The importance of the c subunit in the catalytic cycle has been demonstrated previ-ously, showing that it is probably related to the sequential conformational changes in the ab pairs in
Trang 1stabilization and activity of chloroplast ATP synthase
Zhang-Lin Ni, Hui Dong and Jia-Mian Wei
Shanghai Institute of Plant Physiology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China
ATP synthase occurs ubiquitously on
energy-transduc-ing membranes such as chloroplast thylakoid
mem-branes, mitochondrial inner memmem-branes, and bacterial
plasma membranes This enzyme catalyzes ATP
syn-thesis by a proton motive force across the membrane
formed by the respiratory chain or photosynthetic
elec-tron transport (ATPase in Escherichia coli recently
reviewed in [1,2], and ATP synthase in chloroplasts in
[3,4]) The general structural features of the enzyme
are highly conserved among different organisms The
enzyme in chloroplasts consists of two parts: CF0 and
CF1 CF0, a membrane-spanning complex, conducts
proton flux through the thylakoid membrane and
pro-vides affinity sites for the CF1 complex CF1, extrinsic
to the membrane, contains the nucleotide-binding and
catalytic sites, and can hydrolyze ATP at high rates
after appropriate treatment [4,5] The CF1 complex
consists of five types of subunit with the stoichiometry
a3b3cde
The first high-resolution X-ray structure of ATP synthase was of bovine mitochondrial F1 in 1994 [6] The structure is essentially unchanged in X-ray studies
of bovine F1 inhibited by N,N¢-dicyclohexylcarbodi-imide (Fig 1) [7] Nucleotide bound to all three cata-lytic sites in the aluminum fluoride-inhibited form of bovine F1 [8] The X-ray structures show that the a and b subunits alternate with each other to form a hexamer surrounding a central cavity, where a coiled-coil structure formed by the N-terminal and C-ter-minal helices of the c subunit penetrates The three catalytic sites of F1 are located on the b subunits, where the sites interface subunit a in three different conformational states The importance of the c subunit
in the catalytic cycle has been demonstrated previ-ously, showing that it is probably related to the sequential conformational changes in the ab pairs in addition to being responsible for the generation of a high-affinity nucleotide-binding site on the b subunits
Keywords
ATP synthase; chloroplast; glutathione
S-transferase pull-down assay; abc
assembly; c subunit
Correspondence
J.-M Wei, Shanghai Institute of Plant
Physiology, Shanghai Institutes for Biological
Sciences, Chinese Academy of Sciences,
300 Fenglin Road, Shanghai 200032, China
Fax: +86 21 54924015
Tel: +86 21 54924230
E-mail: wjm@iris.sipp.ac.cn
(Received 25 November 2004, revised 22
December 2004, accepted 17 January 2005)
doi:10.1111/j.1742-4658.2005.04570.x
Five truncation mutants of chloroplast ATP synthase c subunit from spin-ach (Spinacia oleracea) lacking 8, 12, 16, 20 or 60 N-terminal amino acids were generated by PCR by a mutagenesis method The recombinant c genes were overexpressed in Escherichia coli and assembled with ab sub-units into a native complex The wild-type (WT) abc assembly i.e abcWT exhibited high Mg2+-dependent and Ca2+-dependent ATP hydrolytic activity Deletions of eight residues of the c subunit N-terminus caused a decrease in rates of ATP hydrolysis to 30% of that of the abWT assembly Furthermore, only 6% of ATP hydrolytic activity was retained with the
sequential deletions of c subunit up to 20 residues compared with the activ-ity of the abWT assembly The inhibitory effect of the e subunit on ATP hydrolysis of these abc assemblies varied to a large extent These observa-tions indicate that the N-terminus of the c subunit is very important, together with other regions of the c subunit, in stabilization of the enzyme complex or during cooperative catalysis In addition, the in vitro binding assay showed that the c subunit N-terminus is not a crucial region in bind-ing of the e subunit
Abbreviations
CF 0 , the hydrophobic portion of chloroplast ATP synthase; CF 1 , coupling factor one; GST, glutathione S-transferase; WT, wild-type.
