(Received for publication, December 6, 1995; and in revised form, December 28, 1995)
From the
The 60-kDa Ca-ATPase from Flavobacterium
odoratum is kinetically and mechanistically similar to other
P-type ATPases, suggesting its use as a model system for
structure-function studies of ion transport. A portion of the gene was
amplified by polymerase chain reaction of genomic DNA with degenerate
oligonucleotide primers, one based on the N-terminal amino acid
sequence of the purified protein and the other based on a consensus
sequence for the phosphorylation site of P-type ATPases. This gene
fragment was used to screen a
library of F. odoratum 29979 DNA. Clone ``C'' is 3.3 kilobases in length and contains
one complete and part of a second open reading frame, the first of
which encodes a 58-kDa protein containing the exact N-terminal amino
acid sequence of the purified protein. We have named this gene cda, for calcium-dependent ATPase. Escherichia coli,
transformed with clone C, demonstrates high levels of calcium-dependent
and vanadate-sensitive ATP hydrolysis activity, forms a 60-kDa
phosphointermediate, and cross-reacts with antibodies to the purified
Ca
-ATPase. The gene has almost no sequence homology
to even the highly conserved regions characteristic of P-type ATPases
but does possess significant homology to a protein with alkaline
phosphatase activity (PhoD) from Zymomonas mobilis. The
putative phosphorylation site is a Walker A (P-loop) ATP binding
sequence and is modified relative to P-type ATPases, suggesting that
the F. odoratum Ca
-ATPase may represent an
ancestral link between the F- and the P-type ATPases or perhaps a new
class of ATPases.
Calcium is an important component of the signal transduction
process in eukaryotic cells. It is therefore necessary that
intracellular calcium levels are kept low so that even small changes in
concentration are detectable for signal transduction. In prokaryotes,
however, calcium has not been shown to have such a role; nonetheless,
intracellular calcium is maintained in the micromolar range even when
extracellular levels are in the millimolar range. Although most
prokaryotes use secondary transport to remove calcium from the cell,
the Gram-negative Flavobacterium odoratum has been shown to
possess a protein with Ca-ATPase activity (1) similar to the sarcoplasmic/endoplasmic reticulum
Ca
-ATPase (SERCA). (
)More recently, genes
homologous to sequences for other Ca
-transporting
proteins have been cloned from several cyanobacterial
species(2, 3) .
The Ca-ATPases
found in eukaryotes and the genes cloned from prokaryotes all belong to
a family of ATPases called P-type. P-type ATPases transport ions across
membranes at the expense of ATP; besides calcium,
H
(4) , Na
(5) ,
K
(6) , Mg
(7) ,
Cu
(8) , and Cd
(9) have been shown to be transported by P-type ATPases.
Small regions of high sequence homology between all P-type ATPases,
regardless of ion specificity, suggest common structural motifs and
possibly common mechanisms for ion transduction(10) . All
P-type ATPases form a vanadate-sensitive phosphointermediate during the
reaction cycle; the aspartyl phosphate species is acid stable and
alkaline labile. Many of the P-type ATPases are comprised of a single
subunit, although several contain two or three subunits in a
membrane-bound complex. Sizes range from 72 kDa for the KdpB subunit of
the K
-transporting Escherichia coli Kdp
complex (6) to 102 kDa for the MgtB Mg
transporter of Salmonella typhimurium(7) , while
the eukaryotic P-type ATPases tend to be somewhat larger with SERCA at
110 kDa(11) . The 60-kDa size of the protein from F.
odoratum makes it the smallest known P-type ATPase.
The F.
odoratum protein has been extensively characterized (12) and is found to be functionally and kinetically similar to
other P-type ATPases. F. odoratum membrane vesicles transport
Ca in an ATP-dependent and vanadate-inhibitable
manner(1) . The protein forms an acid-stable vanadate-sensitive
phosphointermediate and exhibits K
and K
for calcium, ATP, and vanadate similar
to SERCA, although the enzyme turnover is much faster (12, 13) . This would appear to make it an ideal
candidate to use as a model system for the P-type ATPase, and SERCA in
particular. While much work has elucidated the role of specific
residues and domains in SERCA, the bacterial system offers the
advantage of allowing high levels of expression of a mutant protein in
its native environment with no interference from endogenous wild-type
activity.
