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Topography of the yeast ATP synthase F0 sector
Biochimie ~1998) 80, 793-801 O Soci6t6 ~ranqaise de biochimie et biologie mol6culaire / Elsevier, PreSs
Topography of the yeast ATP synthase F o sector Jean Velours*, Christelle Spannagel, St6phane Chaignepaim Jacques Vaillier, Genevieve Arselin, Pierre Vincent Graves, Gis le Velours, Nadine Camougrand Institut de Biochimie et G~n~tique Cellulaires du CNRS, Universit6 de Bordeaux IL !, rue Camille-Saint-SaChs, 33077 Bordeaux cedex, France
(Received 9 March 1998: accepted 3 June 1998) Abstract ~ The interaction between the hydrophilic C-terminal part of subunit 4 (subunit b) and OSCE which are two cornponents of the connecting stalk of the yeast ATP synthase, was shown alter reconstitution of the two over-expressed proteins and by the two-hybrid method. The organization of a part of the Fo sector was studied by the use of mutants containing cysteine residues in a loop connecting the two N-terminal postulated membrane-spanning segments. Labelling of the mutated subunits 4 by a maleimide fluorescent probe revealed that the sulfhydryi groups were modified upon incubation of intact mitochondria. In addition, non-permeant maleimide reagents labeled .,,ubunit 4D54C, thus showing a location of this residue in the intermembrane space. Cross-linking experiments revealed the proximity of subunits 4 and f. In addition, a disulfide bridge between subunit 4D54C and subunit 6 was evidenced, thus demonstrating near-neighbor relationships of the two subunits and a location of the N-terminal part of the mitochondrially-encoded subunit 6 in the intermembrane space. © Soci6t6 franqaise de biochimie et biologic mol6culaire / Elsevier, Paris ATP synthase I cross-linking / subunit 4 / subunit 6 1 subunit f I. Introduction
The mitochondrial ATP synthase is the major enzyme responsible tbr aerobic synthesis of ATP. ATP synthase exhibits a tripartite structure consisting of a headpiece (catalytic sector), basepiece (membrane sector) and a connecting stalk. However, the enzyme resolves into only two parts. The catalytic sector F~, with subunits tt, [], y, 5 and t:, is a water-soluble unit retaining the ability to hydro!yse ATP when in a soluble form, and a detergent soluble unit, the Fo sector, which is embedded in the membrane and is composed of hydrophobic subunits forming a specific proton pathway. The connecting stalk is composed of components of both F~ and Fo. When the two sectors ate coupled, the enzyme functions as a reversible * Correspondence and reprints Abbreviations: AMDA, 4-acetamido-4'-maleimidylstilbene-2-
2'disulfonic acid; APA-Br, p-azidophenacyl bromide; APDP, N-I4. p-azidosalicylamido)butyi]-3'(pyridyldithio) propionamide; ASIB, i-(p-azidosalicylamido)-4-(iodoacetamido) butane; C-ter4, C-terminal part of subunit 4; DACM, N-(7dimethylamino-4-methyl-3-coumarinyl)maleimide; DSP, dithiobis(succinimidyl propionate); EDTA, ethylenediamine tetraacetic acid; Fo and F~, integral membrane and peripheral portions of ATP synthase; GnCI, guanidinium chloride; HEPES, N-(2hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid); MPB, 3-(N-maleimidylpropionyl) biocytin; OSCP, oligomycinsensitivity-conferring protein; SDS, sodium dodecyi sulfate; su, subunit.
H+-transporting ATPase or ATP synthase [ 1,2]. The model lbr energy coupling by F~Fo ATP synthase that has gained the most general support is the binding change 131. This concept has been strengthened by the crystal structure of the major part of the mitochondrial Fj 141. The affinity change of substrates and products at catalytic sites is coupled to proton transport by the rotation of st, bunits which belong to F~ and Fu (15-81 for reviews). As a result, the ATP synthase could be a rotary motor with a rotor consisting of subunits y, s and the DCCD binding protein oligomer in E. colt, all other subunits being parts of the stator Whereas the E. colt ATP synthase contains eight different subunits, the bovine enzyme is composed of 16 different subunits 191. The Saccharomyces cerevisiae AI"P synthase is composed of at least 13 different subunits that are involved in the structure of the enzyme (table I). The Fo part contains at least eight different subunits. Thtve of them are mitochondrially encoded (subunits 6, 8 and 9) 1101. The other five are subunit 41111, OSCPII21 subtmit d 1131, subunit h 1141 and subunit f l151. Subunit 4 is a component of the stator and is homologous to the b-subunit of beef heart mitochondria 1161. It contains 209 amino acid residues and has a molecular mass of 23 250 Da. The structure of the eukaryotic b-subunit is composed of two domains: the N-terminal part which is predominantly hydrophobic, and the C-terminal part which is charged and hydrophilic. This subunit is considered to traverse the membrane twice via two hydrophobic
Velours el al.