Trang 2by its rotation within the a3b3 core [1–4,6–8] In F1,
rotation of the c subunit coupled with ATP hydrolysis
was confirmed by its direct observation in the
move-ment of a fluorescence-labeled actin filamove-ment, which
was attached to the c subunit of the thermophilic
bac-terial F1 subcomplex, a3b3c [9], e subunit F1 [10,11]
and CF1[12]
The catalytic core of the enzyme is a3b3c, despite ab
exhibiting lower rates of ATP hydrolysis [13] It is
generally accepted that ATP synthase generates
coup-ling between cooperative catalysis and proton
translo-cation during hydrolysis⁄ synthesis processes However,
the precise catalytic mechanism of F1-ATPase is still
unknown [1,14] With respect to CF1, it is also
pro-posed that isolated CF1 operates through a full 360
rotation, like other F1-ATPases [12]. CF1 is unique in
that thiol modulation, the structural basis of which is
an insert of about 20 amino acids including a
regula-tory disulfide bond, is reversibly oxidized and reduced
The N-terminus and C-terminus of c subunits from
dif-ferent organisms are highly conserved [15] Deletion of
the 20 amino acids in the C-terminus of the c subunit
resulted in an active chloroplast enzyme [16] Crystal
structures (Fig 1) reveal that the N-terminal domain
of the c subunit makes contact with the bE
sub-unit C-terminal domain containing the conserved
DELSEED motif, which is thought to be important
for energy-coupling rotation of the c subunit by steric
interaction This indicates the possible importance in
the c N-terminal domain during catalytic cooperativity [4,7,8] cSer8 substitution with a Cys residue resulted
in it being cross-linked with a different b region in the presence of Mg2+-ADP or Mg2+-ATP [17] cMet23 substitution caused ATPase uncoupling [18], which was suppressed by amino-acid replacements between
269 and 280 in the C-terminal domain [19] It has been demonstrated by fluorescence mapping [20] and cross-linking [21] that the e subunit is in close proximity to the c subunit The e subunit interacts directly with the
c subunit [22–24], but does so with a higher affinity when the c subunit is assembled with the a3b3 core [25, 26] The hybrid enzyme from the a,b subunits of a thermophilic bacterium and the mutant CF1 c subunit (D194–230) was insensitive to added e subunit [25] Studies by Gao et al [26], who developed an in vitro reconstitution system by assembling the ab complex with an isolated c subunit, showed that this complex was able to obtain the reconstituted core enzyme com-plex as effectively as the native a3b3c Recently, a hybrid
F1-ATPase from Rhodospirillum rubrum or chloroplast subunits was used to study the mechanism of photosyn-thetic F1-ATPase [27,28] In the present study, we exam-ined the importance of the CF1 N-terminus of the
c subunit during hydrolytic turnover using this reconsti-tution system and the binding of e to c through a gluta-thione S-transferase (GST) pull-down assay To do this,
we selectively deleted 8, 12, 16, 20 or 60 residues from the N-terminus of the c subunit The reconstituted abc assemblies were tested for ATP hydrolytic activity The results show that the c subunit’s N-terminus is very important for stabilization of the enzyme complex
Results
Overexpression and assembly of the c truncated mutants
All the plasmids listed in Table 1 were transformed into the expression strain E coli BL21 (DE3)⁄ pLysS The spinach chloroplast atpC gene constructed in the pET11b expression vector had a high expression level
in E coli More than 100 mg recombinant c protein was obtained per litre of culture medium Overexpres-sion of the cloned polypeptides in E coli resulted in the accumulation of insoluble inclusion bodies The inclusion bodies were solubilized in 4 m urea and recovered by slow dialysis as described previously [16] Like the wild-type and native c subunits [16], all the c mutants tended to aggregate during dialysis when pro-tein concentration was high On SDS⁄ PAGE, wild-type c protein and the truncated polypeptides migrated for distances consistent with the extent of truncation
Fig 1 Schematic representation of the bovine heart mitochondria
F 1 produced from pdb file 1e79 using DEEPVIEW [37] Subunit a is on
the left, and subunit b is on the right The N-terminal 20 residues
of subunit c are depicted in gray.
