(Received for publication, November 1, 1994)
From the
The highly conserved subunit of the Escherichia coli F
F
ATPase was divided into three sections,
each of which was exchanged with the homologous section of the
subunit of the obligate aerobe Bacillus megaterium. Plasmids
coding for the resultant six chimeric
subunits varied in their
abilities to complement two E. coli
mutants as measured
by testing transformed cells for aerobic growth on a nonfermentable
carbon source or anaerobic growth on rich medium containing glucose.
Two chimeras were able to restore both growth on succinate and
anaerobic growth on rich medium. The genetic results corresponded to
increased levels of membrane-bound ATPase and ATP synthase activities.
These chimeric subunits were therefore capable of being assembled into
functional E. coli ATPase complexes. The results indicate that
chimeric
subunits can be used to analyze assembly of the
subunit and that the final 181 amino acids of the
subunit might
contain a region involved in functional energy coupling.
ATP is synthesized by a class of ubiquitous enzymes referred to
as F-type ATPases, or FF
ATPases. In the
cytoplasmic membranes of bacteria, the thylakoid membranes of
chloroplasts, or the inner membranes of mitochondria, these enzymes
utilize the energy of a transmembrane proton gradient to synthesize ATP
from ADP and P
. They consist of two sectors, an F
and an F
. The F
is an intrinsic membrane
protein complex, which forms a transmembrane proton channel. The
F
sector is an extrinsic membrane protein complex, which is
bound to the F
and which contains the catalytic sites for
ATP synthesis or hydrolysis. The F
of Escherichia coli consists of five subunits:
,
,
,
, and
(for review, see (1) ). Of these, the
subunit contains
nucleotide binding sites involved in catalysis. The
subunit has
been strongly conserved evolutionarily.
subunits isolated from
sources as diverse as E. coli and cows contain over 60%
identity of amino acid sequence(2) .
E. coli is a
facultative anaerobe, able to grow both aerobically and anaerobically.
During aerobic growth, the ATPase can act as an ATP synthase, using the
energy of the proton gradient generated by the electron transport chain
to synthesize ATP. Anaerobically, the ATPase can hydrolyze ATP
generated from glycolysis to pump protons out of the cell, restoring
the proton gradient that is required for transport of nutrients. During
an analysis of the ATPase genes of the obligate aerobe Bacillus
megaterium, we demonstrated that the subunit of this
organism can complement E. coli mutants defective in uncD, the gene for
(3) . Therefore, the B.
megaterium
is capable of being assembled into a hybrid E. coli-B. megaterium F
complex. We
subsequently demonstrated that this cross-species assembly did not
require any other B. megaterium subunits. We also demonstrated
that an E. coli
mutant carrying the B. megaterium
gene had become an obligate aerobe as a result of carrying
hybrid ATPases containing the B. megaterium
subunit(4) .
A common approach to dissecting the function of
a polypeptide or enzyme involves site-directed mutagenesis of
individual amino acids suspected of being involved in determining the
structure and function of the protein. The ability of homologous
subunits from different organisms to be assembled into functional
hybrid complexes provides a system for analyzing the effects of large
numbers of evolution-driven amino acid changes on subunit assembly,
structure, and function. A careful comparison of the differences
between homologous subunits and the effects of those differences on
subunit assembly and activity represents an alternative approach to
site-directed mutagenesis for analyzing the significance of amino acid
substitutions on the function of the subunit.
This paper
describes a more detailed analysis of this cross-species subunit
assembly process by dissecting the subunits of the two organisms.
To determine which regions or residues of the
subunit are
involved in assembly, energy coupling, or both, we constructed chimeric
subunits consisting of regions from both E. coli and B. megaterium and analyzed the abilities of those subunits to
complement an E. coli
mutant and to restore
ATPase-dependent energy coupling to that mutant.
