(Received for publication, March 15, 1995; and in revised form, May 12, 1995)
From the
F
The F
The amino acid sequences
of both the
Several important protein-protein interactions are suspected to
involve the amino-terminal region of the
We previously
described (Chen et al., 1992) a bacterial expression system in
which large amounts of chloroplast
Figure 1:
Deduced amino acid sequence of the
spinach chloroplast CF
Figure 2:
Expression clones of chloroplast atpB.A, plasmid pBS(-)
Figure 3:
Complementation of an uncD deletion by cloned
Figure 4:
Wild-type and mutant
To assess the functionality of
these altered
Figure 5:
Dependence of cell growth on
[succinate] and side chain volume at residue 63. A,
dependence of growth on [succinate]. Strain JP17 was
transformed with clones of wild-type atpB (Cys) or
the mutants C63A (Ala), C63V (Val), or C63W (Trp). Cell density was measured as turbidity at 590 nm after
32 h of growth. Each value is the mean of two determinations. B, dependence of cell growth on side chain volume of the amino acid
at position 63. Growth yields for JP17 containing the four plasmids
shown in panelA were determined at 32 h and 6
mM succinate. Absorbances were normalized to that of
JP17/
To determine
whether the defect in ATP synthesis resulted from a defect in substrate
binding, wild-type atpB and the mutant atpB genes
C63A and C63W were expressed in E. coli BL21[DE3]/pLysS (Chen et al., 1992; Rosenberg et al., 1987; Studier et al., 1990), and the
resultant
Figure 6:
Nucleotide binding to over-expressed
wild-type and mutant CF
The hybrid spinach/E. coli enzyme was sufficiently active
in ATP synthesis to allow cells to grow on succinate almost as well as
cells containing the native bacterial enzyme. This indicates that the
protein-protein interactions necessary for proper proton coupling are
essentially intact in the hybrid enzyme. The ATPase activities of the
endogeneous and the hybrid enzymes were also very similar to each
other, and Western blots indicated that the E. coli and
spinach
We examined several mutants at position 63
of the CF
The role of
proton translocation through CF
The phenotype of the CF
The nucleotide
sequence(s) reported in this paper has been submitted to the
GenBank®/EMBL Data Bank with accession number(s)
U23082[GenBank® Link].
We thank Dr. Alan Senior for providing the E. coli
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES.
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
F
-ATP synthases utilize protein
conformational changes induced by a transmembrane proton gradient to
synthesize ATP. The allosteric cooperativity of these multisubunit
enzymes presumably requires numerous protein-protein interactions
within the enzyme complex. To correlate known in vitro changes
in subunit structure with in vivo allosteric interactions, we
introduced the
subunit of spinach chloroplast coupling factor 1
ATP into a bacterial F
ATP synthase. A cloned atpB gene, encoding the complete chloroplast
subunit,
complemented a chromosomal deletion of the cognate uncD gene in Escherichia coli and was incorporated into a
functional hybrid F
ATP synthase. The cysteine residue at
position 63 in chloroplast
is known to be located at the
interface between
and
subunits and to be conformationally
coupled, in vitro, to the nucleotide binding site >40
Å away. Enlarging the side chain of chloroplast coupling factor 1
residue 63 from Cys to Trp blocked ATP synthesis in vivo without significantly impairing ATPase activity or ADP binding in vitro. The in vivo coupling of nucleotide binding
at catalytic sites to transmembrane proton movement may thus involve an
interaction, via conformational changes, between the amino-terminal
domains of the
and
subunits.
F
-type ATP synthases utilize the
energy of a transmembrane proton gradient to drive the conversion of
ADP plus P
to ATP. The enzymes from chloroplasts,
mitochondria, and bacteria exhibit a striking similarity in their
overall structure, being composed of F
(chloroplast
CF
),
(
)a membrane-spanning proton
channel complex, and F
(chloroplast CF
), a
peripheral complex that contains the catalytic site(s) for ATP
synthesis and hydrolysis. The F
portion is composed of five
subunits designated
to
in order of decreasing molecular
weight and with a stoichiometry of
(Nalin and Nelson,
1987). ATP synthesis in vivo is absolutely dependent upon
continued translocation of protons through the F
and
F
complexes. The isolated F
complex, however,
catalyzes only the reverse reaction of ATP hydrolysis. The minimal
subunit assembly that efficiently supports ATP hydrolysis is the
complex (Hu et al.,
1993). The
and
polypeptides are arranged in an alternating
array such that each
subunit makes contact with two
subunits to form a hexameric ring (Boekema and Bottcher, 1992; Abrahams et al., 1994). In addition, the
subunits make direct
contact with the
subunit (Musier and Hammes, 1987) and with the
(Engelbrecht et al., 1986) subunit. Six nucleotide
binding sites are located at
subunit interfaces. The
putative catalytic sites are formed by
residues with a minor
contribution from
subunit residues (Abrahams et al.,
1994). The reverse is true for the remaining three sites, located
predominantly on
subunits and thought to play a regulatory role.
CF
is thus an allosteric enzyme whose catalytic function
requires correct structural communication between
subunits and at
least the
,
, and
subunits.
and
subunits, but especially of the
subunits, are highly conserved among the different ATP synthases
(Walker et al., 1985), strongly suggesting that they are
structurally and functionally homologous. Indeed, we previously
demonstrated (Richter et al., 1986) that the
subunit
isolated from spinach chloroplasts could be reconstituted into
-deficient F
from Rhodospirillum rubrum chromatophores to form a functional hybrid F
F
enzyme. When this evidence is considered along with other
structural and enzymological studies (for review, see Penefsky and
Cross(1991) and Boyer(1993)), little doubt remains that all
F
F
-type ATP synthases share the same basic
catalytic mechanism and essential allosteric intersubunit interactions.
polypeptide
(approximately residues 20-95). For example, sensitivity to the
fungal peptide tentoxin, an inhibitor of allosteric cooperativity in
CF
(Richter et al., 1986), is determined in part
by the side chain of
residue 83 (Avni et al., 1992).
