The GxxxG motif of the transmembrane domain of subunit e isinvolved in the dimerization/oligomerization of the yeast ATP synthase complex in the mitochondrial membrane Genevie`ve Arselin
Trang 1The GxxxG motif of the transmembrane domain of subunit e is
involved in the dimerization/oligomerization of the yeast ATP synthase complex in the mitochondrial membrane
Genevie`ve Arselin, Marie-France Giraud, Alain Dautant, Jacques Vaillier, Daniel Bre`thes,
Be´ne´dicte Coulary-Salin, Jacques Schaeffer and Jean Velours
Institut de Biochimie et Ge´ne´tique Cellulaires du CNRS, Universite´ Victor Segalen, Bordeaux, France
A conserved putative dimerization GxxxG motif located in
the unique membrane-spanning segment of the ATP
syn-thase subunit e was altered in yeast both by insertion of an
alanine residue and by replacement of glycine by leucine
residues These alterations led to the loss of subunit g and the
loss of dimeric and oligomeric forms of the yeast ATP
syn-thase Furthermore,as in null mutants devoid of either
subunit e or subunit g,mitochondria displayed anomalous
morphologies with onion-like structures By taking
advant-age of the presence of the endogenous cysteine 28 residue in
the wild-type subunit e,disulfide bond formation between
subunits e in intact mitochondria was found to increase the stability of an oligomeric structure of the ATP synthase in digitonin extracts The data show the involvement of the dimerization motif of subunit e in the formation of supra-molecular structures of mitochondrial ATP synthases and are in favour of the existence in the inner mitochondrial membrane of associations of ATP synthases whose masses are higher than those of ATP synthase dimers
Keywords: ATP synthase; oligomerization; subunit e; GxxxG motif; yeast
The F0F1-ATP synthase is a molecular rotary motor that is
responsible for the aerobic synthesis of ATP It exhibits a
headpiece (catalytic sector),a basepiece (membrane sector)
and two connecting stalks The sector F1 containing the
headpiece is a water-soluble unit retaining the ability to
hydrolyse ATP when in a soluble form F0is embedded in
the membrane and is mainly composed of hydrophobic
subunits forming a specific proton conducting pathway
When the F1 and F0 sectors are coupled,the enzyme
functions as a reversible H+-transporting ATPase or ATP
synthase [1–4] The two connecting stalks are constituted of
components from F1 and F0 The central stalk is a part
of the rotor of the enzyme The second stalk,which is part
of the stator,connects F1 and hydrophobic membranous
components of the enzyme High resolution X-ray
crystal-lographic data have led to solving the structure of the F1
[5–8] from different sources Stock et al [9] reported
the 3.9 A˚ resolution X-ray diffraction structure of the
Saccharomyces cerevisiae F1 associated with the c10-ring
oligomer
In Escherichia coli, F0is composed of subunits a,b and c only The mitochondrial F0of mammals is composed of 10 different subunits [10] The same 10 components have been identified in the S cerevisiae enzyme [11–13] It has been shown that the yeast ATP synthase exists as dimeric and oligomeric forms in Triton X-100 and digitonin extracts and that the subunits of F0,e,g and 4(b) are essential for such a process [12,14,15] In addition, the existence of the dimeric form in the inner mitochondrial membrane has been recently demonstrated [16]
Under its mature form,the yeast subunit e is composed of
95 amino-acid residues and displays a mass of 10 744 Da It
is an integral membrane protein anchored to the inner mitochondrial membrane by its unique membrane-spanning segment at its N-terminus,and which adopts,such as the mammalian ATP synthase subunit e,an Nin–Couttopology [11,17] A stoichiometry of 2 mol of subunit e per mol of rat liver ATP synthase has been estimated [18] In mammals, expression of the gene encoding subunit e is regulated in tissues and cells in response to physiological stimuli,thus suggesting that subunit e plays a regulatory role in the ATP synthase [19–21] In yeast,subunit e is involved in the dimerization/oligomerization of ATP synthases,prob-ably in association with subunit g [12] Surprisingly, mutant mitochondria devoid of either subunits e or g were found to have numerous digitations and onion-like struc-tures,thus suggesting a link between dimerization/oligo-merization of the ATP synthase and cristae morphology [14,15]
The purpose of the present work was to provide information on the involvement of subunit e in the dimerization/oligomerization of yeast ATP synthases in the inner mitochondrial membrane Mutations were intro-duced into a putative membranous dimerization motif