Having divided both
subunits into three interchangeable sections, we constructed
chimeric
genes in pSRM103, pSRM104, and pRAS009, by modification
of pMAS112, which consists of the entire B. megaterium
gene cloned into pUC19(6) . The appropriate section or sections
of the B. megaterium
gene were replaced with the
homologous section from the E. coli
gene by standard
recombinant techniques. In like fashion, chimeric constructs pSRM107,
pRAS007, and pRAS008 were constructed by modification of pWSB39,
consisting of the entire E. coli
gene cloned in pACYC184 (7) .
Fig. 1shows a schematic of the E. coli
subunit (M
= 50286), indicating the
locations of SalI and PstI restriction enzyme
recognition sites, which divide the protein into three sections of 22%,
39%, and 39%. Using site-directed mutagenesis, we created SalI
and PstI sites in the identical locations of the gene for the B. megaterium
subunit. We were then able to create genes
coding for chimeric
subunits containing all combinations of these
three regions from the two different
subunits, as shown in Fig. 1. The vectors for these constructs consisted of either
pUC19 (6) (ampicillin-resistant; for pMAS112, pSRM103, pSRM104,
and pRAS009) or pACYC184 (7) (chloramphenicol-resistant; for
pWSB39, pSRM107, pRAS007, or pRAS008).
Figure 1:
Chimeric subunits. The location
of the SalI and PstI sites in the E. coli
gene, and engineered into the B. megaterium
gene, are indicated at the top of the figure. These two restriction
sites divide
into three fragments containing 22%, 39%, and 39% as
shown. The designations for the plasmids containing each chimera are
shown on the left, and the identity of each section is
designated by either a BM (B. megaterium) or EC (E. coli). Plasmid pWSB39 contains the intact E. coli
gene, and plasmid pMAS112 contains the intact B.
megaterium
gene.
Each of these plasmids was
transformed into two E. coli mutants, strain AN818, an uncD mutant that fails to assemble an F
on the
membrane(8) , and JP17, a chromosomal deletion of uncD(9) . The transformants were tested both
genetically and biochemically for ATPase function. Genetically, cells
were analyzed for their ability to grow either aerobically on minimal
succinate medium or anaerobically on LB medium containing glucose.
Aerobic growth on succinate is a test for the ability of the ATPase to
synthesize ATP from a proton gradient. Similarly, without an ATPase,
cells will not grow anaerobically, presumably because they are unable
to couple ATP hydrolysis to proton pumping. For biochemical assays,
cells were grown in rich medium to an A
of 1,
and membranes were isolated and assayed for ATPase activity and
respiration-driven ATP synthesis activity. The results are shown in Table 1. The intact E. coli
gene in pWSB39 and the
intact B. megaterium
gene in pMAS112 complemented both
mutants well for growth on succinate. Two of the chimeric
subunit
constructions, pRAS007 and pRAS008, also complemented both mutants. As
was the case for pWSB39, both chimeric constructions allowed both
mutants to grow aerobically on succinate and anaerobically on rich
medium.
Biochemical assays of ATPase and ATP synthase activities also showed that the four plasmids that complemented the mutants (pWSB39, pMAS112, pRAS007, and pRAS008) also restored measurable ATPase activities to membranes isolated from the plasmid-bearing mutants. The activities measured in the uncD deletion strain JP17 were, for most of these plasmids, lower than those assayed in the point mutant AN818 carrying the same plasmid.
The hybrid subunit coded for
by pRAS007, consisting of the center section from B. megaterium between the N- and C-terminal sections from E. coli,
acted the most like the E. coli
subunit alone,
indicating that assembly of a
subunit into an F
complex is affected very little by the differences in amino acid
sequence between the center sections of the two
subunits.
The
opposite construction in pSRM104, consisting of the N- and C-terminal
sections from B. megaterium combined with the center section
from E. coli, did not complement the AN818 mutant as well as
pMAS112. Growth on succinate was very poor, as indicated by the
+/- in Table 1. This growth was only marginally better
than that produced by the negative control culture carrying pUC18.