7~ Tal14eI, Yeast ATP s~,ntha.~ subunits. Subunit Ft
Fo
Associat¢~ proteins
Amino acids
Mass (Da)
get~e
ct I]
5 !2 490
55 394 52 800
ATP1 ATP2
3 3
[441 [45l
y
278 138 61 249 48 76 209 !95 173 95 88 63 63 83 96
30 649 14 555 6612 27 943 5850 7761 23 253 20 874 19 722 I0 567 9953 7384 7287 9455 10 876
ATP3 ATPI6 ATPI5 OLI2 (ATP6) AAP! (ATP8) OLli (ATPg) ATP4 ATP5 ATP7 ATP 17 ATP!4 INH I STFi STF2 TIM I I
I i i
1461 [471 [48~ 1381 I491 150, 511 l! II I 12I [I 31 ! 15] [ 14I 1521 [53 ! 1541 155, 561
su.6 su.8 su.9 su.4 oscp su.d su.f su.h IF1 9 kDa 15 kDa su.e
Stoichiomets3'
I 9-12 I I ! n.d. n.d. I n.d. n.d. n.d.
Ref~,re~z~'e
n.d,, not determined.
stretches of amino acids, with N- and C.terminal parts emerging from the membrane on the Ft side 1171. Like its c o u n t e ~ r t subunit b in E, coll, the euka~),otic b-subunit appears necessary for a tight coupling between proton flux and ATP synthesis 118~231. This study was designed: i) to examine the relationships between OSCP and the hydrophilic Cqerminal part of subunit 4; and ii) m examine the accessibility and the environment of the N-termina! part of the e ukaryotic subunit.
2, M a l ¢ ~
aml methods
APA-Br, DACM and oligomycin we~ p u ~ h a ~ d from Sisma, APDP and ASIB we~ from Pierce, AMDA and MPB were obtained from Molecular Probes. All other chemicals were of reagent grade quality,
2,1, Yeast strains and nucleic acid techniques "rb~ Saccht~romyces ce~visiae strain D273- !0B/A/H/U (MATot met6, ur~3, his3)1181 was the wild type strain. Wild type ~ d mutant strains were obtained after complementation of the deleted-disrupted yeast strain PVYI0 (MATer, met6, urn3, his3, ATP4::URA3) by the low copy shuttle ~ t o r pDRI containing the wild tyt~ ATP4 gene ~ mutated ve~ions of ATP4 gene, respectively 1241, M u ~ t s were n ~ as (wild type residue)(residue num~r)(mutant residue) where the residues are given a single-letter code, The two-hybrid system of Fields and Song | ~ 1 was u ~ , The coding sequences were introd~ into the unique Smal or BamHl sites in plasmids
pGBT9 and pGAD424 which were transformed into the yeast strain SFY526. The I~-galactosidase assay was used to determine the interaction 1261.
2.2. Biochemical procedures Cells were grown aerobically at 28 °C in a complete liquid medium containing 2% lactate as carbon source [27l and harves!ed in logarithmic g~x~wth phase. Mitochondria were prepared according to Lang et al. 1281 and Gu~rin et al. 1291. Protein amounts were determined ~tccording to Lowry et al. 130,1in the presence of 5% SDS. Bovine serum albumin was used as standard protein, The s~eific ATPase activity of the purified F~Fo ATP synthase was measured in the presence el" exogenous phospholipids [141. DACM-labelling, MPB-labelling, AMDA protection and cross-linking experiments were performed as descried in [241, !mmunoprecipitation of the yeast ATP synthase was done according to 1311.