Recently, Colvert et al.(1992) determined that Cys
is inaccessible in the assembled CF
complex but that it is
accessible to covalent modification in the isolated
subunit. The
simplest explanation was that Cys
is buried in an
interface. This conclusion was confirmed from the
crystal structure of the mitochondrial F1 complex, which indicated that
the residue that is equivalent to Cys
of CF
is located at an interface between adjacent amino-terminal
-barrel domains of neighboring
and
subunits. Recent
studies of Miki et al. (1994a, 1994b) have indicated that
conformational processes involving these contacting domains are
important for the coupling of transmembrane proton movement to ATP
synthesis. Mills et al. (1995) have shown that binding of ADP,
but not ATP, to the isolated
subunit induces a significant change
in the conformation of the amino-terminal domain. This change was
detected as altered fluorescence of probes attached directly to
Cys
, greater than 40 Å from the ADP binding site.
These observations suggest that allosteric interactions between the
different nucleotide binding sites, as well as proton-driven changes in
nucleotide binding affinities, may be mediated by the amino-terminal
-barrel structures of adjacent
and
subunits. To
further examine such interactions, we have examined the effects of
mutations at
residue 63 on nucleotide binding and catalytic
properties of spinach chloroplast CF
.
subunit can be produced to
facilitate site-directed mutagenesis investigations of assembly and
conformational coupling among CF
subunits. To address
questions of subunit interaction among either homologous or
heterologous F
subunits in situ and to determine
whether mutant
polypeptides are functional, we have developed an in vivo complementation system in which a cloned spinach
chloroplast atpB gene, encoding the CF
subunit, complements an Escherichia coli chromosomal deletion
of the cognate uncD gene. In this communication we describe
the properties of the in vivo complementation system and its
application to investigating mutations in codon 63 of the spinach
chloroplast atpB gene.
Materials
E. coli strain JP17, obtained
from A. Senior (Lee et al., 1991), contains a deletion of the uncD gene corresponding to amino acids 20-332 of the
F
subunit. The genotype of JP17 is
uncD, argH, entA, pyrE, recA::Tn10, tet
. Plasmid pRPG31
(Gunsalus et al., 1982) contains an internal portion of the E. coliunc operon, including uncD (
subunit) and uncC (
subunit), inserted into pBR322 and
transcribed from an endogenous plasmid promoter. Spinach chloroplast
CF
and
subunit were prepared as described elsewhere
(Hu et al., 1993; Richter et al., 1986). Antibiotics
(ampicillin, tetracycline, and tentoxin) were purchased from Sigma.
Tryptone and yeast extract were from Difco. All other chemicals were of
the highest quality reagent grade available.
Computational
Putative ribosome binding sites were
scored using results from the Perceptron neural network analysis of
Stormo et al.(1982). The DNA sequence for 71 nucleotides
surrounding each initiating AUG was represented as a 4 71
binary matrix, and the 71
4 weight matrix W71 was taken from
Stormo et al.(1982). The scalar evaluation score is the inner
product of the sequence matrix and the weight matrix. The software used
is available from the authors. Oligonucleotide primers were designed
with the aid of software from Scientific and Educational Software
(State Line, PA); software from this source was also used for
simulation, analysis, and graphic presentation of cloning
manipulations. Sequence alignments were performed via the
``PhD'' profile alignment facility (Rost and Sander, 1993)
and refined manually.
Mutagenesis
Oligonucleotide-directed mutagenesis
was accomplished essentially as described before (Chen et al.,
1992) using single-stranded DNA produced from plasmid
pBS(-)XB3a in E. coli CJ236. Mutagenic primers were
20-23-mers with altered sequence (in lower case) at codon 63:
-C63A, 5`-gcT;
-C63V, 5`-gtT;
-C63W, 5`-TGg. Mutations
were confirmed by partial sequencing. For overexpression, an XbaI-SstI fragment containing each mutation was used
to replace the corresponding region of expression plasmid
pET3a-
NE3 (Chen et al., 1992).
Plasmid Construction
Plasmids pBS(-)XE0
and pET3a-
NE2 have been described previously (Chen et
al., 1992). pBS(-)
XE0 contains an XbaI-EcoRI fragment of spinach chloroplast DNA
bearing the atpB gene (
subunit) and the proximal 50
nucleotides of the overlapping atpE gene (
subunit).
pET3a-
NE2 contains atpB fused to the gene 10 ribosome
binding site and translational enhancer of coliphage T7 (Olins and
Rangwala, 1990) in the expression vector pET3a (Studier et
al., 1990). The atpB insert and T7 ribosome binding site
of pET3a-
NE2 were excised with XbaI and BamHI
and inserted into similarly cleaved pBS(-) (Stratagene) to
produce plasmid pBS(-)
XB1. The plasmid was digested with HindIII for 4 h at 37 °C in 50 mM KOAc buffer
(1-4-All Plus buffer, Pharmacia Biotech Inc.) and filled in by
the addition of E. coli DNA polymerase Klenow fragment (25
units/ml plus 0.5 mM dNTP for 50 min at 20 °C). The
reaction mixture was extracted with phenol/chloroform,
ethanol-precipitated, and self-ligated with T4 DNA ligase (Life
Technologies, Inc.) overnight at 18 °C. DNA was transformed
according to Hanahan(1985) into E. coli DH5
(Life
Technologies Inc.) and was selected on LB medium (1% (w/v) NaCl, 1%
(w/v) Tryptone, 0.5% yeast extract, pH 7.5) plus 100 µg of
ampicillin/ml. Two (of about 50) of the recovered colonies were chosen
and designated pBS(-)
XB3a and -b. As
documented below, both of these plasmids could express a functional
subunit in E. coli. All subsequent experiments and
constructions reported here as
XB3 were performed with
XB3a.