Correspondence to J Velours,Institut de Biochimie et Ge´ne´tique
Cellulaires du CNRS,UMR 5095,Universite´ Victor Segalen,
Bordeaux 2,1,rue Camille Saint Sae¨ns,
33077 Bordeaux cedex,France.
Fax: + 33 5 56999051,Tel.: + 33 5 56999048,
E-mail: jean.velours@ibgc.u-bordeaux2.fr
Abbreviations: BN/PAGE,blue native polyacrylamide slab gel
electrophoresis; F 0 and F 1 ,integral membrane and peripheral portions
of ATP synthase; NEM, N-ethylmaleimide.
(Received 3 February 2003,revised 3 March 2003,
accepted 5 March 2003)
Trang 2GxxxG of subunit e We demonstrate that such a motif is
involved in both the edification of supramolecular ATP
synthase species and in correct mitochondrial morphology
In addition,cross-linking experiments involving the
endo-genous Cys28 residue of subunit e provided data in favour
of the oligomerization of the yeast ATP synthase in the
mitochondrial membrane
Experimental procedures
Materials
Digitonin was from Sigma Oligonucleotides were
pur-chased from MWG-BIOTECH All other reagents were of
reagent grade quality
Yeast strains and nucleic acid techniques
The Saccharomyces cerevisiae strain D273–10B/A/H/U
(MATa, met6, ura3, his3) [22] was the wild-type strain
The null mutant DTIM11 was constructed by a PCR-based
mutagenesis and the kanr gene was removed [23] The
mutations 19A,G15L,G19L and C28S were introduced by
a PCR mutagenesis procedure [24] into a plasmid pRS313
bearing an insert encoding the wild TIM11 gene and the
kanr gene The strains containing modified versions of
subunit e were obtained by integration of the mutated
versions of TIM11 gene at the chromosomic locus in the
deleted-disrupted yeast strain and were selected for their
resistance to Geneticin The yeast mutants with a point
mutation were named as (name of the subunit)(wild-type
residue)(residue number)(mutant residue) The strain
con-taining the subunit e(His)6was constructed according to the
following strategy Two partially complementary
oligo-nucleotides 5¢-CGCGGAATTCTTAGTGATGGTGATG
GTGATGTGTTGAAGCTTCCTTCAGGG-3¢ and 5¢-CAT
CACCATCACCATCACTAAGAATTCCGCGATAGAA
GCTTCAACATAAATAGGATACTA-3¢ were used to
introduce the (His)6sequence into the C-terminus of subunit
e by the PCR mutagenesis procedure
Biochemical procedures
Cells were grown aerobically at 28C in a complete liquid
medium containing 2% lactate as carbon source [25] and
harvested in logarithmic growth phase The rho– cell
production in cultures was measured on glycerol plates
supplemented with 0.1% glucose Mitochondria were
prepared from protoplasts as previously described [26]
Protein amounts were determined according to Lowry et al
[27] in the presence of 5% SDS using bovine serum albumin
as standard Oxygen consumption rates were measured with
NADH as substrate [28] Phosphorylation rate was
mea-sured in the respiratory buffer supplemented with 1 mM
ADP by ATP formation measured by a bioluminescence
technique [29] ATP synthesis rate and oxygen consumption
rate were measured at the same time in the oxygraph
chamber All reactions were stopped by adding an aliquot of
the medium to perchloric acid The ATP/O ratio
stoichio-metries were determined from the yield of ATP synthesis
rate vs state 3 respiratory rate [30] The ATPase activity was
measured at pH 8.4 [31]
Cross-linking experiments Mitochondria isolated from wild-type and mutant cells were washed by centrifugation in 0.6Mmannitol,50 mMHepes
pH 7.4 containing 0.25 mM of phenylmethylsulfonyl fluo-ride The pellet was suspended at a protein concentration of
5 mgÆmL)1 in 0.1M mannitol,50 mM Hepes pH 7.4 containing either 5 mM of EDTA and 5 mM of N-ethyl-maleimide (NEM) for the control experiment or 2 mM
CuCl2 for the cross-linking experiment Incubation was perfomed at 4C for 30 min The reaction was stopped upon addition of 5 mM EDTA and 5 mM of NEM Mitochondrial membranes were then dissociated in the presence of 20 mMof NEM for SDS/gel electrophoresis and Western blot analysis For BN/PAGE analyses of cross-linked products,mitochondrial membranes were centri-fuged at 10 000 g for 10 min at 4C after incubation with either 5 mMof NEM or 2 mMof CuCl2,Then,the digitonin solution containing 5 mM NEM was added to the mito-chondrial pellet to extract the ATP synthases