2.3. F~xpression and purification of C-ter4 and OSCP The DNA sequence encoding the C-terminal part of subunit 4 (residues 76 to 209) was cloned into the expression vector pTT-7. One L of liquid medium containing 0. ! mglmL of ampicillin and 0. I mg/mL of kanamycin wcrc inoculated by an overnight culture of the E. coil strain JMI01 containing the pT7-7C-ter4 and the pGPl-2 plasmids 1321. Cell growth was monitored at 30 °C to an A6oo of I. The cells were placed for 3 h at 42 °C and then centrifuged for 5 min at 10 000 g. The cellular pellet was suspended in 6 mL per g wet weight of
Topography of the yeast ATP synthase Fo sector lysis buffer conlaining 50 mM Tris-HCI, 100 mM NaCi, 2 mM EGTA, 2 mM EDTA, |0 mM p-aminobenzamidine, 10 mM e-aminocaproic acid, 0,5 mM PMSE pH 7.5. Cell lysis was performed with lyzozyme ~1 mg/mL, 30 rain at 4 °C) and four sonications of 30 s. DNase was added (10 mg/mL) and incubated for 40 min at room temperature. The inclusion bodies containing C-ter4 were recovered by centfifugation (15 min at 750 g) and washed with lysis buffer. After centrifugation, the pellet was dissolved in the lysis buffer containing 8 M GnCi. The extract was separated by gel permeation chromatography on a Sephadex-G75 column (95 x 2.5 cm) that was eluted in the same buffer at a flow rate of 12 mL/h, and C-ter4 containing fractions were chromatographed by reverse phase HPLC on a Vydak C40.5 mm (0.46 × 25 cm) column. Proteins were eluted with an acetonitrilc gradient in the presence of 0.1% triflu0roacetic acid. OSCP was over-expressed by using the pET3a RV vector in BL21(DE3) E. coil strain. Purilication of the recombinant protein was described previously 1331.22 rng of pure OSCP were obtained from 1 L of culture medium.
2.4. Solubilisation of C-ter4 and interaction of OSCP and C-ter4 Four l.tL of 20 mM Tris-HCl, 1 mM EDTA, 0.001% PMSE pH 7.5, were added to 200 lag of purified C-ter4. Six laL of the same buffet" containing 6 M GnCi were then added to solubilise the sarnple. After 10 rain at room temperature, 10 lxL of the interaction buffet" containing 100 mM Tris-HCI, l m M EDTA, 20% glycerol, 2 mM MgCI,, pH 7.5 were added. After centrifugation (15 rain, 100 000 g), the supernatant was added to 165 l.tL of the interaction buffer containing OSCP or not. The mixture was incubated Ibr I h at room temperature lbr !0 rain at 100 000 g. For cross-linking experiments of C-tcr4 and OSCP by DSP, the Tris bu!'!¢r was replaced by 100 mM HEPES buffer, pH 7.5. Aliquots of 10 M, were incubated for 30 rain and analysed by Western blot.
795 lbllowed by successive dilutions was tested. It resulted in a soluble fraction which chromatographed by molecul~x sieving in the range of 20-10 kDa like subunit OSCP. Incubation of C-ter4 and OSCP led to a new peak located ahead of each subunit. SDS-PAGE analysis of this peak revealed the presence of the OSCP and C-ter4 in a molecular mass range corresponding to the sum of the two partners, thus showing a likely interaction (not shown). Confirmation of the interaction was provided by a crosslinking between OSCP and C-ter4 (figure 1). The bifunctional reagent DSP was used to make a bridge between the two partners. The cationale of this experiment is to maintain the interaction during analysis. Incubation of DSP with C-ter4 probably aggregated or oligomerized the protein which disappeared from the slab gel. OSCP spontaneously dimerized in our experimental conditions by the unique cysteine group. This was reversed under reducing conditions (not shown). DSP incubation of the mixture containing OSCP and C-ter4 led to a new band corresponding to the heterodimer of the two proteins, as shown by the relative molecular mass and by twodimensional electrophoresis. This band was absent when GnC! or SDS were added before cross-linking. The fact that a sharp band was obtained upon cross-linking is in favor of a unique structure of the partners with a limited number of cross-links, which otherwise could lead to a
12
34
5 6 78
9
10
kDa 94-
67-
p ~.~, ~.
4330-
~
o -oscpdimer .- o s c p + C - t e r 4
~
2.5. SDS-PAGE and Western blot analyses SDS-PAGE was perlbrmed as described previously i lll. The slab gel was washed with methanol/ water/acetic acid (5/5/1) for 15 min. Fluorescent bands were photographed under ultraviolet light, and the slab gel was silver-stained1341. Two-dimensional gel electrophoresis was described in 1241. Western blot analyses were described previously 1141. Antibodies raised against subunits 4 and f were used with a i: 10 000 dilution. 3. Results and discussion
3. !. Interaction of OSCP and the C-terminal part of subunit 4 in a preliminary experiment a renaturating method consisting in the solubilization ot" C-ter4 by 6 M GnCI
211--
-- ---
- C-ter4
14"
Figure 1. Cross-linking of subunits OSCP and C-ter4. SDSPAGE of over-expressed OSCP and the C terminal part of subunit 4 incubated with 0.38 mM (lanes 3-5) and 0.9 mM (lanes 6-10) of DSP (see Materials and methods). Lane !, standards; lane 2, OSCP + C-ter4; lanes 3 and 6, C-ter4; lanes 4 and 7, OSCP; lanes 5 and 8, OSCP + C-ter4; lane 9, OSCP + C-ter4 + SDS; lane 10, OSCP + C-ter4 + GnCI. Standard~ were ct-lactalbumin, soybean trypsin inhibitor, carbonic anhydrase, ovalbumin, bovine serum albumin and phosphorylase b.