ATPase and ATP synthase activities produced by cells carrying this
construction, however, were about half of those produced by cells
carrying pMAS112, which carries the gene for the intact B.
megaterium subunit. The deletion strain JP17 carrying
pSRM104, however, produced no measurable ATPase or ATP synthase
activity and did not grow at all aerobically on succinate medium or
anaerobically on rich medium.
The other construction that was
capable of genetically or biochemically complementing both of the
mutants was the chimera produced by pRAS008, consisting of the first
two sections from B. megaterium and the C-terminal section
from E. coli. Cells carrying pRAS008 grew both aerobically on
succinate and anaerobically on rich medium, but not as well as cells
carrying pRAS007. The assays of ATPase and ATP synthase activities
produced by membranes isolated from cells carrying pRAS008 contained
about half the activity of membranes isolated from cells carrying
pRAS007 or pMAS112 and 15-20% of the activity in membranes from
cells carrying pWSB39, the intact E. coli
plasmid.
Membranes isolated from the deletion strain JP17 carrying pRAS008 had
the same ATP synthesis activity as JP17 cells carrying pRAS007 but had
virtually no measurable ATPase activity. All other plasmids carrying
chimeric
genes produced no genetic complementation and no
measurable ATPase or ATP synthase activities in either of the
mutants.
The subunit of the ATPase has been the subject of much
investigation, as it contains nucleotide binding sites and the
catalytic site for ATP synthesis or
hydrolysis(10, 11) . It is the most evolutionarily
conserved of the ATPase subunits(2) . The effects of many
naturally occurring and site-directed mutations in the
subunit
gene have been analyzed. Chemical modification studies on the ATPase
have been focused largely on reactive residues in the
subunit(12) . The recent structural determination of the bovine
mitochondrial ATPase has provided the most detail about the structure
and function of the
subunit(13) . Since we demonstrated
that the B. megaterium
subunit could substitute for the E. coli
subunit(3) , we have attempted to
develop a system for constructing chimeric
subunits to analyze
the effects of the many amino acid differences between the two
subunits on the assembly and function of
. Of particular note is
the energy-coupling defect seen in the E. coli
mutant
complemented with the B. megaterium
subunit. The
resultant E. coli are capable of aerobic growth on succinate,
but cannot grow anaerobically on rich medium, and have therefore become
obligate aerobes as a result of carrying hybrid ATPases containing B. megaterium
subunits(4) . A goal of our
chimeric
studies is to localize this coupling defect to a region
and possibly to a small number of amino acid differences between the
subunits.
We tested the abilities of these plasmids to complement
two different mutants. Our initial studies had been performed on
AN818, which codes for an assembly-defective
subunit(8) .
One possible interpretation of positive results from those studies is
that the B. megaterium
subunit, or the chimeric
subunits, were acting as chaperones for the AN818
subunit rather
than, or in addition to, being assembled into a hybrid B.
megaterium-E. coli ATPase complex
themselves(14) . We therefore repeated the AN818 studies on
JP17, a strain carrying a complete deletion of the uncD gene(9) , thus avoiding this potential problem. The
genetic results were identical in both strains. Two chimeras, coded for
by pRAS007 and pRAS008, complemented both mutants. The results of the
biochemical assays, however, supported the concerns about possible
chaperone function. ATPase and ATP synthase activities produced by
complementation of JP17 by pRAS007 or pRAS008 produced lower ATPase and
ATP synthase activities than in AN818. Additionally, several
non-complementing plasmids, specifically pSRM103, pSRM104, and pSRM107,
produced measurable ATPase activities in AN818 but not in JP17. These
differences in measured activities might have been caused by a
secondary effect of the foreign, chimeric subunit on assembly of the
AN818
subunit, producing functional ATPases containing either the
AN818
subunit alone or together with the chimeric
subunit.
However, the observed complementation and activities produced by
pMAS112, pRAS007, and pRAS008 in JP17 must result from functional
ATPase containing exclusively the plasmid-encoded
subunit.