Trang 3(Fig 2A) Each of the c constructs reacted with c
anti-serum on immunoblots (data not shown) The
trun-cated polypeptides were designated cDN8 to cDN60
according to the number of amino-acid residues
dele-ted from the c subunit N-terminus The N-terminus
amino-acid sequence of c subunit from chloroplasts is
shown in Table 1
ATP hydrolytic activity of the mutant assemblies
We tested ATP hydrolytic activity of the reconstituted
assemblies Incubation of the native c protein from
chloroplasts with isolated ab subunits (Fig 2B)
resul-ted in their assembly into a stable, highly active abc
complex under optimal conditions The cloned c
poly-peptide was identical with the native c subunit in its
ability to form a fully active core enzyme complex
[16,26] Mg2+-ATPase activity was measured in the
presence of sodium sulfite, a strong stimulator of ATP
hydrolysis [16] The relative rates of ATP hydrolysis
of these reconstituted assemblies in the presence of
either Ca2+ or Mg2+ as the bivalent cation substrate
were compared (Fig 3) The wild-type assembly
exhibited the maximum activity [14.4 lmol PiÆ(mg
pro-tein))1Æmin)1), consistent with previous results showing that assembling the cloned c polypeptide with the iso-lated ab subunits resulted in a fully active core enzyme complex [26], although there were some differences among the hydrolytic rates Deletion of eight residues from the c subunit N-terminus impaired the ATP hydrolytic ability, despite differences between Mg2+ -ATPase and Ca2+-ATPase The deletion of 12 residues resulted in a greater decrease in Ca2+-ATPase activity About 6% of ATP hydrolytic activity retained on dele-tion of 20 residues When 60 residues were deleted, the rate of ATP hydrolysis of the reconstituted assembly was similar to the ab complex containing no c subunit
Interaction of subunits c and e in vitro GST pull-down assays were used to detect c–e interac-tion The full-length cDNA encoding the e subunit was fused to the C-terminus of the GST gene in the expres-sion plasmid, and the GST-fuexpres-sion protein was over-expressed in E coli As shown in Fig 4, GST alone did not bind to the wild-type c subunit (cWT); in trast, GST–e was able to bind directly to all the c con-structs It was also able to bind to the cDN8, cDN12
Table 1 Amino-acid and primer sequences of truncated mutants Listed below are the amino-acid sequences and PCR primers of truncation mutants (D) of the c subunit of spinach chloroplast ATP synthase The numbers in the plasmids indicate the numbers of residues deleted fol-lowed by N designating N-terminus truncation, respectively The truncations begin with deletion of eight residues with successive deletion
of four residues up to 20 residues from the N-terminus, and N-terminal mutants with deletion of 60 residues (cDN60).
Plasmid Amino-acid sequences Forward primers (5¢-3¢) Reverse primers (5¢-3¢) pET11-cWT ANLRELRDRIGSVKNTQKITEAMKLVAAAK-31 TTTGTCATATGGCAAACCTCCGTGAGC
pET11-cDN8 …8RIGSVKNTQKITEAMKLVAAAK-31 GGCCATATGCGGATCGGATCAGTCAAA ATTCCGGAC pET11-cDN12 …12VKNTQKITEAMKLVAAAK-31 TCGGCCATATGGTCAAAAACACGCAGAAG ACGGATCCA pET11-cDN16 …16QKITEAMKLVAAAK-31 TTGGCCATATGCAGAAGATCACCGAAGCA ATTAATCTC pET11-cDN20 …20 EAMKLVAAAK-31 TCCGGCATATGGAAGCAATGAAGCTCGTC
Fig 2 Gel electrophoresis profiles of
prepa-rations of inclusion bodies and the purified
ab complex Preparations were analyzed by
SDS ⁄ PAGE on 15% polyacrylamide gels,
and the proteins were stained with
Coomas-sie Brilliant Blue R250 Each lane contained
6 lg protein (A) Lane 1, CF1; lanes 2–7,
inclusion body preparations of cWT, DcN8,
DcN12, DcN16, DcN20 and DcN60,
respect-ively (B) Lane 1, CF1(–de); lane 2, isolated
ab complex.