Electrophoretic and Western blot analyses SDS-gel electrophoresis was performed as described in [32,] Western blot analyses were described previously [33] Nitrocellulose membranes (Membrane Protean BA83, 0.2 lm from Schleicher & Schuell) were used Polyclonal antibodies against subunits e and g were raised against amino-acid residues 69–82 and 31–45,respectively Anti-bodies against subunits e, g and i were used with dilutions of
1 : 10 000 Membranes were incubated with peroxidase-labeled antibodies and visualized with the ECL reagent of Amersham Pharmacia Biotech Molecular mass markers (Benchmark Prestained Protein Ladder) were from Invitro-gen BN/PAGE experiments were performed as described previously [34,35] Mitochondria (1 mg of protein) were incubated for 30 min at 4C with 0.1 mL of digitonin solution with the indicated digitonin/protein ratio The extracts were centrifuged at 4C for 15 min at 40 000 g and aliquots (40 lL) were loaded on the top of a 3–13% polyacrylamide slab gel After electrophoresis the gel was incubated in a solution of 5 mMATP,5 mMMgCl2,0.05% lead acetate,50 mM glycine/NaOH pH 8.4 to reveal the ATPase activity [36,37]
Ultrastructural studies Freezing and freeze-substitution of yeast cell pellets were performed as previously described [14]
Results
Presence of a dimerization motif in the membrane-spanning segment of subunit e The supernumerary subunit e is a component of the mitochondrial ATP synthase which is involved in the dimerization of ATP synthase [11,12] Subunit e has been identified in many organisms The multiple alignment of subunits e of different sources shows that five amino-acid residues are fully conserved (Fig 1) These are Arg8,Ser10, Leu12,Gly15 and Gly19 The five conserved amino-acid
Trang 3residues lay in a domain predicted to be a
membrane-spanning segment beginning with Asn5 and ending at
Leu26 Subunit e has an Nin–Coutorientation [11,17] which
exposes the main part of the subunit to the intermembrane
space,with the unique cysteine residue (Cys28) at the
frontier between the membrane and the intermembrane
space The predicted membrane-spanning segment of
sub-unit e displays the dimerization motif GxxxG of
glyco-phorin A [38,39] at position Gly-Leu-Phe-Phe-Gly(15–19)
The highly conserved Gly15 and Gly19 suggested their
involvement in a transmembrane helix–helix interaction To
address whether the GxxxG motif and the two glycine
residues are critical in a dimerization process which could be
the basis of the dimerization/oligomerization of
mito-chondrial ATP synthases,three mutants were constructed
An alanine residue was inserted after Phe18 in order to
disrupt a helix–helix packing interface involving the
amino-acid residues on both sides of the insertion [40] In the other
two mutants,the small amino-acid residues Gly15 and
Gly19 were replaced by leucine residues
Phenotypic analyses of mutant strains and oxidative
phosphorylation properties of isolated mitochondria are
reported in Table 1 The three mutants displayed a slight
increase in the doubling time with lactate as carbon source
An interesting point was the low amount of rho–cells in cultures in comparison with the null mutant DTIM11, which is devoid of subunit e From these data,there appears
to be a correlation between the increase in spontaneous rho– cell conversion (rho–cells are unable to grow with lactate as carbon source) and the increase in the generation time of yeast strains The ATPase activity of mutant mitochondria displayed a low sensitivity toward the F0 inhibitor oligo-mycin,thus showing a decreased stability of F0under the experimental conditions of ATPase activity measurements (pH 8.4 and Triton X-100) In contrast,under oxidative phosphorylation conditions,the ATP/O ratio value of e19A mitochondria indicated that the efficiency of the oxidative phosphorylation machinery was not altered,as in mutants devoid of either subunits e or g [14]
Loss of dimerization/oligomerization of e19A, eG15L and eG19L ATP synthases
We next sought whether the mutations in the dimerization motif of subunit e affected the dimerization/oligomerization
of the ATP synthases Therefore,the presence of
Table 1 Phenotypic analysis of yeast strains used Yeast cells were grown at 28 C on complete medium containing lactate as carbon source rho– production was measured on glycerol plates supplemented with 0.1% glucose Mitochondria were prepared from protoplasts ATPase activities and the sensitivity to oligomycin (6 lgÆmL)1) were measured at pH 8.4 in the presence of Triton X-100 to remove the F 1 inhibitor ATP/O ratios were determined with NADH as substrate ND,not determined.