Complementation Assay
Plasmids were transformed
into E. coli JP17 and plated onto LB agar plus 100 µg of
ampicillin/ml and 12.5 µg of tetracycline/ml. Vector pBS(-)
was also transformed into E. coli DH5 and selected
without tetracycline. After growth overnight at 37 °C, colonies
were streaked onto a prewarmed minimal medium plate containing
succinate (Hawthorne and Brusilow, 1986; Gibson et al., 1977)
(100 mM potassium phosphate (pH 7.1), 1 mM MgCl
, 0.2 µM ZnSO
, 0.2
µM CuSO
, 0.5 µM MnSO
,
2 nM CoCl
, 0.5 µM FeSO
,
10 µM CaCl
, 0.1% (w/v) thiamine HCl, 40
µM 2,3-dihydroxybenzoate, 0.8 mM arginine, 0.2
mM uracil, 0.2% (NH
)
SO
,
0.06% casein, 0.4% sodium succinate). For growth yield determinations,
cells containing plasmids were inoculated into 3 ml of LB medium plus
100 µg of ampicillin/ml and (except for DH5
) 12.5 µg of
tetracycline/ml and grown overnight at 37 °C. A loop of each
culture was transferred to 15 ml of prewarmed minimal medium containing
succinate at 3, 6, or 12 mM plus appropriate antibiotics as
above. Cell densities were measured as turbidity at 590 nm after growth
for varying times at 37 °C.
ATPase Assays
Cells were grown on LB medium
containing 30 mM glucose. Starter cultures (3 ml) were grown
at 37 °C to an optical density at 590 nm of 0.7 then
inoculated into 1 liter of the same medium and grown to midexponential
phase. Cells were harvested by centrifugation for 15 min at 10,000
g, resuspended, and washed once in TM buffer (50
mM Tris-HCl (pH 8), 10 mM MgCl
). The
cells were finally suspended to 40% (wet w/v) in TM buffer and lysed at
4 °C with 0.1-mm glass beads in a Mini-Bead Beater (Biospec
Products) at full speed. Unbroken cells and cell debris were removed by
centrifugation at 10,000
g for 15 min. Plasma
membranes were collected by centrifugation at 100,000
g for 3 h at 4 °C and resuspended to
10 mg of protein/ml.
ATPase activity of washed membranes was determined by measuring release
of P
from ATP at 37 °C (Taussky and Shorr, 1953) in an
assay mixture (1 ml) containing 50 mM Tris-HCl (pH 8), 4
mM ATP, 2 mM MgCl
, and membranes
equivalent to 50 µg of protein. The protein concentration of
membrane preparations was measured by the BCA method (Pierce Chemical
Co.) (Smith et al., 1985).
Immunoblot Analysis
From 10,000 g supernatants prepared as above, plasma membranes were obtained by
centrifugation at 100,000
g for 1.5 h and washing once
in 4 ml of TM buffer. The F
complex was released by
resuspending membranes in 7.5 ml of stripping buffer (1 mM Tris-HCl, 0.5 mM Na-EDTA, 2 mM dithiothreitol
(pH 8)) and mixing gently overnight at 4 °C. The membranes were
sedimented at 100,000
g for 1 h. Proteins were
precipitated from the supernatant by the addition of trichloroacetic
acid to 8% (Schumann et al., 1985). Proteins were solubilized
in 1 ml of sample buffer (1.25% (w/v) sodium dodecyl sulfate, 7.5%
sucrose, 125 mM dithiothreitol, 125 mM
Na
CO
), and polypeptides were separated on 12%
polyacrylamide gels (Schumann et al., 1985). One of a set of
duplicate gels was stained with Coomassie Blue G-250; the other was
electrotransferred to polyvinylidene difluoride membranes (Immobilon,
Millipore) in a Hoefer TE apparatus following the manufacturer's
instructions. The membrane was decorated overnight with a mouse
monoclonal antibody (1:2500 dilution) raised against the purified
chloroplast
subunit. Decorated protein bands were reacted for 1 h
with goat anti-mouse peroxidase-conjugated antibody (1:1000 dilution;
Sigma).
Nucleotide Binding Assays
polypeptide was
over-expressed in E. coli, purified, and refolded essentially
as before (Chen et al., 1992). 0.5 mg of inclusion body
protein, suspended at 0.1 mg/ml in TE50/2 buffer (50 mM Tris-HCl (pH 8), 2 mM Na-EDTA), was mixed with 150 ml of
refolding buffer (20 mM Tris-HCl (pH 8), 2 mM Na-EDTA, 2 mM dithiothreitol) containing 4.3 M urea for 90 min at 4 °C, and then dialyzed successively
against (i) 150 ml of refolding buffer plus 3 M urea, 12%
glycerol for 5 h at 4°; (ii) 150 ml of refolding buffer plus 1.5 M urea, 10% glycerol for 7 h at 4°; (iii) 200 ml of
refolding buffer plus 3 mM ATP, 8% glycerol for 6 h at
20°. Isolated
polypeptides were titrated with
trinitrophenyl-ADP (TNP-ADP). The net fluorescence enhancement was
corrected for inner filter effect, and binding constants were
determined as before (Chen et al., 1992; Mills and Richter,
1991) by least-squares nonlinear curve-fitting to the untransformed
data. Since each subunit has one ADP binding site, the fraction of
polypeptide which was correctly refolded was taken as the
calculated fractional number of binding sites.