Velours et al~
7% ~I!. ~ocL
OSCP-C4er4 interaction by the yeast two-hybrid
F~ion proteinsa Gal4-bd + Gal4,ad Cat + Gal4-nd ~ b d + Oal4-ad-OSCP Gat4-bd + Ga14A-ad42ter4 G~4.bd43SCP ~ad~SCP Oal4.ad.C-lel~
~bd.OSCP
+
Gal4-ad-C-ter4
fl.galactosidase activi~, b 0.53 0.46 0.34 0.42 0.21 0.19 0.21 752
e x ~ s i o n plasmids carrying the genes for the indicated fusionpix~(ns were cotran~fected into yeast strain SFY5261251. *'VM~ f~ [3~galactosidaseactivity are the mean of at least two trans~ each assayed at lea~ three dines. The activity is shown according to Miller [261 with a slight modification:the optical density at 420 nm of o~nitrophenoi released by the ~ y m ¢ was normalizedby the proteins measured by the Lowry procedure 1301,
and AMDA labelled su4D54C and to a lesser extent su6C23 (not shown), whereas su4N47C and su4E55C were not accessible (L49C mutant was not tested). This was shown by an increase in the relative molecular mass of subunit 4 (fgure 2(7). AMDA protected su4D54C from MPB-binding (figure 2C, D), thus showing that the two maleimides were competitive towards the sulfhydryi group of Cys54. From these data, we propose that the hydrophilic loop containing the amino acid residues 46 to 56 is directed toward the mitochondrial intermembr~ae space. The highly hydrophilic maleimide AMDA efficiently protected su4D54C from MPB-labelling, thus showing that Cys54 is located in a hydrophilic environment. On the contrary, the absence of MPB-labeling of su4N47C and su4E55C (data not shown) is in favor of a location of these residues in a more hydrophobic environment. As a result, the predicted loop connecting the two membrane-spanning segments should expose only a few
A
broad band. ~o-hybrid experiments with a combination of OSCP fused to Gal4-bd and C-let4 fused to Gal4-ad r e s u l ~ in a strong expression of the reporter gene, thus showing that interaction between OSCP and C-ter4 also o e c u ~ d u n d e r in vivo conditions (tab& !1).
B
wt 47 49 54
55
wt47
49 54 55
3,2, Accessibi!ity of subunit 4 oscl
The location of a loop region (residues 46 to 56) connoting the two Notermina! ~stulated membrane~ ~ n a t n g ~ g m e n t s !16, 351 was investigated by using four eysleine sub~tituti,~n mu~nts, each containing a sole cysteine refidue in subunit 4 at locations 47, 49, 54 and 55 (lhe wild type ~,~nit 4 is devoid of ¢ysteiae ~sidue), !~e m u ~ t s t t a i m 8 f e w wi~ l a c ~ as c a ~ n souse, thus ~owin$ that ~ phosphotylation process was not altered in the cysteine m a ~ t s , Furthermore, the mitochondfial oligomycin-~nsitiv¢ ATPase ~tivifies were not significandy modified (not shown), • :cessibility of the eysteine residues of mutated subun~ 4 was examin~ by t cu~uon of intact mito¢hondfia with the fluorescent maleimide hydrophobic t ~ n t DACM, which forms a stable, s~ngly fluore~ent adduct with thiol g ~ p s [361, ATP syntha~s were extracted, perifi~ by immunoptccipitalion and analysed by $DS-PAGE, Of all the putative DACM-labelled subunits, only subunit 6, su4N47C, su4L49C, su4D54C and ~a4ESGC we~ fluorescent, Figure 2 shows the migration of subunits 4, 6, OSCP and d, As DACM is a hyd~obic ~nt, more ~finit¢ evidence for the Iotalion of ~ hydrophilic loop was p m v i ~ by b i l k i n g sulfhydryi ~ with water-soluble maleimides, Freshly isolated ~ int~t D S ~ mitocholldria were reacted with M P B ~ AMDA, The water-soluble maleimides MPB
MPB AMDA
°°
C * ~
-
*
-
+
+
-
D + -
-
+
+
+
Figure 2, Labellingof subunit4. A, B. SDS-PAGEof wild type control and mutant immunoprecipitatedATP synthases isolated from intact mit~hondfia labelledby DACM. A. Silver-staining. B, Analysis for fluorescenceunder ultravioletlight, wt, wild type control (no mutation).The numbersat the top of the figurerefer to the I~ation of the mutations in subunit 4. C, D. Western blot analyses of D54C immunoprecipitatedATP synthases isolated from intact D54C mitochondria labelled by MPB and AMDA (see Materials and methtxh'). Blots were revealed either with antibody raised against subunit 4 (C) or with streptavidin horse radi~ peroxidase (D).