Fig. 2shows an alignment of the two sequences,
indicating the locations of the SalI and PstI sites
used to construct the chimeras. The center section contains the
residues involved in nucleotide binding and catalysis. These residues
compose the so-called Walker homology sequences A and B, two sequences
found by Walker et al.(15) to be common to
nucleotide-binding proteins. Sequence A,
GGAGVGKT
, also called the P-loop, is
believed to actually bind to the phosphoryl groups of ATP, and is
identical in E. coli and B. megaterium. The lysine
residue at position 155 can be modified by pyridoxyl
5`-diphosphadenosine (16) and 7-chloro-4-nitrobenzofurazan (17) and has been implicated in catalysis and coordination of
the
-phosphoryl group of bound ATP(18) . The recent
structural determination of the bovine mitochondrial F
has
demonstrated that this residue, together with threonine 156, does
indeed coordinate the
- and
-phosphoryl groups of the bound
nucleotide in that ATPase(13) . Homology sequence B from E.
coli,
RDEGRDVLLFVD
, is slightly
different from the homologous sequence of B. megaterium. The
arginine residue found at position 235 is replaced by a glutamine
residue in B. megaterium, and leucine 239 is replaced by a
phenylalanine. Additionally, there is an insertion of a glutamine
between aspartate 233 and glycine 234. As shown by the results with
chimeric
construction pRAS007, this section, between the SalI and PstI sites, can be moved from B.
megaterium to E. coli with little apparent change in
assembly or function of the resultant ATPase. Although during the
course of evolution there have been 51 amino acid changes in this
region, none destroys the ability of the chimeric subunit containing
the N- and C-terminal regions from E. coli to assemble into a
functional ATPase. The chimera in pSRM104 contains the reverse
construction, in which the E. coli section was transferred to
a B. megaterium
gene. This chimeric
subunit does
not complement either E. coli mutant as well as the intact B. megaterium
gene alone, although it does restore a
significant amount of ATPase activity to the E. coli
mutant AN818. As discussed above, the ATPase activity produced in AN818
may be the result of interactions between the AN818
subunit and
pSRM104 chimeric
subunit, resulting in the assembly into an
ATPase of the AN818
subunit in addition to, or instead of, the
chimeric
subunit.
Figure 2:
Comparison of the primary structures of
the subunits from E. coli and B. megaterium.
Identical amino acids located in the same position are boxed.
The locations of the SalI and PstI restriction sites
used in the construction of the chimeras is indicated on the sequence.
The amino acids composing Walker homology sequences A and B are shown
in boldface type and labeled near the start of each sequence.
Sequence A is also referred to as the
P-loop.
The other chimera that complemented both
mutants, coded for by pRAS008, differs from the chimera coded for
by pRAS007 by the additional replacement of the first 22% of the E.
coli
with the homologous section from B. megaterium and differs from pMAS112 only in the C-terminal section. However,
whereas pMAS112 allowed either mutant to grow aerobically on succinate
but not anaerobically on rich medium, cells carrying pRAS008, differing
only in this C-terminal section, did grow anaerobically. The amino acid
differences that cause the coupling defect may therefore be localized
to this last section, containing 124 identical amino acid position out
of 181 (E. coli) or 185 (B. megaterium).
Systematically converting the B. megaterium section to the E. coli section may therefore define certain
-specific
determinants of energy coupling.
Chimeric proteins consisting of
domains assembled from regions of homologous proteins have been used to
analyze the function of large sections of proteins in catalysis and
assembly (for examples, see (19, 20, 21, 22) ). The construction
of chimeras represents an alternative approach to site-directed
mutagenesis in the analysis of structure-function relationships in
enzymes. The studies described here demonstrate that the evolutionarily
conserved subunit of F-type ATPases can be divided into
interchangeable regions, and chimeras of
can be constructed for
the analysis of the
structure and function.