We have therefore set out to clone the gene for the
Ca-ATPase from F. odoratum to analyze the
structure and function of this prokaryotic ATPase. The approach
employed relies on polymerase chain reaction (PCR) amplification of
short regions from genomic DNA using degenerate primers based on both
the N-terminal amino acid sequence of the purified protein (12) and highly conserved sequences in other P-type ATPases.
The PCR products were then used to probe a genomic library for a
full-length clone. The cloned gene, which we have called cda for calcium-dependent ATPase, has been expressed in E. coli and is functionally identical to the purified
Ca
-ATPase from F. odoratum(12) .
However, sequence analysis of the cda gene reveals that,
despite its functional similarities to the P-type ATPases, it is
structurally distinct. Surprisingly, the sequence contained almost none
of the highly conserved regions characteristic of all known P-type
ATPases.
ATP
phosphorylations were performed as described for ATP hydrolysis except
that the reactions were carried out on ice using 5 µl of a 100
µM [-
P]ATP (at 1000-2000
cpm/pmol). Reactions were terminated by the addition of 750 µl of
10% trichloroacetic acid, the samples were centrifuged (12,000
g for 5 min), and the pellet was solubilized and analyzed by
SDS-PAGE followed by autoradiography as described(12) .
The first open reading frame (Fig. 1) contained the
N-P probe sequence; however, the N-terminal serine did not immediately
follow a methionine, suggesting the presence of a signal sequence,
which would target the protein to the membrane. Several in-frame
methionines upstream would produce signal sequences of 11, 21, or 42
amino acids. The EGCG program ``sigcleave'' analyzed the
entire sequence and predicted the presence of a signal sequence that
cleaves at the N-terminal serine. The translation start site is likely
to be the methionine at either -21 or -42 since both
contain the hydrophobic helical region (from amino acids -13 to
-1) typical of signal sequences. Starting from the N-terminal
serine, the gene consists of 1581 bp, coding for a protein of 527 amino
acids with a molecular mass of 58,805 Da, very close to the 60-kDa size
predicted from the protein's mobility on SDS-PAGE. We therefore
suggest that the first open reading frame of clone C encodes the
Ca-ATPase protein purified from F. odoratum.
We propose to call this gene cda, for calcium-dependent
ATPase.
Figure 1:
Nucleotide sequence of the F. odoratum Ca-ATPase gene. The figure
includes the 5`-untranslated sequence, the open reading frame of cda, and the intergenic sequence between cda and the
beginning of the second open reading frame contained in clone C.
Residue and nucleotide numbers are labeled from 1 at the N-terminal
serine residue (as determined by the purified protein). A second open
reading frame begins at nucleotide 1701 and continues to the end of the
clone. The putative phosphorylation site from residues 158-167 is underlined.
The second open reading frame continues for 1310 bp to (and
probably past) the end of the clone, coding for a protein of at least
49,906 Da (436 amino acids). The GCG program ``motifs''
revealed homology to the insulinase family of proteins, divalent
cation-dependent proteases that process peptides including
insulin(17) . Comparison of the predicted amino acid sequence
to the GenBank and PIR protein sequence data banks revealed homologies
to a number of proteins in the insulinase family, including
insulinase(18) , E. coli pitrilysin(19) , a
mitochondrial protein processing enzyme(20) , and its homolog
in Bacillus subtilis(21) . It is therefore doubtful
that this protein has a direct function in Ca-ATPase
activity; however, it may be that this protein is responsible for
cleavage of the signal sequence from the Ca
-ATPase.
The F. odoratum Ca-ATPase exhibits moderate sequence homology
(30%) with an alkaline phosphatase (phoD) from Zymomonas
mobilis(23) . Despite weak homology to short regions of
several Ca
-ATPases, including the Synechococcus pacL, which has been hypothesized to be a
Ca
-ATPase, the Z. mobilis sequence contains
none of the highly conserved regions found in other P-type ATPases,
including the phosphorylation site.
The Kyte-Doolittle hydropathy
plot of the F. odoratum Ca-ATPase (Fig. 2) definitively indicates only one transmembrane helix in
the signal sequence that is cleaved off in the isolated protein. This
result is confirmed by the program PredictProtein from the
EMBL-Heidelberg(24, 25) . However, many other regions
of the protein are weakly hydrophobic, and the protein partitions with
the membrane during cellular disruption ( (1) and data not
shown), suggesting that it is a peripheral membrane protein, possibly
part of a multi-subunit membrane complex.