Strains
Doubling time (min)
Rho –
cells in cultures (%)
ATPase activity
ATP/O
lmol PiÆmin)1Æmg protein)1
Inhibition – Oligomycin + Oligomycin
Fig 1 Multiple alignment of subunits e from
different sources The conserved amino-acid
residues between subunits e from human
(H s.) (P56385,Swiss-Prot),pig (S s.)
(Q06185,Swiss-Prot),bovine (B t.) (Q00361,
Swiss-Prot),hamster (C l.)
(P12633,Swiss-Prot),mouse (M m.) (Q06185,Swiss-Prot),
rat (R n.) (P29419,Swiss-Prot),Drosophila
(D m.) (AY060656,GenBank),Neurospora
crassa (N
c.,AW710731,GenBank),Botryo-tinia fuckelians (B f.,AL111090,EMBL) and
Saccharomyces cerevisiae (P81449,Swiss-Prot)
are in bold The numbering of the yeast
sub-unit e begins at the initiating methionine The
star indicates the position of the unique
cys-teine residue of the yeast subunit e (position
28) The putative transmembrane segment of
subunit e (TM) is boxed.
Trang 4supramolecular species of the ATP synthase in the
mito-chondrial digitonin extracts of mutant strains was examined
by BN/PAGE The digitonin extracts were loaded on a 3–
13% acrylamide slab gel and the mitochondrial complexes
were separated under native conditions The gel was
incubated with ATP-Mg2+and Pb2+to reveal the ATPase
activity (Fig 2) The wild-type digitonin extracts contained
the dimeric and oligomeric forms of the enzyme that were
destabilized upon increasing the digitonin-to-protein ratio,
as shown previously [14] The mitochondrial digitonin
extracts of mutant strains did not display any oligomeric
forms of the ATP synthase Whatever the
digitonin-to-protein ratio used,the monomeric form of the enzyme was
predominant,although a small amount of dimeric form was
found,as already observed for null mutants devoid of either
subunit e or subunit g [14,15]
It was previously shown that in the absence of subunit e,
subunit g is not present in mitochondrial membranes [12]
As a consequence,the presence of both subunits in strains
mutated in subunit e was checked by Western blot analyses
of SDS-solubilized mitochondrial membranes Despite the
presence of altered subunits e,the amount of subunit g was
highly decreased in e19A mitochondrial membranes and the
subunit was not detectable in eG15L and eG19L
mito-chondrial membranes (Fig 3)
The e19A, eG15L and eG19L mutants are defective
in the mitochondrial morphology
It has been previously reported that the null mutants in
either TIM11 or ATP20 genes have anomalous
mitochon-drial morphologies [14,15] Thus, transmission electron
microscopy of yeast cell sections was performed to examine
the effect of mutations in the dimerization motif of subunit e
on the ultrastructure of mitochondria Figure 4 shows that
cells of e19A,eG15L and eG19L strains had abnormal mitochondria such as onion-like structures similar to those observed in mutant cells devoid of either subunits e or g
The subunit e of the e19A mutant dimerizes spontaneously via Cys28 in a form which is loosely
or not associated to the yeast ATP synthase
To gain more insight into the behaviour of mutant subunits e, Western blot analyses of SDS-dissociated wild-type and mutant mitochondria were performed Figure 3 shows that polyclonal antibodies against subunit e revealed the pre-sence of subunit e and a 21.4-kDa band in e19A,eG15L and eG19L mutant mitochondria The 21.4-kDa band was observed upon oxidation of wild-type mitochondria with CuCl2 (Fig 5A) but it was absent in a mutant devoid of Cys28 (not shown),thus indicating the involvement of Cys28 in the formation of the adduct The 21.4-kDa band corresponded to a homodimer of subunit e resulting from the formation of a disulfide bond between two subunits e This result was obtained upon incubation with CuCl2 of wild-type mitochondria complemented with a pRS313 shuttle vector bearing a gene encoding a subunit e having
a (His)6sequence at its C-terminus (wild-type + eHis6) In this case,Western blot analysis of CuCl2-treated mitochon-dria displayed three bands in the 21.4-kDa region which could be attributed to e + e,e + eHis6and eHis6+ eHis6
dimers because of their respective apparent molecular masses (Fig 5A) This result is in full agreement with that
of Brunner et al [41]
Despite the presence of NEM during solubilization of mutant mitochondria by SDS,the amount of e + e dimer was large in mutant mitochondria (Fig 3),whereas wild-type mitochondria did not display such a dimer,thus showing that pre-existing e + e dimers were present in mutant mitochondrial membranes It was possible
to increase considerably the e + e dimer formation by oxidation with CuCl2 As shown in Fig 5B,incubation of intact mutant mitochondria with CuCl led to nearly full
Fig 2 Lack of dimerization/oligomerization of the yeast ATP synthase
upon alteration of the GxxxG dimerization motif of subunit e
Mito-chondria were isolated from wild-type,e19A,eG15L and eG19L
strains Digitonin extracts were obtained with the indicated digitonin/
protein ratios and analysed by BN/PAGE The gels were incubated
with ATP-Mg2+and Pb2+to reveal ATPase activity.
