing the
histidine/cysteine-rich region of the N-terminal domain did appear to
bind Ni
or Cu
(data not shown).
All P type ATPases are polytopic membrane proteins. Whereas the mammalian and fungal P type ATPases have similar hydropathy profiles, these are distinct from the bacterial type. In the case of this bacterial P type ATPase, various algorithms predict some transmembrane sequences in common and some different transmembrane sequences based on the deduced amino acid sequence. As compiled in Table 2, we used the algorithms of Rao & Argos(35) , Klein et al.(37) , and Eisenberg et al.(38) for prediction of putative membrane-spanning sequences of the H. pylori P type ATPase to guide selection of sequences to be inserted in the fusion vectors. The membrane insertion properties of the predicted segments were assayed using inserts into the M0 and M1 vectors for in vitro transcription/translation. As shown in this study, in vitro translation of the various M0/M1 vectors carrying various possible transmembrane sequences of the H. pylori enzyme led to the identification of eight transmembrane sequences along the polypeptide chain. The even number of transmembrane segments and the location of the phosphorylation and ATP binding region places both the N- and C-terminal amino acids in the cytoplasmic domain.
The experimental data presented above show that the regions
corresponding to H1 and H2 acted as signal anchor and stop transfer
sequences, corroborating the predictions of the different algorithms
for this region of the enzyme. The region corresponding to H3 acted as
signal anchor sequence, whereas the region of H4 acted only as a stop
transfer sequence, consistent with the predictions derived from the
Rao/Argos algorithm. The vectors containing the H5 and H6 regions acted
as signal anchor and stop transfer sequences as predicted by all the
algorithms. The first half of the enzyme behaves as if it has four
segments. The vectors containing the putative H7 and H8 regions
beginning at Ile and ending at Arg
again
were characterized by signal anchor and stop transfer properties,
respectively. The seventh and eighth sequences predicted by the
Eisenberg algorithm (PTMS X and Y; Table 2)
were not found to have significant membrane insertion properties. The
agreement between the experimental results found here is best with the
Rao/Argos algorithm predicting six transmembrane segments and a seventh
longer sequence that splits into two domains, giving the segments
discovered here.
The hydropathy profile of this P type ATPase is
similar to the various Cu P type ATPases including
the mammalian types and the Cd
-ATPase of S.
aureus. The phosphorylation site, as shown in Fig. 4, is
predicted to be between the sixth and seventh transmembrane segments in
these pumps. In contrast, the mammalian and fungal ATPases have their
phosphorylation site between the fourth and fifth transmembrane
segments. Alignment of these various pumps suggests that the H.
pylori pump and other homologous bacterial ATPases have an
additional pair of membrane sequences preceding the first pair of
membrane segments that are present in the mammalian and yeast enzymes
as well as in the Mg
-ATPase of S. typhimurium(32) . The H. pylori pump and other similar
bacterial P type ATPases, on the other hand, do not have sequences
following the eighth transmembrane segment. Hence P type ATPases fall
into two groups, one containing eight transmembrane segments acting as
heavy metal cation transporters and the other containing 10
transmembrane segments, which transport monovalent cations as well as
Ca
and Mg
. The last six
transmembrane segments of the heavy metal transporters and the first
six transmembrane segments of the monovalent cation transporters could
therefore form a central core, flanked by one or two pairs of
additional transmembrane segments depending on the nature of the pump.
Although bacterial membrane-inserted proteins are often inserted
post-translationally and bacteria lack the endoplasmic reticular system
that characterizes eukaryotic cells, the translation/transcription
system that had been applied to the mammalian gastric
H,K
-ATPase and SERCA 2
Ca
-ATPase (33, 49) was successful in
finding eight transmembrane segments in a bacterial P type ATPase as
shown in this study. All signal anchor sequences had stop transfer
properties, even though the sequence when used for stop transfer is
usually of opposite orientation as compared with the assembled protein.
It is also of interest to note that when a transmembrane segment showed
only stop transfer properties, this property was retained in longer
constructs, and signal anchor properties did not appear. Therefore,
this method appears to be generally applicable to defining
membrane-spanning
-helices in ATPases. Varying the length of the
insert also provided information on the number of amino acids required
for efficient membrane insertion. For example, truncating H4 or H6
prevented their acting as stop transfer sequences.
The membrane
sequences predicted for this P type ATPase vary considerably in their
content of charged or hydrophilic residues. The TM2 segment is
predicted to have a lysine, a glutamic acid, and an aspartic acid; TM3
a glutamic and aspartic acid; TM4 three glutamic acids and a lysine;
TM7 a lysine; and TM8 an aspartic acid residue. The presence of
membrane-embedded carboxylic acids is frequent in mammalian P type
ATPase that have been analyzed, but lysines are not often present in
the membrane domain although an arginine is predicted in the membrane
domain of the SERCA Ca-ATPase(51) . There are
enough carboxylic acids present in the predicted membrane domain of the H. pylori ATPase to form ion pairs with the lysines. The
extracytoplasmic domain has several carboxylic acids, and there is a
CPC sequence in TM6. Whereas the composition of the transmembrane
segments does not allow any firm conclusion as to the nature of the
ion(s) transported by this enzyme, it seems highly likely that the
enzyme is a cation transporter. The number of histidines and the CPC
motif suggest either a role for divalent cation in transport or in fact
transport itself of a divalent cation.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) L46864[GenBank].