Figure 2:
Kyte-Doolittle hydropathy plot of the F. odoratum Ca-ATPase. The plot starts at
the first possible initiation methionine with residue 1 corresponding
to the N-terminal serine residue (as determined by the purified
protein). Positive values represent hydrophobic regions. A potential
transmembrane helix is apparent near the N terminus, while there are
several other regions of weak hydrophobicity throughout the
sequence.
Figure 3:
ATP hydrolysis activity of the pBK-C genes
expressed in E. coli. Calcium-dependent ATP hydrolysis
activity of the 90% fraction from E. coli XL1 Blue cells
transformed by the vector pBK with (pBK-C) or without (pBK) clone C
were compared to F.odoratum on a per protein basis. ATP
hydrolysis was carried out with the 90% fraction incubated in 45 µl
of buffer A (plus 0.5 mM DCCD) with 0.1 mM CaCl (+ calcium), 2 mM EGTA (- calcium), or 0.1
mM CaCl
plus 100 µM vanadate (+
vanadate) for 10 min, and the reaction was initiated with 5 µl of a
1 mM [
-
P]ATP mixture (final
concentration, 100 µM at 100-200 cpm/pmol). After 10
s, the reaction was terminated by the addition of 150 µl of 30%
trichloroacetic acid, 1 mM K
HPO
.
Inorganic phosphate was extracted and counted as
described(12) .
Figure 4:
Phosphointermediate formation by the F. odoratum Ca-ATPase. Calcium-dependent ATP
phosphorylation of the 90% fraction from F. odoratum and E. coli XL1 Blue cells transformed by the vector pBK with
(pBK-C) or without (pBK) clone C are compared by SDS-PAGE. Assays were
performed in buffer A (plus 0.5 mM DCCD) with and without
calcium on the 90% fraction, and gel electrophoresis was performed as
described under ``Experimental Procedures.'' The gel was
loaded with sufficient protein to ensure equal amounts of
Ca
-dependent ATP hydrolysis activity for the F.
odoratum (50 µg) and E. coli (10 µg). The fixed
gels were exposed to x-ray film. Bands represent proteins labeled with
P. Both F. odoratum and E. coli with
pBK-C demonstrate a 60-kDa phosphointermediate only in the presence of
calcium.
Western blots of vesicles from pBK-C cells cross-react with
antibodies prepared against the purified F. odoratum Ca-ATPase, while cells with pBK (no insert) do
not show a reaction (Fig. 5). The band labeled by the antibodies
migrates at 60 kDa, as does the phosphorylated protein produced by the F. odoratum and the clone.
Figure 5:
Western blot analysis of the the F.
odoratum Ca-ATPase. Polyclonal antibodies
prepared against the purified F. odoratum Ca
-ATPase were incubated with a blot containing
approximately 10 µg of each the purified F. odoratum Ca
-ATPase (lane A) prepared by the
method of Desrosiers et al.(12) and the 90% fraction
of E. coli XL1 Blue cells transformed by the pBK vector with
(pBK-C, lane B) or without (pBK, lane C) the clone C
insert. Antibody preparation and blots were performed as described
under ``Experimental Procedures.'' The antibody reacted with
a 60-kDa protein in the lanes with fractions of F. odoratum and E. coli with pBK-C but not with
pBK.
The pBK vector did not tightly
control the expression of the cda gene, and induction with
isopropyl-1-thio--D-galactopyranoside often resulted in
levels of expression that adversely affected cell viability. Therefore,
the clone C insert was ligated into the pET-21 vector (pET-C), which
more tightly controls expression, allowing us to regulate levels of
expression. In addition, the sequence beyond the cda open
reading frame of pET-C (including the second open reading frame of
clone C) was deleted (pET-cda) so that only the Cda protein
would be expressed. Importantly, vesicles from E. coli transformed with pET-cda demonstrated high levels of
calcium-dependent, vanadate-sensitive ATP hydrolysis (Fig. 6).