Fig 3 Mitochondria isolated from e19A, eG15L and eG19L strains are deficient in subunit g Mitochondria isolated from wild-type (lane 1), e19A (line 2),eG15L (lane 3) and eG19L (lane 4) were treated with NEM as described in the Material and methods section to prevent disulfide bond formation during the dissociation with SDS Aliquots (30 lg of protein) were analysed by Western blot The blots were incubated either with antibodies raised against subunits e and i or with antibodies raised against subunits g and i.
Trang 5conversion of mutant subunits e into the dimeric form This
was not the case with wild-type mitochondria,as
densito-metric analyses revealed that 45 ± 8% (mean of five
experiments) of wild-type subunit e led to the dimeric form
in the presence of CuCl This result reflected a different
behaviour of mutant and wild-type subunits e,thus suggesting different relationships between subunit e and the other F0 components of wild-type and mutant ATP synthases To verify this point,BN/PAGE analyses were performed with mitochondrial digitonin extracts obtained with a digitonin-to-protein ratio of 0.75 gÆg)1 (Fig 6) Under these conditions,the wild-type ATP synthase displayed only dimeric and oligomeric forms in BN/PAGE analysis [14] After migration,the slices of gel were cut and submitted to an SDS/gel electrophoresis in the second dimension The proteins were transferred onto a nitro-cellulose membrane which was probed with polyclonal antibodies directed against subunit e Polyclonal antibodies directed against subunit i were also used as control to detect the position of the different forms of ATP synthases,as subunit i is strongly associated to the yeast enzyme at a digitonin-to-protein ratio of 0.75 gÆg)1 When NEM-treated
or copper-treated wild-type mitochondria were solubilized with digitonin,subunits e and i comigrated with the dimeric and oligomeric forms of the ATP synthase during native electrophoresis (Figs 6A,B) However, the e + e dimer, which resulted from the incubation of wild-type mitochon-dria with CuCl2,was found in the oligomeric form of the ATP synthase but not in the dimeric form The subunit e and the dimer of subunit e of NEM- and copper-treated e19A mitochondria were observed mainly at a position corresponding to the front of the native gel (right side of the SDS-gel electrophoresis) (Fig 6D) However,upon oxida-tion of e19A mitochondria,a faint amount of e + e dimer was also observed at molecular masses higher than that of the remaining dimeric form of the ATP synthase (Fig 6D) Owing to the destabilization of supramolecular ATP synthase forms of e19A mitochondria,a continuous band
of subunit i was observed stretching from the top of the native gel to the position of the monomeric form of the enzyme From these data,it was concluded that the mutation e19A altered the relationship between subunit e and the other F0components of the ATP synthase and that under the experimental conditions used,subunit e was highly dissociated from the mutant enzyme
Subunit e is involved in the oligomerization
of the yeast ATP synthase
An interesting result shown in Fig 6 is the presence of
e + e dimers in the oligomeric forms of the wild-type ATP synthase upon incubation of wild-type mitochondria with CuCl2,thus suggesting that subunits e participate in an interface allowing ATP synthase oligomers to exist To test this hypothesis,wild-type and eC28S mitochondria were incubated either in the presence or absence of CuCl2 BN/ PAGE analyses of digitonin extracts (Fig 7A) revealed the presence of the oligomeric form of the CuCl2-treated wild-type ATP synthase migrating at an acrylamide concentra-tion of 4.8%,despite a digitonin-to-protein ratio of 2 gÆg)1, i.e conditions which highly destabilize the oligomeric forms
of wild-type mitochondria With the same digitonin-to-protein ratio of 2 gÆg)1,the eC28S extract did not display this oligomeric form This result indicates an increased stabilization of the wild-type oligomeric form by the disulfide bond formation between two subunits e The monomeric,dimeric and oligomeric forms of ATP synthase
Fig 4 Mitochondria isolated from e19A, eG15L and eG19L strains are
defective in mitochondrial morphology Transmission electron
micro-scopy of yeast cell sections of e19A (A),eG15L (B) and eG19L (C)
strains m,mitochondria The bar indicates 0.5 lm.