Trang 4and cDN16 molecules with similar affinity to cWT.
Deletion of 20 residues resulted in slight impairment of
the binding activity
Inhibitory effects of the e subunit Given that the N-terminal deletions of the c subunit barely inhibited the interaction between c and e, fur-ther studies were performed to confirm the responses
of the mutant assemblies to the inhibitory e subunit The inhibitory responses of the Ca2+-ATPase activity
of the different mutant assemblies were examined after the addition of the e subunit (Fig 5) The abcWT assembly that exhibited the highest Ca2+-ATPase activity was inhibited by 84% The inhibitory effects
of the e subunits on hydrolytic rates in the mutant (cDN8, cDN12, cDN16 and cDN20) assemblies varied greatly, ranging from 35% to 63% reduction in hydro-lysis
Discussion
The studies presented here focused on the importance
of the N-terminus of the c subunit during ATP hydro-lysis in addition to examining the role of binding and inhibition of the e subunit A schematic representation
of bovine heart mitochondria F1 is shown in Fig 1 The conserved N-terminal region of the c subunit forms an antiparallel left-handed coiled coil with the C-terminal part, which penetrates into a cavity formed
by the a3b3 hexamer. The c subunit’s N-terminus makes contact with the C-terminal domain of the bE subunit, which contains the conserved DELSEED motif [7,8] It is generally accepted that the c subunit confers the asymmetric properties of the catalytic sites
by interacting with the a and b subunits, resulting in catalytic cooperativity The permanent asymmetry of
Fig 3 Relative ATPase activity of reconstituted abc assemblies.
Mg 2+ -ATPase (black columns) and Ca 2+ -ATPase (gray columns)
activities were analyzed as described in Experimental procedures.
The ATPase activities [lmol P i Æ(mg protein))1Æmin)1] of the cWT
assembly (100%) were: Mg 2+ -ATPase, 13.8; Ca 2+ -ATPase, 14.4.
The activities of both the ab complex and the abDcN60 assemble
were about 0.3.
Fig 4 Interactions of wild-type c and the engineered c with e
in vitro GST pull-down assays were performed as described in
Experimental procedures GST or GST–e fusion proteins were
bound to glutathione–Sepharose 4B beads 6 lg of each of the c
construct proteins were incubated with 20 lL of the beads bound
to GST–e After extensive washing, proteins eluted from the beads
were subjected to Western-blot analysis with c antiserum GST
alone was used as a negative control by incubating 20 lL of the
beads bound to GST with 6 lg of the wild-type c recombinant.
Fig 5 Inhibition of the Ca 2+ -ATPase of the reconstituted abc assemblies by addition of e subunit Ca 2+ -ATPase activities were analyzed as described in Experimental procedures Assemblies were incubated with the e subunit at the molar ratio 10e:1ab The inset shows the Ca 2+ -ATPase activities [lmol PiÆ(mg pro-tein))1Æmin)1] in the absence of e subunit, which are set as 100%.