Sequence of the Spinach Chloroplast atpB Gene
An
initial alignment of the spinach chloroplast amino acid sequence
deduced from the gene sequence (Zurawski et al., 1982)
(Genbank entry SPICPATBE, accession J01441) revealed several
alterations in residues completely or highly conserved in all other
F
F
-type
polypeptides (see also Chen et al., 1992). To resolve these discrepancies, we resequenced
most of the remainder of atpB. Three additional errors in the
published sequence were located at positions G
(was C),
G
(was C), and G
(was A). These amendments
change three amino acids: Ala
(was Pro), Gly
(was Arg), and Asp
(was Asn). Each change results
in greater sequence conservation with other
polypeptides. The
deduced amino acid sequence is presented in Fig. 1A,
aligned with representative chloroplast, mitochondrial, and bacterial
sequences. A more detailed comparison of the amino-terminal
domain is given in Fig. 1B. The revised DNA sequence is
GenBank accession U23082, locus SOU23082.
subunit. A, the
deduced amino acid sequence (GenBank accession U23082) is aligned over
representative sequences of CF
from a monocot gramineae (Oryzasativa), a bryophyte (Marchantiapolymorpha), a green alga (chlorophyte, oocystacea; Chlorellaellipsoidea, Swiss-Prot entry ATPB_CHLEL),
and a cyanobacterium (Synechococcus PCC 6301), and of F
ATP synthase from a purple non-sulfur bacterium (E.
coli), and bovine mitochondrion. An initial alignment was
performed with all chloroplast, mitochondrial, and bacterial F
sequences in the SWISS-PROT data base; a representative
subsampling is shown. Swiss-Prot entries not given are listed below. In
sequences other than spinach, upper case letters denote
nonconservative changes, whereas lower case letters indicate
conservative or semiconservative changes (see Bordo and Argos(1991)). B, alignment of spinach
residues 19-95
corresponding to the amino-terminal
-barrel of bovine
mitochondrial F
(residues 9-80). The spinach
chloroplast sequence is aligned with sequences from organisms
representative of a uniformly broad range of taxa. The uppernumbers designate positions in the spinach chloroplast
sequence, and the lowernumbers correspond to the
bovine mitochondrial sequence. The unshadedareas represent the approximate extents of the six
-strands
identified in the crystal structure of bovine mitochondrial F
(Abrahams et al., 1994). Sequences are identified
by their Swiss-Prot entry names (given in parentheses below). Kingdom
Eucarya: Tracheophytes - Angiosperms, Dicot, Solanaceae, tobacco Nicotianatabacum (ATPB_TOBAC) and Nicotianaplumbaginifolia (ATPB_NICPL); Dicot, Fabaceae, pea Pisumsativa (ATPB_PEA); Monocot, Gramineae,
rice Oryzasativa (ATPB_ORYSA);
Monocot, Poaceae, Aegilopscolumnaris (ATPB_AEGCO). Gymnosperm, Pinaceae, black pine Pinusthunbergii (extracted from Genbank accession
PINCPTRPG). Pteridophyte, filicophyta, turnip fern Angiopterislygodiifolia (ATPB_ANGLY). Bryophyte:
Hepaticopsida, liverwort Marchantiapolymorpha (ATPB_MARPO). Phycophytes: chlorophyte (green
alga), chlamydomonadaceae, Chlamydomonasreinhardtii (ATPB_CHLRE); phaeophyte (brown alga) Pylaiellalittoralis (ATPB_PYLLI);
``prochlorophyte'' Prochlorondidemni (PIR
entry A42697); ``euglenophyte'' Euglenagracilis Z (ATPB_EUGGR). Kingdom Bacteria: Cyanobacterium Synechococcus PCC 6301 (ATPB_SYNP6); Green
sulfur bacterium Chlorobiumlimicola (ATPB_CHLLI); Purple sulfur bacterium Rhodospirillumrubrum; Purple non-sulfur bacterium E.coli (ATPB_ECOLI), plant
mitochondrion (N. plumbaginifolia, ATP2_NICPL). (Mitochondria arose from the purple
non-sulfur lineage of bacteria.)
Complementation of an E. coli uncD Deletion by the
Spinach Chloroplast atpB Gene
To establish an in vivo system in which chloroplast could be produced in soluble
form, we first examined the relevant sequences of spinach chloroplast atpB for potential E. coli promoters or ribosome
binding sites. No likely matches to these sequences were found. In
particular, atpB does not have a good E. coli Shine-Dalgarno sequence upstream. We applied the neural network
predictor of Stormo et al.(1982) to estimate whether
translational initiation would occur at the first AUG of atpB.
Evaluation with weight matrix W71 (Stormo et al., 1982)
predicted that atpB would be translatable in E. coli.