Trang 6of CuCl2-treated wild-type mitochondria extracted with a
digitonin-to-protein ratio of 2 gÆg)1were cut from the BN/
PAGE slab and the proteins they contained were separated
by SDS-gel electrophoresis The gel was transferred to a
nitrocellulose sheet which was probed with polyclonal
antibodies raised against subunits i and e (Fig 7B) An
intense band corresponding to the e + e dimer was found
only in the oligomeric form of the ATP synthase as in
Fig 6B,whereas the monomeric subunit e was present only
in the dimeric forms As described in [12],subunit e was
absent from the monomeric form of the yeast ATP
synthase In control experiments,incubation of wild-type
digitonin extracts (digitonin-to-protein ratio of 2 gÆg)1) with
CuCl2did not promote oligomer formation and in addition,
the CuCl2-treated eC28S mitochondria extracted with either
a digitonin-to-protein ratio of 0.75 or 2 gÆg)1displayed only
the monomer of subunit e in the oligomeric and dimeric
forms of the yeast ATP synthase (not shown)
Discussion
Yeast mutants altered in the dimerization motif
of subunit e are devoid of subunit g and ATP synthases
neither dimerize nor oligomerize
The subunits e,g and 4 are three components of the yeast
ATP synthase F0which are involved in the dimerization/
oligomerization of ATP synthases The purpose of the
present paper was to provide information on the
involve-ment of a putative dimerization motif located in the
membranous domain of subunit e in the dimerization/ oligomerization of yeast ATP synthases From the analysis
of yeast mutants altered in this motif,we show that this conserved dimerization motif of subunit e has an essential role in the cohesion of an interface between ATP synthases,
as shown by BN/PAGE analysis However,subunit e was still present in mutant mitochondria,as shown by Western blot analysis of whole mitochondrial membranes,but subunit e was loosely or not bound to the ATP synthase,
as observed by SDS/gel electrophoresis followed by Western blot analysis of the mitochondrial digitonin extracts separ-ated by electrophoresis under native conditions This result indicates an alteration of the relationships between subunit e and other F0components For instance,an interesting point was the absence of subunit g,a small hydrophobic protein
of F0,which has been identified as a near neighbour of subunit e in bovine submitochondrial particles [17] It has also been shown that in the absence of subunit e,subunit g is not present in mitochondria whereas the absence of subunit
g in the null mutant DATP20 does not preclude the presence
of subunit e The lack of subunit g has also been described in
a mutant devoid of the first membrane-spanning segment of subunit 4,whereas subunit e was still present On the basis
of cross-linking data,the absence of subunit g in the latter mutant was attributed to the loss of interaction between subunits 4 and g at their membranous levels [15] Taken together,these observations indicate (a) a close relationship
of subunits e,g and 4 in the membranous F0domain and (b) that subunit g is a very unstable protein which disappears from the ATP synthase upon alterations of either subunits e
Fig 5 Oxidation of cysteine 28 promoted the dimerization of subunit e of wild type and mutant mitochondria (A) Mitochondria iso-lated from wild-type,eHis 6 cells and wild-type cells complemented with the plasmid pRS313 encoding eHis 6 (wild-type + eHis 6 ) were incubated in the presence or absence of CuCl 2 as described in the experimental pro-cedure The control experiment (in the absence of CuCl 2 ) was performed in the presence of NEM instead of CuCl 2 (B) Mito-chondria isolated from wild-type,e19A, eG15L and eG19L strains were incubated in the presence or absence of CuCl 2 The control experiment (in the absence of CuCl 2 ) was performed in the presence of NEM instead of CuCl 2 Cross-linking conditions are described
in the experimental procedure After dissoci-ation of samples with SDS in the presence of
20 m M of NEM,aliquots (30 lg of protein) were analysed by Western blot The blots were incubated with polyclonal antibodies raised against subunit e.