The cda gene product also formed a 60-kDa phosphointermediate
in the presence of calcium and cross-reacted with our anti-F.
odoratum Ca
-ATPase antibody (not shown). These
data verify that cda encodes the F. odoratum Ca
-ATPase. Calcium transport assays were
performed with vesicles treated with 0.1 mM DCCD and 0.5
mMN-ethylmaleimide, which inhibited the endogenous
Ca
antiporters but not the heterologously expressed
Ca
-ATPase. Unfortunately, we were unable to detect
ATP-driven calcium uptake (data not shown). This may be due to
insufficient inhibition of the endogenous Ca
antiporters, the membrane being leaky to calcium, or the possible
requirement of a second transmembrane protein for calcium transport
activity.
Figure 6:
ATP
hydrolysis activity of the cda gene expressed in E.
coli. Ca-dependent ATP hydrolysis activity of
the 90% fraction from E. coli NovaBlue (DE3) cells transformed
by the vector pET-21 with the clone C (pET-C) or with the cda gene alone (pET-cda) was compared to F. odoratum on a per protein basis. The transformed E. coli were
grown to an A
of 0.2, induced with 0.1 mM isopropyl-1-thio-
-D-galactopyranoside, and incubated
for 8 h at 30 °C. ATP hydrolysis was carried out with the 90%
fraction incubated in 45 µl of buffer A (plus 0.5 mM DCCD)
with 0.1 mM CaCl
(+ calcium), 2 mM EGTA (- calcium), or 0.1 mM CaCl
plus
100 µM vanadate (+ vanadate) for 10 min, and the
reaction was initiated with 5 µl of a 1 mM [
-
P]ATP mixture (final concentration,
100 µM at 100-200 cpm/pmol). After 10 s, the
reaction was terminated by the addition of 150 µl of 30%
trichloroacetic acid, 1 mM K
HPO
.
Inorganic phosphate was extracted and counted as
described(12) .
A 1581-bp gene from F. odoratum has been cloned and
sequenced. The gene encodes a protein with the same 60-kDa molecular
mass and N-terminal amino acid sequence as the purified
Ca-ATPase. The expressed gene product exhibits
calcium-dependent and vanadate-sensitive ATP hydrolysis, forms a
phosphointermediate in the presence of calcium and ATP, and is
immunologically related to the F. odoratum Ca
-ATPase. We therefore conclude that the first
gene of clone C, which we call cda for calcium-dependent
ATPase, encodes the Ca
-ATPase, that we have
previously purified.
This gene, however, appears to code for a
protein very different than other P-type ATPases, despite its
functional similarities. Most of the highly conserved regions found in
all other P-type ATPases are missing, including the TGES transduction
domain, the KGAPE fluorescein isothiocyanate binding site, and the
MTGDGVNDAPAL ATP binding domain. Only the putative phosphorylation site
shares homology with other P-type ATPases, and it is altered relative
to them. All other P-type ATPases conserve the 7-amino acid sequence
DKTGT(I/L)T, in which aspartate is the residue phosphorylated by ATP
during the reaction cycle. In contrast, the F. odoratum Ca-ATPase contains the sequence DGKTGDWIT,
notably with a glycine inserted between the positive and negative
charges of the aspartate (Asp-158) and the lysine (Lys-160). In the
yeast plasma membrane H
-ATPase, insertion of a glycine
in this position affected the assembly and/or stability of the
H
-ATPase and resulted in no H
-ATPase
being detected in secretory vesicles(26) . Moreover, the
addition of an extra aspartate (Asp-163) after the second glycine makes
it unclear which aspartate (if either) is phosphorylated. The sequence
GKT (preceded by an A/G five residues earlier), however, is a common
motif for ATP binding in many, if not most, ATPases other than the
P-type ATPases(22) . This Walker A or P loop sequence is found
in proteins as varied as the
and
subunits of the
F
-ATPase, the myosin head protein, the ras oncogene, and many other kinases. It is puzzling that this motif
is present in the F. odoratum Ca
-ATPase
rather than the standard P-type ATPase sequence, given that other
proteins using the Walker A sequence do not form an aspartylphosphate
intermediate as part of their reaction mechanism.