Munn et al.(1991) had previously demonstrated that spinach
chloroplast atpB was translated in E. coli minicells
but that translation yields were increased by introducing a consensus E. coli Shine-Dalgarno sequence. We therefore tested an
existing construct pBS(-)
XB0 (Chen et al., 1992) in
which atpB is preceded by native chloroplast DNA sequences. We
also placed atpB under control of the E. colilacP promoter and provided it with a strong bacterial
translation start signal; since we had also constructed a
high-expression plasmid, pET3a-
NE3, consisting of atpB fused to the bacteriophage T7 ribosome binding site and
translational enhancer (Chen et al., 1992), we subcloned this
T7-atpB fusion into plasmid pBS(-), giving plasmid
pBS(-)
XB1. Fig. 2shows the relevant features of
these constructs. Plasmids XE0 and XB1 are high-copy-number derivatives
of pUC19 and exhibit constitutive expression of the lac promoter as a result of titration of endogenous lac repressor. Cells containing these plasmids, particularly when
grown in the absence of glucose, thus synthesize
polypeptide
constitutively (data not shown). In order to prevent read-through
translation from lacZ into atpB, we modified plasmid
pBS(-)
XB1 by filling in a HindIII site at lacZ codon 18 to introduce in-frame termination 63 base pairs upstream
of atpB. Upon further examination of the resultant plasmids
pBS(-)
XB3a and -b, however, we found that each
contained, instead of the expected fill-in, a deletion extending from
the HindIII site to one of two upstream HindIII*
sites (AtGCTT and AgGCTT respectively) located within the lacP promoter. This region is depicted in Fig. 2B, and the sequences are shown in Fig. 2C. The deletion in plasmid
XB3a starts just
upstream of the -10 box of the lacP (P1) promoter
(Reznikoff and McClure, 1986), whereas the deletion in
XB3b also
removes the -35 region of lacP. Transcription of atpB in these plasmids is evidently driven by constitutive
promoters in the vector, such as the upstream P
in the
colE1 ori region (Balbas et al., 1988).
XE0. Hatchedbars represent vector sequences; darkshading represents chloroplast atpB coding
sequences; lightshading represents chloroplast atpE sequences; and openbars or arrows represent noncoding chloroplast insert sequences. Also indicated
are the positions of HindIII and HindIII* restriction
recognition sites, the pBS-derived lacP promoter, and the
codon for Cys
, discussed in the text. B, plasmids
pBS(-)
XB1 and pBS(-)
XB3a and b. Shading is the
same as in A, with the heavysolidline representing phage T7 sequences. The italicizedrestrictionenzymesites are derived
from T7 or adaptor sequences. The figure shows the extent of the two
deletions in plasmids XB3a and b. Plasmid pBS(-)
XB1 is
identical but lack the deletion; the leftmost vector region is intact
up to XbaI, as in panelA. Position of the
pET3a-derived T7 ribosome binding site (RBS) is indicated. C, sequence of the lac promoter region and deletions.
Nucleotide and inferred amino acid sequences are shown for the lacP1 promoter,
-galactosidase
-peptide, and atpB translation initiation region from plasmids
pBS(-)
XB1 and the deletion derivatives pBS(-)
XB3a
and XB3b. Restriction site sticky ends are underlined;
sequences created by filling-in are lowercase; and
promoters and translation initiation codons are doubleunderlined.
Expression
of atpB in cells transformed with these plasmids was monitored
indirectly by an assay that requires formation of a functional ATP
synthase in vivo. E. coli cells lacking ATP
synthase are unable to respire and hence cannot use citric acid cycle
intermediates such as succinate as an energy source. The genetic
complementation assay thus measures the ability of an introduced gene
to support bacterial growth on minimal medium containing succinate as
the sole carbon source. The ATPase host is E.
coli JP17 (Lee et al., 1991), which has a targeted
deletion of most of the uncD gene encoding the F
subunit. We transformed JP17 with plasmids either lacking
an insert, containing the E. coli uncD gene, or containing the
various constructs of spinach chloroplast atpB described
above. After growth overnight on LB agar to allow expression of the
inserted gene, cells were transferred to minimal agar containing 0.4%
succinate. Complementation was scored as cell density after 1-3
days at 37 °C. As shown in Fig. 3, the
uncD strain transformed with vector only (pBS(-)) fails to grow.
As a positive control, cells containing E. coli uncD (plasmid
pRPG31) (Gunsalus et al., 1982) grow almost as well as the
wild-type strain DH5
transformed with vector. The chloroplast atpB gene in plasmid pBS(-)
XB0, with constitutive
transcription but native chloroplast translation control sequences,
supports weak but detectable growth on solid media. Finally, Fig. 3shows that plasmid pBS(-)
XB3, containing atpB with a weak plasmid promoter but a strong bacteriophage
translational control region, supports growth almost as well as does
the native E. coli gene. We were surprised to find, however,
that the constitutively-transcribed, translationally-enhanced gene in
plasmid pBS(-)
XB1 could not complement the uncD deletion (not shown). Further investigation revealed that although
this plasmid directs synthesis of reasonable amounts of
polypeptide, the protein aggregates entirely into insoluble inclusion
bodies (data not shown).
subunit genes. Colonies of JP17
(
uncD) or DH5
cells transformed with the indicated
plasmids were streaked onto succinate minimal plates as described under
``Experimental Procedures'' and grown for
3
days.
Assembly and Activity of Hybrid ATP Synthase
To
provide a more quantitative assessment of ATP synthesis capability in
bacteria carrying a chloroplast CF
subunit gene, we
determined the yield of cells grown on limiting concentrations of
succinate, which is an indirect measure of ATP synthesis efficiency. To
confirm that active ATP synthase had been assembled in these cells, we
determined the specific ATPase activity of partially purified ATP
synthase, and we identified chloroplast
by Western blot analysis.
The results shown in Table 1demonstrate that all strains capable
of growth possess comparable levels (within one standard deviation) of
Mg
-dependent membrane-bound ATPase activity.
Membranes from the uncD deletion mutant JP17 transformed with
vector only exhibited undetectable ATPase activity, whereas cells
transformed with E. coliuncD exhibited 86% of
wild-type Mg-ATPase activity. Strikingly, chloroplast atpB restored Mg-ATPase activity to about 75% of the level achieved
with the E. coliuncD gene or to about 65% of
wild-type activity. Both the hybrid and the endogeneous enzymes
exhibited only low levels of Ca
-dependent ATPase
activity (Table 1).