Trang 7or 4 This has two consequences; the loss of dimerization/
oligomerization of ATP synthases as revealed by BN/
PAGE analysis and the presence of anomalous
mitochon-dria with onion-like structures This underlines the
relation-ship between the dimerization/oligomerization of the
mitochondrial ATP synthase and the yeast mitochondrial
morphology,a point which has been reported in previous
papers [14,15] As the above phenotypes have a common
feature,i.e the absence of subunit g,we propose that by its
presence,subunit g exerts a central role in the interfaces
allowing the dimerization/oligomerization of the yeast ATP
synthases This role and these interfaces will be more
precisely studied by site-directed mutagenesis of the ATP20
gene encoding subunit g to identify the interaction domains
between subunit g and subunits e and 4
The oligomeric forms of the yeast ATP synthase
in the inner mitochondrial membrane
The unique cysteine residue of the wild-type subunit e,
which is located in the intermembrane space,is an accessible
target to chemical reagents that allows the environmental
study of this subunit in the inner mitochondrial membrane
Western blot analysis of proteins separated by SDS-gel
electrophoresis originating from native complexes separated
by BN/PAGE allowed us to examine the behaviour of subunit e in the different forms of the yeast ATP synthase extracted by digitonin (at a digitonin-to-protein ratio of 0.75 gÆg)1) The dimeric and oligomeric forms of ATP synthase of NEM-treated wild-type mitochondria contained only monomeric subunit e In contrast,CuCl2incubation of wild-type mitochondria led to the formation of the e + e dimer that was found only in the oligomeric form of the yeast ATP synthase migrating at an acrylamide concentra-tion of 4.8%,i.e at an apparent molecular mass which corresponds to at least a tetrameric form of the enzyme [14]
In addition,while a digitonin-to-protein ratio of 2 gÆg)1 destabilized the oligomeric forms of the enzyme,the disulfide bond formation between two subunits e via the Cys28 residues increased the stability of the oligomeric form migrating at an acrylamide concentration of 4.8% The existence of supramolecular structures of the yeast ATP synthase in Triton X-100 and digitonin extracts and in the inner mitochondrial membrane has been well documented Biochemical evidence has shown that the dimeric forms of the yeast ATP synthase are not due to the aggregation of the monomeric form of the enzyme as (a) dimerization and oligomerization of the ATP synthase are dependent on the
Fig 6 Upon oxidation, the subunit e dimer of e19A mitochondria was loosely associated with the ATP synthase, whereas the subunit e dimer of wild-type mitochondria was associated only with the oligomeric forms of the wild-wild-type enzyme Wild-wild-type and e19A mitochondria were incubated either with NEM or with CuCl 2 as described above Mitochondrial digitonin extracts were obtained with a digitonin-to-protein ratio of 0.75 gÆg)1and analysed by BN/PAGE A part of the gel was revealed by the ATPase activity and slices of the BN/PAGE are shown on the top of each figure (first dimension) Corresponding slices were cut,incubated with 1% SDS and submitted to SDS/gel electrophoresis (second dimension) The proteins of the gel were transferred onto a nitrocellulose membrane,which was probed with polyclonal antibodies raised against subunits e and i NEM-treated wild-type (A) and NEM-treated e19A (C) mitochondria (control experiments) CuCl 2 -treated wild-type (B) and CuCl 2 -treated e19A (D) mito-chondria.