Trang 5isolated CF1 was found in labeling experiments with
Lucifer Yellow [29], which possibly indicated that the
N-terminal part of subunit c remains in contact with
the aE and bE subunits during the complete catalytic
turnover without a full 360 rotation [4] However,
direct observation of the movement of subunit c in
iso-lated CF1 was also determined recently to resemble
that of E coli F1 or thermophilic bacterial F1
subcom-plex, thereby favoring multisite catalysis with a full
360 rotation
The data presented here reveal that deletion of 60
residues eliminated almost all hydrolytic activity of
the reconstituted assembly; moreover, the removal of
eight residues abolished most of the activity When
up to 20 residues were deleted, very low ATP
hydro-lytic activity was retained (Fig 3) There are two
possible explanations for the above results (a) The
deletions impaired the stability of the reconstituted
assemblies and the efficient assembly of the ab
com-plex with recombinant c constructs; or (b) the
struc-tural change in the truncated c construct altered the
asymmetry conformation of a3b3c, thereby affecting
transmission of conformational signals between
cata-lytic sites, which resulted in impaired normal catacata-lytic
cooperativity These observations indicate that the
N-terminal part of subunit c is indispensable and
functions with other regions of c during stabilization
of the abc complex and rotational catalysis
Consis-tent with previous studies, we also found that Ca2+
-dependent and Mg2+-dependent ATP hydrolytic
activities were different, indicating different catalytic
mechanisms [28]
It is well established that the e subunit, a regulatory
protein of ATPase, binds to c directly and rotates with
the c and c (homologous to CF0-III in chloroplasts)
subunits as a part of a rotor (cec10) [1,2] The
inter-action of c and e increases when c penetrates into a
a3b3hexamer [26] The e subunit binds to CF1 with an
apparent dissociation constant of < 10)10m [20] In
the e subunit, the sites of c interaction with e were
mapped to between R49 and R70, and the C-terminal
part beyond K199 [24] The regulatory c regions of
CF1 seem to be very important for e subunit binding
[30] Our results also show that the e subunit stably
binds to c, which is consistent with earlier studies
[23–25] Deletion of 20 residues from the N-terminal
region did not markedly decrease the binding affinity
between the c and e subunits in vitro (Fig 4)
The engineered c subunit bound to the e subunit
with almost identical affinity, although the inhibitory
effects of e subunit varied with the number of
resi-dues removed The high binding affinity of a3b3c for
the e subunit is essential for inhibition of ATPase
catalysis, which is much higher than that of binding
e to individual c [26] The truncation seemed not to change the binding affinity between c and e (Fig 5), but the altered asymmetrical conformation of a3b3c resulting from deletions in c is enough to decrease the binding of e to a3b3c Meanwhile, impaired cata-lytic cooperativity may not be efficiently inhibited
by the e subunit Both of the above may partially explain the varied inhibitory effect of e on ATP hydrolysis
Taken together, we have shown that the N-terminal region of the c subunit is important for stabilization of
a3b3c or cooperative catalysis of isolated CF1. The N-terminal region of subunit c in CF1 is not crucial for binding of subunit e in vitro Further studies are needed to determine candidate residues participating in transmission of conformation signals and efficient energy coupling of the c subunit N-terminus
Experimental procedures
Materials Restriction endonucleases, T4 DNA ligase, Klenow frag-ment, and Pfu and Taq DNA polymerase were purchased from Takara (Dalian, China) and Promega (Shanghai, China) Sephadex G-50 was purchased from Pharmacia (Uppsala, Sweden) DEAE-cellulose was obtained from Whatmann (Uppsala, Sweden) and hydroxyapatite from Bio-Rad (Hercules, CA, USA) Other reagents were all standard AR grade
Generation of c truncation mutants and GST-fusion protein