To confirm that this ATPase activity was
that of a hybrid F species, washed membranes from selected
transformants were treated with EDTA to release the F
portion of ATP synthase. The presence of bacterial or chloroplast
polypeptides in the released protein fraction was assayed by
polyacrylamide gel electrophoresis and Western immunoblotting, as shown
in Fig. 4. Strain JP17 transformed with vector only has no
membrane-bound
-reactive material (Fig. 4B, lane3), whereas when transformed with E. coli
uncD it contains the 50.3-kDa E. coli
(lane2). Membranes from JP17 transformed with clone
XB3 (lane4) contain substantial amounts of the 53.6-kDa
chloroplast
polypeptide.
subunit
assembly into membrane-bound ATP synthase. Soluble protein released
from washed membrane preparations was prepared, and polypeptides were
fractionated by denaturing SDS-polyacrylamide gel electrophoresis, as
under ``Experimental Procedures.'' Lane1,
20 µg CF
; lanes2-6,
60 µg of membrane-eluted protein from E. coli JP17
transformed with the following plasmids: lane2,
pRPG31; lane3, pBS(-), lane4, pBS(-)
XB3; lane5,
pBS(-)
XB3-C63A; lane6, C63V; lane7, C63W. A, polypeptides were visualized by
staining with Coomassie Blue. B, an identical gel was
transferred to polyvinylidine difluoride membrane, and
polypeptide was detected with an anti-
monoclonal antibody. The upperband corresponds to spinach chloroplast
(M
= 53,571), and the lowerband corresponds to E. coli
(M
= 50,331).
Previous studies (Richter et
al., 1986) showed that the CF
subunit, when
reconstituted in vitro with the
-less F
of R. rubrum chromatophores, conferred upon the hybrid enzyme
sensitivity to the CF
-specific inhibitor tentoxin. The
hybrid enzyme also exhibited an enhanced dependence of Mg-ATPase
activity on the oxyanion sulfite, compared with that of the R.
rubrum F
. We therefore examined the activity of the
hybrid chloroplast-E. coli F
in the presence of
tentoxin or sulfite. Table 2demonstrates that neither the
bacterial nor the hybrid membrane-bound ATPase preparation is inhibited
by tentoxin concentrations well above those required to give almost
total inhibition of native CF
(see Hu et
al.(1993)). Table 2also shows that low concentrations of
sulfite, 2-10 mM, slightly stimulate (by 20-25%)
the activity of the hybrid enzyme but not that of the bacterial enzyme.
Higher concentrations of sulfite lead to significant inhibition of both
enzymes.
Hybrid ATP Synthases with Amino Acid Replacements at
CF
The region surrounding
Cys Residue 63
is of particular interest because it is exposed
only upon removal of the
polypeptide from the CF
complex and is thus a candidate for a residue involved in
intimate protein-protein interactions at the subunit interface (Colvert et al., 1992). The amino acid sequence from 60 to 70 (spinach
numbering) is virtually identical among all chloroplasts and
cyanobacteria and exhibits strong conservation with purple bacteria and
mitochondria; these sequences are presented in Fig. 1B.
To investigate the properties of this region, residue 63 in plasmid
pET3a-
XB3 was changed from Cys to Ala, Val, or Trp by
oligonucleotide-directed mutagenesis to produce plasmids
pET3a-
XB3/C63A, C63V, and C63W.
subunits in situ, the mutant genes were
tested for their ability to complement the uncD deletion in E. coli strain JP17. Complementation was scored initially by a
plate assay (as in Fig. 3) and then by measuring growth yields
in succinate-limited medium. These in vivo data were
corroborated with ATPase activities of isolated bacterial membranes. As
seen in Table 3, the
-less strain JP17 transformed with a
plasmid encoding
-C63A grows somewhat better than JP17 containing
wild-type CF
, and exhibits identical ATPase activity.
JP17 containing
-C63V grows less well, and JP17 containing
-C63W exhibits essentially no growth after 32 h, a time by which
JP17 transformed with wild-type
is at stationary phase. (Some
growth of JP17/
-C63W was evident after 48 h.) In contrast to its
poor growth, mutant
-C63W shows near normal in vitro ATPase activity (
90% versus wild-type). The native
chloroplast DNA clone
XE0, which supported growth on solid medium (Fig. 3), did not support growth in liquid medium (Table 3). A plot of growth yield versus [succinate], shown in Fig. 5A, reveals a
linear dependence for JP17 transformed with every plasmid except the
mutant
-C63W, indicating that for
-C63W a factor other than
succinate is growth-limiting. We infer that in JP17/
-C63W, ATP
synthesis is growth-limiting. Furthermore, decreased growth yield is
also directly correlated with increased bulk of the amino acid side
chain of residue 63. As seen in Fig. 5B, a plot of
growth yield versus side chain volume yields an inverse linear
relationship.
C63A. Amino acid side chain volumes were calculated directly
from the crystallographic data compiled by Richards(1974); the volume
of cysteine was taken as one-half cystine plus an average hydrogen
volume of 7 Å
. Very similar curves were obtained for
different growth times and succinate
concentrations.
That a true hybrid F had been assembled in
these strains was confirmed by immunoblot analysis of F
isolated from washed bacterial membranes. As seen in Fig. 4, lanes5-7, JP17 transformed with
plasmids containing
-C63A, -C63V, or -C63W contains substantial
amounts of authentic membrane-bound spinach chloroplast
polypeptide, comparable with the amount present in JP17 transformed
with wild-type chloroplast
(lane4) or with E. coli
(lane2).
polypeptides were refolded from purified inclusion
bodies. Each subunit preparation was assayed for ADP binding by a
standard titration with the fluorescent ADP analogue TNP-ADP. The
dissociation constant, K
, and binding
site occupancy were calculated from duplicate or triplicate titrations.