Topography of the yeast ATP synthase Fo sector
797
residues (at least Cys54) to the aqueous phase. In addition, the fluorescent labelling of tile sole cysteine (Cys23) of mature subunit 6 is also in |avour of an intermembrane space location of the N-terminal part of subunit 6, as proposed previously [37].
3.3. Cro,~'s-linking of sub~nits 4, 6 and f The mutated subunits 4 were used for determining neighboring proteins in Fo. In a preliminary experiment, crude mitochondrial Triton X-lO0 extracts of the four mutants were reacted with ASIB and exposed to UV light. Two cross-link products migrating with relative molecular masses of 33 000 and 46 000 were found in D54C extract, but not in N47C extract. L49C and E55C mitochondrial extracts contained only the 33 kDa product (figure 3A). The identilication of the two neighboring proteins wax undertaken from D54C mitochondria. As subunit 4 has a molecular mass of 23 250 Da, the two neighboring proteins displayed molecular masses of 10 and 23 kDa, respectively. Similar cross-links were obtained by incubation of intact D54C mitochondria with ASIB. Wild type subunit 4 was not modified by the reagent, whereas three additional bands with relative molecular masses of 50 000, 46 000 and 33 000 were recognized by our anti-su4 antibodies in D54C mitochondria (¢igure 3B). A 50 kDa band was detected without incubation with the cross-linking reagent. This band was present only in D54C and E55C mitochondria. It disappeared upon reduction with 2% fl-mercaptoethanol and was insensitive to a 10 mM NEM incubation prior to dissociation (not shown). Experiments are under way to identify this product. Polyclonal antibodies raised against most yeast F, subunits were tested. We found that the 33 kDa product
A v.'" 47
49 54
55
B + + + wT 47 49
+ 54
+ 55
ASIB
was a heterodimer of subunits 4 and f, which is in agreement with the sum of the respective molecular masses. When cross-linking experiments were performed with APA-Br, APDP and AS|B, the band of 46 kDa was obtained with the three reagents whereas subunits 4 and f were not cross-linked by APA-Br which has the shorter arm (9 k,) (not shown). Oxidation of crude D54C mitochondrial Triton X-100 extracts and intact D54C mitochondria by cupric 1-10 phenanthrolinate also gave the immunoreactive band of 46 kDa (figure 4A). This band was present in high amoun!s in the purified D54C F:F o ATP synthase (ligure 4B, lane 1). As anti-OSCP and anti-subunit d antibodies did not react with the 46 kDa cross-linked product, we took the disulfide bond formation between su4D54C and the cysteine residue of another yeast Fo subunit to identify the neighboring subunit. Purified D54C ATP synthase was incubated under oxidative conditions. The intensity of the 46 kDa cross-linked product increased under oxidative conditions and dis~ppeared under reducing conditions (figure 5A). In figure 5B the subunits of a CuCl~-induced cross-linked D54C ATP synthase were separated under non-reducing conditions. The gel was then reduced and submitted to a separating slab gel gradient. Silver-staining of the slab gel clearly revealed that subunits 4 and 6 were linked during the first electrophoresis. Since Cys23 is the sole cysteine residue of subunit 6 [38], the disulfide bridge between su4D54C and su6C23 indicates a close proximity of the hydrophilic loop ol subunit 4 and the N-terminus of subunit 6. Despite the presence of light chain immunoglobulins migrating in the 25-30 kDa range, a 4-6 dimer and a 44 dimer were also evidenced by two-dimensional SDS-PAGE of an ATP synthase immunoprecipitate isolated from a crude D54C
C
-+-+
WTWT5454
wr 5454
- 50 -46 ,~
33-
~'~P"
-" + - -
,..- - 3 3
f
'qi,WmmD
-.. 4"-
-23
kDa
f-
-10
kDa
Figure 3. Crossolinking between sub° units 4 and f. Western blot analysis of mitochondrial proteins (30 l,tg). Yeast mitochondria were incubated with ASIB or not in the dark for 2 h at morn temperature. The reaction was blocked with t~-mercaptoethanol, samples were illuminated at 365 nn~ Ibr 10 rain and dissociated. Blots were reacted with antibodies raised against subunit 4 (A, B) and against subunit f (C). wt, wild type control (no mutation). The number 54 at the top of the figure refers to the location of the mutation in subunit 4,
798
Velours et al.