Trang 8presence of subunits e,g and the first membrane-spanning
segment of the b-subunit (subunit 4) and (b) inter-ATP
synthase cross-linking with a bis-maleimide reagent and
involving a cysteine residue introduced into the C-terminal
part of subunit i located in the intermembrane space has
been reported both in detergent extracts which preserve the
dimeric form of the ATP synthase and in intact
mitochon-dria,thus showing the existence of such dimers in the inner
mitochondrial membrane [16] Whether oligomeric forms of the ATP synthase exist in the inner mitochondrial mem-brane is still a matter of discussion Using freeze-fracturing, deep-etching and replicates,Allen et al [42] showed the presence of double rows of ATP synthases on cristae of Paramecium multimicronucleatum mitochondria Subse-quently,Allen proposed a model that described the association of ATP synthase dimers as generating the tubular cristae [43] In yeast,oligomeric forms of ATP synthase have been found in mitochondrial digitonin extracts obtained with digitonin-to-protein ratios of 0.75–
1 gÆg)1,but they were absent at higher ratios However,by the formation of disulfide bonds between subunits e in intact wild type mitochondria,we have now found that it is possible to increase slightly the stability of an oligomeric form of the ATP synthase in digitonin extracts The association of ATP synthases in supramolecular structures higher than dimeric forms by oxidation could result from the Brownian lateral diffusion of proteins in the inner mitochondrial membrane However (a) the experiments were performed at 4C to decrease the diffusion,(b) no other cross-links between the ATP synthase and other mitochondrial complexes have been identified and (c) these oligomeric structures exist without cross-linking in mito-chondrial digitonin extracts Therefore,we consider that these data are in favour of the existence in the inner mitochondrial membrane of oligomeric forms such as those observed by Allen et al [42] Such oligomeric forms of ATP synthase imply the existence of two different interfaces between ATP synthase monomers On the basis of cross-linking data on mitochondrial membranes [44],it was proposed that two subunits 4 belonging to two neighbour-ing ATP synthases participate at one interface [14] The second interface involves subunits e and g [12] and it appears from the above data that the dimerization motif in subunit e
is essential for the stability of this interface
Subunits e have different environments While oxidation of mutant mitochondria led to nearly full conversion of altered subunit e under a dimeric form,only 50% of wild type subunit e was converted under its dimeric form in the presence of CuCl2 On the other hand,whatever the digitonin-to-protein ratio used,the e + e dimer was only found in wild type ATP synthase oligomer upon oxidation,whereas the monomeric form of subunit e was associated with both oligomeric and dimeric forms of the enzyme at a digitonin-to-protein ratio of 0.75 gÆg)1 With a digitonin-to-protein ratio of 2 gÆg)1,which destabilizes the ATP synthase oligomers,the monomeric form of subunit e was removed,while an e + e dimer was still present in the CuCl2-induced ATP synthase oligomer As a stoichiometry
of two subunits e has been established in rat ATP synthase,
it appears that the two subunits e of each wild type enzyme react differently and thus likely have different environment
in F0 It has not yet been established whether the dimeri-zation motif of subunit e mediates homodimer formation between subunits e of each enzyme or between subunits e of two interacting enzymes or heterodimer formation with another component of F0in the wild type ATP synthase The combination of BN/PAGE,SDS-gel electrophoresis
of isolated supramolecular complexes and cross-linking
Fig 7 The disulfide bond formation between two subunits e stabilizes an
oligomeric form of the yeast ATP synthase Mitochondria isolated from
wild-type and eC28S cells were incubated in the absence or in the
presence of CuCl 2 (A) Digitonin extracts obtained with the indicated
digitonin-to-protein ratios were submitted to BN/PAGE The gel was
incubated with ATP-Mg 2+ and Pb 2+ to reveal the ATPase activity.
NEM was added in the control experiments instead of CuCl 2 (B)
Mitochondria isolated from wild-type cells were incubated with CuCl 2
and solubilized with a digitonin-to-protein ratio of 2 gÆg)1 After BN/
PAGE analysis the bands were revealed by the ATPase activity The
oligomeric form (lane 1),the high band (lane 2),the low band (lane 3)
of the dimeric forms and the monomeric form (lane 4) of the ATP
synthase were cut and submitted to SDS-gel electrophoresis The slab
gel was transferred onto nitrocellulose which was probed with
poly-clonal antibodies against subunits i and e T,acrylamide concentration.
Trang 9experiments with engineered targets in subunits e,g and 4
will allow the identification of contact areas of the different
partners involved in the interfaces between ATP synthases
Acknowledgements
We are grateful to Drs C Napias and R Cooke for their contribution to
the editing of the manuscript This research was supported by the Centre
National de la Recherche Scientifique (Programme Dynamique et
Re´activite´ des Assemblages Biologiques),the Universite´ Victor Segalen,
Bordeaux 2 and the Etablissement Public Re´gional d’Aquitaine.
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