The
three-dimensional structure of the F. odoratum Ca-ATPase may differ significantly from other
P-type ATPases. The Kyte-Doolittle plot predicts only one transmembrane
helix, that of the signal sequence, with the remaining protein being
hydrophilic or only weakly hydrophobic. This, taken together with
experiments suggesting that at least some of the protein's
activity can be removed from inside-out membrane vesicles by high salt
wash (data not shown), suggests that the protein is a peripheral, not
an integral, membrane protein. It is highly unlikely that a peripheral
membrane protein alone could transport calcium across a membrane, and
therefore a second, transmembrane, component would be required. The
fact that we have been unsuccessful in demonstrating calcium transport
by the reconstituted protein and by E. coli expressing cda supports the theory that the Cda protein is part of a multisubunit
membrane complex. The subunits of the F. odoratum Ca
-ATPase may be analogous to the subunits of
the plasmid-mediated E. coli arsenate transporter (although
not a P-type ATPase), in which soluble ArsA is capable of
arsenate-dependent ATP hydrolysis(27) , but ArsB (transmembrane
protein) and ArsC are required for transport(21, 28) .
Similarly, potassium transport in E. coli is mediated by the
multisubunit P-type ATPase called Kdp, in which KdpA is postulated to
be responsible for K
transport (29) while KdpB
binds ATP and is phosphorylated(30) . In this case, both KdpA
and KdpB are transmembrane proteins(6) . In contrast to the F. odoratum Ca
-ATPase, it is not known
whether KdpB alone is active since it has never been isolated to
homogeneity.
Although it has no significant homology with P-type
ATPase sequences, the F. odoratum Ca-ATPase
does show a moderate homology (overall 30%) to an alkaline phosphatase (phoD) from Z. mobilis(23) , which is itself
weakly homologous to short regions of several P-type ATPases, in
particular Ca
-ATPases. The F. odoratum protein is not highly homologous to the Z. mobilis protein in most of those regions. Interestingly, those regions
homologous between the Z. mobilis and the
Ca
ATPases are not in the most highly conserved
regions of P-type ATPases, and the Z. mobilis is missing an
obvious phosphorylation site or ATP binding site. The PhoD protein
demonstrates high phosphatase activity at alkaline pH using p-nitrophenylphosphate as a substrate, as does the F.
odoratum Ca
-ATPase(12) , as well as
other P-type ATPases including SERCA(31, 32) . The Z. mobilis PhoD protein is found in the cytosol and does
hydrolyze ATP but at rates that are dramatically below that of the F. odoratum Ca
-ATPase(23) .
The Z. mobilis enzyme has a similar hydropathy profile to the F. odoratum protein in that there is only one predicted transmembrane helix, at the N terminus (it is not clear whether there is a cleaved signal sequence), with weakly hydrophobic regions throughout the remainder of the protein. Given the ease of removal from the membrane, the PhoD protein has been postulated to contain only one transmembrane helix(23) . Further experimentation will reveal whether our protein is structurally similar to the PhoD protein whose sequence it conserves, or to the P-type ATPases whose function it conserves.
Perhaps because it is a peripheral and not an integral
memebrane protein, the F. odoratum Ca-ATPase
is easily expressed in E. coli, in contrast to SERCA, which
has only been expressed at relatively low levels in mammalian cell
cultures (33) , and baculovirus-infected insect
cells(34) , which both contain wild-type
Ca
-ATPase. Molecular biological techniques have
greatly increased our understanding of the molecular mechanism of the
P-type ATPase, but unfortunately the level of expression of the ATPase
in most eukaryotic expression systems is too limited to allow for
extensive biochemical characterization of site-directed mutants.
However, functional expression of the F. odoratum Ca
-ATPase in E. coli at very high
levels will allow for easy purification and biochemical
characterization of any site-directed mutant. This makes the F.
odoratum Ca
-ATPase a very promising system for
structure-function studies.
In conclusion, the F. odoratum Ca-ATPase appears to be a most unique ATPase.
Functionally, it behaves like a P-type ATPase, forming an
alkaline-labile phosphointermediate, and displaying vanadate-sensitive
activity with K
values for ATP and Ca
similar to those for SERCA. The primary structure, however, is
different from the P-type ATPases, containing none of the highly
conserved regions that appear to be involved in ATP binding and
hydrolysis. The putative phosphorylation site is similar but not
identical to that of P-type ATPases, resembling the Walker A/P loop
motif found in other ATP binding proteins including V- and F-type
ATPases. The F. odoratum Ca
-ATPase may
possibly represent an ancestral link between the F-type and the P-type
ATPases or a new class of ATPases. Further study is anticipated to
clarify these structural puzzles and to elucidate how such apparently
different structures can accomplish the same functional goals.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) L42816[GenBank].