A 2
B 3
2 3 4
.......
|
A 2
B 3
567
kDa -50
°li9°mer" 6
/'-~ .
I'-~
4÷. t
..
-
~v ~ "|"
W
oso : _ __-
i
°
• ,,,
cp
-23 h~
F ~ r ~ 4 , Cross-links with CuCI~ and APDE A. Western blot analyses of crude D S ~ mitochondrial extracts 130 ~tg) incubated without (lane 1), with 1.5 mM (lane 2) and with 3 mM CuCI: (lane 3), B, Purified D54C ATPa~ syntha~ (3 ~tg) was reacted with APDP (lane 1. control; lane 2, 20 taM; lane 3, 50 ~M; lanes 4 and $, I ~ I~M. Lanes 6 and 7, crude D54C mitochondrial extract wa~ cro~olink~ with 2 ~ t~M APDP. Lanes 5 and 6, 2% ~me~pt~lhanol was added to the samples be!bre electropho~ r~sis, Blots were reacled wilh antise~ raised against suhunit 4,
mil~hondrial exlracl crOssolinked with APDP (//gm~:, 5C). As subunit 6 and su4D54C were cnassolinked when using either intact mit~hondria or 1~iton Xo I ~) c-st.rac'u,,sa. .close ... p~sim~ty of t ~ two subunits in the same comples was likely, ~ i not a cross-link ~ e u ~ n g ~ t w e e n two subunils of two d i f t ~ n t complexes, From these results we conclude that subunits 6 and 4 are in contact, like the c o u n t e ~ subunits a and b of the I~, ¢oli ATP synthase 139, 401, Tbe hydrophobic plot of the yeast subunit 6 predicts a hydrophilic N-terminal part that is longer than that of the t ~ v i ~ counterpart (not shown), and a location of Cys23 at the beginning of the fi~t membraae~spanning segment, As a result, the N-terminal of subunit 6 should be in the intermembrane comparlment which leads to an i n v e ~ location to that of the prokaryotic counterpart subunit a 141 I, We did not find any significant ATPase activity decrease in the D54C purified enzyme having either sponta~sly or induced 6--4 cross, link, Specific ATPase ~tivifies of 49,3 ± 2,2 and 50,5 ± 0,3 pmol of Pi rain ling protein ~ in the ab~nce of oligomycin and 29,5 ± 1,5 and 27,3 ± 2,5 lamol of Pi rain ~ mg protein '~ in the presence of oligomycin for the wild type and D54C purified
A-
.b
~,f
,
C Figure $. Disullide bridge between subunit 6 and su4D54C. A. SDS-PAGE of D54C ATP synthases 18 ltg) incubated without (lane 1) or with !,SmM CuC!2 (lanes 2 and 3). Before dissolution and electmphoresis, 2% [~-mercaptoethanol was added to the sample in lane 3. Tile slab gel was silver-stained. The arrow indicates the 4-6 cmsso!ink product. B, C. qkvodhuenniona! gel elec|ropho~sis of D54C ATP synthasc. B. 18 ttg of puri|icd entyme were incubaled with i.5 mM CuCl~ alld submitted to a 15% SDS~PAGE without i~omercaptoethanol treatment (that dimension from left to righ!). Second dinlension, 13~18% gradient SDSoPAGE° The second dimension was done after incubation of the rod gel with 2% ~-me~apt(~thanol (see M(ller6ds amt metlu,ls). C. A crude D54C mitochondrial Triton X°I00 extract was cross-linked with 200 itM APDP. The ATP synthase was immunoprecipitated and analysed as in (B).
enzymes were measured, res~ctively. As a consequence, subunits 4 and 6 do not display large relative movements during catalysis, which is in agreement with the fact that these two subunits are part of the enzyme stator.