A typical result is shown in Fig. 6. As summarized in Table 4, both the C63W and C63A mutant subunits refolded to
essentially the same extent as wild-type
. The C63A subunit binds
TNP-ADP with an affinity very similar to that of wild-type, whereas the
C63W subunit binds TNP-ADP about twice as tightly. Since the C63W
mutant subunit has close to normal nucleotide binding ability as well
as ATP hydrolysis activity, the defect in ATP synthesis in vivo is unlikely to result from any intrasubunit abnormality.
. Binding titrations were
performed and analyzed as under ``Experimental Procedures.''
One representative trial is shown. The corrected fluorescence
enhancement was normalized to 180 µg (3.36 nmol)
polypeptide.
Assembly In Vivo of a Functional Chloroplast-Bacterial
Hybrid ATP Synthase
Although we had shown previously that
high-level bacterial expression of the spinach chloroplast atpB gene leads to production of insoluble protein (Chen et
al., 1992), we expected that moderate rates of expression might
yield soluble protein in vivo. Furthermore, we had reason to
hope that interspecies assembly of hybrid F ATP synthase
would ensue, as functional ATP synthase has been produced in vitro by introducing chloroplast
subunits into R. rubrum ATP synthase (Richter et al., 1986). Our
results demonstrate that such a hybrid system can be formed and that it
functions well enough to support vigorous bacterial growth.
Surprisingly, every clone that appropriately expressed soluble
chloroplast
polypeptide to support cellular growth contained
either an active promoter and a very weak ribosome binding site or a
weak promoter and a strong ribosome binding site. In contrast, a clone
in which atpB was transcribed from a strong (lacP)
promoter and translated from a strong (phage T7) ribosome binding site
expressed atpB only as an insoluble polypeptide. From such a
construct, two spontaneous deletions of the lac promoter were isolated
that allowed functional expression of atpB as soluble
protein. Consistent results were obtained by Engelbrecht and co-workers
(Lill et al., 1993; Burkovski et al., 1994), who
found that spinach atpB expressed behind a phage
promoter yielded entirely insoluble
polypeptide and could not
complement E. coliuncD
or
uncD mutations. These results all support the hypothesis
that low-level expression from weak, endogenous plasmid promoters
permits formation of correctly folded, functional CF
polypeptide. Likewise, functional E. coli
polypeptide is
produced from plasmid pRPG31 by transcription of the E. coliuncD gene from a constitutive weak vector promoter, most
likely the P1 promoter of tetR (Balbas et al., 1988).
polypeptides were assembled into the F
F
complex to an approximately equal extent. The spinach
subunit is thus a remarkably effective substitute for the native E.
coli
polypeptide. The failure of an earlier complementation
attempt (Munn et al., 1991) can be attributed to use of an E. coli host in which residual assembly-defective F
could block entry of chloroplast
into the functional
F
complex. Gatenby and Rothstein (1986) found that a
protein containing the first 365 amino acids of maize chloroplast
CF
fused to
-galactosidase could be synthesized
in E. coli and could associate with the bacterial membrane
F
complex. This fusion protein could not, however, assemble
into an intact F
complex (Gatenby and Rothstein, 1986).
Subunit Interactions and Cooperativity
Some recent
studies have indicated that the amino terminus of the
polypeptide is in contact with the
subunit and that this contact
is functionally important. First is the observation that
Cys
is buried within the CF
complex and
is accessible to modifying reagents only upon isolation of the
subunit from the other CF
subunits (Colvert et
al., 1992). Second, the positions cognate to 63 and 64 in E.
coli
are sites of assembly mutants, some of which block
assembly of both
and
subunits into the ATP synthase (Noumi et al., 1986; Miki et al., 1994a, 1994b). Third,
these residues in E. coli are also proposed to lie at an
interface, since they are recognized by a specific
monoclonal antibody in the isolated but not the assembled
(see
Miki et al. (1994a, 1994b). Finally, this hypothesis is
supported by the 2.8-Å crystal structure of beef heart
mitochondria F
(Abrahams et al., 1994). In the
crystal structure, the amino-terminal region of the
and
subunits (mitochondrial
amino acids
9-80,
corresponding to 19-96 for CF
) is organized
into a separate
-barrel domain connected by a short hinge to the
nucleotide-binding central domain.
strand d from each
subunit appears to contact strand a of the adjacent subunit,
joining the hexameric ring of
and
subunits via their
amino-terminal domains. Chloroplast
residue 63 (47 in beef heart
mitochondria) lies in
strand d, and is thus implicated
in subunit interaction by the structural evidence. These sequences, and
the approximate location of the
strands, are shown in Fig. 1B.
subunit. The conservative change (in terms
of the size of the side chain) of Cys
to Ala had little
effect on the function of the
subunit in the complementation
assay. However, substitution of the bulkier side chains of Val or Trp
reduced or blocked ATP synthesis in vivo. Indeed, ATP
synthesis, as measured by succinate-dependent cell growth, was directly
correlated with the volume of the residue at position 63, with the
smallest residue being the most active, and the larger residues
progressively less so. This result is consistent with Cys
being located at a site of interaction between
and
subunits, and further demonstrates that this interaction is
functionally important. Replacing Cys
with the bulkier
amino acids Val or Trp did not, however, prevent assembly of the
subunit into the F
F
complex since washed
membranes from both mutants contained approximately the same amount of
subunit as did membranes from cells containing wild-type
chloroplast or E. coli
subunit. Moreover, ATPase
activities of washed membranes from wild-type cells and from the C63A
and C63W mutants were essentially identical. This result is further
corroborated by the finding that these two mutant
subunits, when
over-expressed and refolded, bound nucleotides with close to the same
affinity as native, wild-type spinach
subunit.