Plasmid pJLA503-pchlc, a gift from S Engelbrecht (Uni-versity of Osnabruck, Germany), contains the atpC genes encoding the ATP synthase c subunit [31] The wild-type c fragment was PCR amplified from pJLA503-pchlc, and five truncation mutants were PCR generated with the mutagen-esis primers (Table 1) The PCR products were digested with NdeI and BamHI, and subsequently subcloned into the pET11b expression vector The resulting plasmids were confirmed by DNA sequencing For construction of the GST–e fusion protein, full-length cDNA encoding the
e subunit was amplified by PCR using pJLA-eWT [23] as
a template with the following primers: 5¢-GACGGATCC CCATGACCTTAAATCTTTGT-3¢ as the 5¢ primer and 5¢-ATAGTCGACCTGGTTACGAAGAAATCG-3¢ as the 3¢ primer The PCR products were cleaved with BamHI and EcoRI, and cloned into plasmid pGEX-5X-1 (Amer-sham-Pharmacia Biotech, Shanghai, China) The resulting plasmid pGEX-5X-e was confirmed to be inframe with the GST cassette by DNA sequencing
Trang 6Solubilization and folding of overexpressed
c mutants and GST-fusion protein
The resulting pET11b plasmids containing the atpC gene
were transformed into the expression strain E coli
BL21(DE3)⁄ pLysS The resulting E coli cells were grown
at 37C in Luria–Bertani medium containing l-ampicillin
Cells were induced with 0.4 mm isopropyl
thio-b-d-galacto-side in mid-exponential phase, incubated for 7 h, and
har-vested as described previously [32] Solubilization and
folding of the insoluble c polypeptide were performed
according as described previously [16] Overexpression and
collection of GST or GST–e fusion protein in E coli were
carried out as previously described [23]
Preparation and reconstitution of an ab complex
and c mutants
An ab complex was isolated from CF1(– de) as described
previously [26] Reconstitution of the complex and
c mutants was carried out as described previously [26] The
incubated mixture was assayed directly for ATP hydrolytic
activity
In vitro binding assays of e with c subunit and
immunoblotting analysis
Equal amounts of GST or GST–e fusion proteins were
bound to glutathione–Sepharose 4B beads in binding buffer
[50 mm Tris⁄ HCl, 100 mm NaCl, 1 mm EDTA, 1% (v ⁄ v)
Triton X-100, 1 mm phenylmethanesulfonyl fluoride and
10% (v⁄ v) glycerol, pH 8.0] at 4 C for 2 h The bound
glutathione–Sepharose 4B beads were washed three times
with binding buffer to remove unbound fusion proteins
These beads were incubated with equal amounts of the
c constructs for 4 h at 4C in binding buffer and washed
four times with binding buffer to remove unbound proteins
Subsequently, the beads were suspended in 2· SDS loading
buffer and boiled for 3 min Proteins released from the
beads were analyzed by SDS⁄ PAGE (15% polyacrylamide
gel) [23], transferred to nitrocellulose membrane, and
detec-ted by western immunoblot analysis using an ECL Western
Blotting Detection System (Amersham) and c antiserum
CF1and CF1(–de) preparation and measurement
of ATPase activity
CF1and CF1(–d) were prepared from fresh market spinach
as described previously [33,34] Before use, the proteins
were desalted on Sephadex G-50 centrifuge columns [35]
ATPase activities were determined by measuring phosphate
release for 5–10 min at 37C The assay was performed
in 1 mL volumes of assay mixture containing 50 mm
Tricine⁄ NaOH, pH 8.0, and 5 mm ATP Ca2+-ATPase was
carried out in the presence of 5 mm CaCl2, and Mg2+ -ATPase in the presence of 2 mm MgCl2 and 20 mm
Na2SO3.The reaction was stopped by adding 200 lL 20% trichloroacetic acid c Antiserum was raised by subcuta-neous injections into rabbits Inclusion bodies containing recombinant c polypeptide were subjected to SDS⁄ PAGE Proteins were recovered from Coomassie Brilliant Blue R250-stained gel bands and used for the immunization of rabbits Protein concentration was measured by the method
of Bradford [36]
Acknowledgements
This work was supported by the National Natural Sci-ence Foundation of China (30170078) and State Key Basic Research and Development Plan (G1998010100)
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