4. C o n c l u s i o n
A representation of a part of Fo is shown in .Figure 6. This scheme derives from the above data. OSCP interacts with the C-terminal part of subunit 4. This result is in agreement with those of Collinson el al. [17] who recently
Topography of the yeast ATP synthase Fo sector reported such an imemction between the hydrophilic part of bovine b°subunit and OSCP In lhe N-terminal part of the subunit, two lransmembrane et-helices are linked by a short hydrophilic segment, The incubation of intact mitochondfia with maleimides having different permeability properties led to the conclusion that subunit 6 and the mutated subunits 4 of the yeast ATP synthase display accessible ta~ets from the inte~xnembrane space. The cross-linking reagents AS1B and APDP with spacer arms of 18,8 and 21 A, respectively [42] linked subunits f and 4 and subunits 4 and 6, but the absence of cross-linking with APA-Br indicates that the sulfhydryl group of su4D54C and subunit f should be at least 9 A apart. Belogmdov et al. [43] reported that dissuccinimidyl mr|rate (cross-linking distance of 6.4 ,/~) produced f-b crosslinks in bovine ATP synlhase preparations but not in submitochondrial particles. We also observed a 1"-4crosslink but only in intact mitochondria and crude mitochondrial Triton X-100 extracts with cysteinyl reagents having arms longer than 9/k. The specificity of the reagents could explain this discrepancy. The orientation of subunit f was taken from [43]. By using the cysteine substitution strategy, recent experiments performed with mutated subunits f were in agreement with the orientation of the bovine subunit f (unpublished data). The three cross-linking reagents bound su4D54C and subunit 6, thus indicating a close proximity of the two subunits. In addition, the disulfide bridge induced by oxidation revealed that Cys54 of subunit 4 and Cys23 of subunit 6 are in contact. As a consequence, the disulfide bridge occurring between su4D54C and su6C23 indicates a similar location of both sullhydryl groups on the outer phase of the inner mitochondrial membrane. The N-terminal part of the eukaryotic subunit 6 is placed in the intermembrane compartment which leads to an inverse location to that ol" the prokaryotic counterparl subunil, We cannol exclude a slight difference between the structure of the cystcineo containing hydrophilic loop and the wild type loop. However, this alteration did not significantly disturb the overall structure of the enzyme, as oxidative phoshoryla° tions were conserved. Thus, a modification of the orien° tation of subunit 4 was excluded, since its C-terminal part is involved in the binding of F~ I181. Therefore, data provided by cysteine residues should reflect the global structure of the wild type hydrophilie loop, and validate the existence of the two postulated membrane-spanning segments linked by a short hydrophilic loop located near the surface of the membrane.
Acknowledgments We are grateful to Dr Guy Lauquin and Dr. David M. Mueller for stimulating discussions. Wc thank Dr. Ray Cooke for his contribution to the editing of the manuscript. This work was supported by the Centre National de la Recherche Scientifique,
799
,ace
coott
i n t e r m e m b r a n e space Figurc6. S¢licmatic represcnlatmn of stibumls 4, {~, I and OSCR
the Ministate de ia Recherche el de l'Enseignement sup~ricur. the Universit6 de Bordeaux II and the Etablissement Public R6gional d'Aquitaine. C.S. holds a research grant from the Minist~re de la Recherche et de la Technologic.
References 111 PcderscnRL,, Fromiers in XI'P synthasc re,,+earch:tlndt'rstandint~' the relationshipbetweensubunitmovenlentsand ATPsynthesis+J. Bioenerg. Bi,,memb. 28 (1996) 389-395. 121 Weber" J.. Senior A.E.. Catalytic mechanism o/ F-I-ATPase. Biochim. Biophys. Acla 1319 (1977) 19-58. 131 Boyer P.D., The binding change mechanism Ik)rATP symhasc some probabilitiesand possibilities, Biochim. Biophys.Acla I1~10 (1993) 2~ ~-250.