is to release bound product
ATP (reviewed by Penefsky and Cross(1991)). On the other hand, ATP
hydrolysis can drive reverse proton pumping, but may not be
obligatorily coupled to it. The observation that the Cys
Trp replacement appears specifically to block ATP
synthesis but not ATP hydrolysis is particularly intriguing as it
suggests that the bulky side chain interferes with cooperative
subunit-subunit interactions required for coupling proton translocation
to ATP synthesis but not with those involved in the catalytic cycle for
ATP hydrolysis (reviewed by Penefsky and Cross(1991)). A complementary
observation with E. coli F
(Miki et
al., 1994a) supports the independence of proton coupling and
ATPase activity. A mutation from Glu to Asn at position
-41
(cognate to CF
-63) has 100% wild-type ATP synthase
activity (judged by succinate-dependent growth), but only 4% wild-type
ATPase activity.
mutant
-C63W indicates that specific subdomains of the
interface may couple proton movement to ATP synthesis. Functional
communication between Cys
and the catalytic site is
suggested by the following evidence. We determined (Colvert et
al., 1992) that Cys
is 42 Å away from the
nucleotide binding site on the
subunit. More recently, we have
demonstrated that binding of ADP (but not ATP) to isolated
CF
-
induces a shift in the conformation or position of
the amino-terminal domain, forcing residue 63 into a more hydrophobic
and less solvent-accessible environment (Mills et al., 1995).
This change is detected as an increase in fluorescence quantum yield
and a decrease in acrylamide-induced fluorescence quenching of pyrenyl
maleimide attached to Cys
. Taken together, these
observations present functional evidence for a critical role of the
amino-terminal domain of the
subunit surrounding Cys
in mediating cooperative interactions between the
and
subunits of CF
, which are essential for ATP synthesis.
Functional Alterations in Hybrid ATP Synthase
We
showed earlier (Richter et al., 1986) that reconstitution of
the spinach chloroplast subunit into
-less R. rubrum F
resulted in a hybrid enzyme that was
fully sensitive to the cyclic tetrapeptide tentoxin. Since tentoxin is
a specific noncompetitive inhibitor of chloroplast F
, this
result meant that the
polypeptide had conferred tentoxin
sensitivity upon the hybrid enzyme. A recent report (Avni et
al., 1992) suggested that sensitivity to tentoxin is determined by
the nature of the amino acid residue at position 83 near the amino
terminus of the
subunit. Chloroplast
residues 63-91
are identical (except for conservative changes at positions 77 and 83)
in all sequenced tobacco species and in spinach. Tentoxin-resistant
tobacco species contain Glu at residue 83, whereas tentoxin-sensitive
tobacco and spinach possess Asp at this position (Fig. 1B) (Avni et al., 1992). Furthermore,
conversion of Chlamydomonas reinhardtii
residues
74-91 to the corresponding tobacco sequence produced algae that
were tentoxin-resistant if residue 83 was Glu and tentoxin-sensitive if
it was Asp (Avni et al., 1992). This result implied that
Asp
alone could direct binding of tentoxin. Wild-type E. coli F
, on the other hand, is completely
insensitive to tentoxin (Table 2), even though its
subunit
has an aspartate residue at the position (residue 60) corresponding to
chloroplast position 83. Our results, furthermore, demonstrate that
introduction of the tentoxin-sensitive spinach
subunit into E. coli does not confer tentoxin-sensitivity to the
transformed bacteria. Thus Asp
alone is insufficient to
determine tentoxin sensitivity. Instead, tentoxin binding probably
requires a unique conformation of F
resulting from an
interaction between the amino terminus of the
subunit and some
other part or parts of the enzyme. We found recently (Hu et
al., 1993) that tentoxin inhibits cooperative interactions between
different nucleotide binding sites on CF
and suggested that
the toxin binds at an interface between the
and
subunits.
However, residue 83, the putative tentoxin interaction site, is located
at the top of
strand e in the crystal structure of
mitochondrial F
(Abrahams et al., 1994) (see Fig. 1B), and may not be directly involved in subunit
interactions. Binding of a molecule the size of tentoxin (packed volume
= 426 Å
; d
10 Å) at this
site might well distort the
interface, leading to loss
of catalytic cooperativity. A more detailed hypothesis concerning the
mechanism of tentoxin interaction can be formulated following
publication of the atomic coordinates of the mitochondrial F
crystal structure.
Conclusion
We have demonstrated that the
chloroplast CF
gene can be expressed in vivo in E. coli cells and assembled along with other E.
coli F
F
subunits to form an active hybrid
ATP synthase complex. In addition to allowing direct study of
interactions between heterologous F
subunits, the genetic
complementation system will be of value in screening mutant spinach
chloroplast
subunits for their ability to fold correctly and to
assemble into functional ATP synthase enzymes. Using this system, we
have presented evidence consistent with the hypothesis that select
amino-terminal subdomains of the
subunit are engaged in important
interactions that may play a critical role in the
coupling of proton translocation to ATP synthesis but which are not
essential for ATP hydrolysis.
, chloroplast coupling factor 0;
CF
, chloroplast coupling factor 1; LB medium, Luria-Bertani
bacterial growth medium; TNP-ADP,
2`(3`)-O-(2,4,6-trinitrophenyl)adenosine 5`-diphosphate.
deletion strain JP17 and plasmid pRPG31. The University of
Kansas Biochemical Research Service Facility provided oligonucleotide
synthesis and DNA sequencing services. We also thank Jing Ou for
sequence confirmation of mutant plasmids and members of the Richter and
Gegenheimer labs for helpful discussions.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.