Velours et al. 141 A ~ J.E, Leslie A.G.W., Lutter R.. Walker J.E.,Structure at 2,8 an~trom re~tuti~ of FoloATPa~ from bovine heart mitt~ c~a. Nmure 37iI (19941 621~628, 151 S ~ D.. Engelbrecht S,. Junge W.. Functional and idling rolatory motion within F- 1-ATPa.~, Prec. Natl. Acad. Sci. USA 94 (19971 440l'-4405. 161 ~ Y,. Dm~a~ T.M., Cross R L Subanit re.ion in Escherichia F O F I - ~ s~ad,~s¢ daring oxidative phosphorylation. Proc. NaIL A-cad. SoL USA 94 (1997) 10583-10587. 17] I ~ i H~, Yasuda R., Yo~hida M., Kino~ita K., Direct observation of tl~ rotation of F l o A ~ Nature 386 (19971 299-30Z Iill ~ P,D~, The ATP s y n ~ - a spkndid molecular machine, Annu. ~ . B i ~ . 66 (1997) 717-749. 191 C~.liastm I.R, Runswick M J.. Buchanan S.K.. Fearnley I.M., Ske,l~l J.M.. V a n R ~ MJ.. G¢iffiths D.E.. Walker J.E.. F-0 m ~ donna of ATP s y n ~ from bo~'in¢ heart mitochondeia: Purification. sabunit composition, and r¢constitution with F-I oATPa~. B i o c ~ s t r y 33 (1994) 7971-7978. II0t Nagley P., F.,ukaryote ~ b r a n e genetics. The Fo sector of mitochondriai ATP symha~e. Trends Genet. 4 (19881 46-52. fill Vetou~ J,.A~,.elin de Chateaubodeau G., Galante M., Gu6rin B.. Sabunit 4 of ATP syntha~ (POFI) from yeast mitochondria. Puri~caticm, amino-acid ~ i t i o n and partial N-terminal sequance, Ear. J, Bio¢l'~m. 164 (19871 579.-584. !12! Uh M.. Jones D.. MueUer DM.. The gone coding fi~r the oli;gomycin ~nsilivity-conferring prolein, J. Biol, Chore. 265 (1990) 19047-190.52, !131 Norris N.. Prom~ D.. Velours J, ATP syntha~ of yeast mitochono dfla, Characterization of sabunit d and sequence analysis of the structural gone ATPT, J, Biol. Chore, 266 (19911 16541~16549, II,ll A ~ ! i n G,, Vaillier J,. Graves P.V, Velours J,, ATP synth~¢ of y ~ mitoeho~fia, l~datio~ of suhunit h and disruption of the ATPI4 ge~, J, Bio!, Chore, 271 ( ! ~ ) 20284~20290, l~_Sl Sl~nnag¢l C,, Wilier J, Ars~lin G,. Gr~ves Per., Velours J., The sub,nit f of mi!~bondfia! yeas! ATP syntha~, Characterization of the p~o~¢in ~d all,up!ion of the stru¢tura! gone ATPt7, Eur~ L Bioeh¢:m, 247 (19~7) !1!!~1!17, It01 W~!ker J,E,, R.u.n~wick, M.J., Paulter L., ATP synthase from bovine n!lt~hondl'ifl, The eh~teri~.ation and ~.quonset, ~na!ysis of t~,~ n~mhrane~as~i~t~ sah:units and of the corresponding ~DNA,' ~ J, Mol, BIoL 197 11987) 89~1(X). 1171 Cotlinsoa I,R,. V~neaaij M J.. Rullgwtek MJ,, Feamley I M.. Skeh~l J,M,, O~ss G,L,, Mirous B,, W~lker J.E.=/~I'P synthase from bovi~ henri mih~hondfla, In vitro as.~emhly of a stalk ~|¢~ in Ih~ t ~ of F t~ATP~ ~nd in it..~absence, J, Mul0 lliol,242 ( ! ~ ) ~ 2 1 , h a l M,F,, G ~ n B,, Velo~ J,, 1 ~ Cotem ~" fmal region of subuail~ (subunit b) is ¢s\~ntial t°ot as~mbly of lh~ Fo l ~ i o n of yea~ m i i ~ f a l ATP synlha~, I~ur, J, Biod~m, ~5 (1992) 10=172, l lgl th~stek J,, K ~ k y J,, 7 ~ x t i F,, Guerrieri E, Jirillo E,, Capo~za G,, ~ S,~ Topological and fut~tionai ch~teri~ation of the F,,! sul~i~ of Ih~ n~n~rane meaty of the mitoehondria! H'~ATP sy~th~, E~, J, Bioc~m, 173 (1988) 1~8, I~1 ~ f i F,, G ~ t F,, C a ~ . ~ G,, Houstek J,, Roach) S., Papa S,, I ~ liit~ ! lk~ of ~u¢leus~¢at~d i~I pt~ein of bovine heart m i l o c ~ a l H*oATPa~ as ~ functiona~ part of Fo moiety, FEBS Left, 237 (1988) 9~14, t211 Ra~k~4olly D,, Rigoulet M,, Gu~rin B., Vdours J,, Mutation in the, hydtophobie domain of ATP syntha~ subunit 4 (subunit b) of ~¢a~ mit~bo~kia disturbs c~pling betw~n proton transloeation a ~ eala!ysis, Bi~hemistr~ 33 11994) 9684~9691, [22t SI¢ffens K,, Seh~i~¢ E., ~k¢~s-Ilct'~sltcit G,, Alten&~f K.. !:,, ! t ~ i ~ of g,w/~t~ich/a co//ATP syatha,~, Furtlvzr resolution of t1~I~in-~r~rat~d l~1~nts fr~n subunit b, J, Biol, Chem, 262
t231 Take)~a~ M,, N~mi I",, Maoda M,, Fatal M,, Fo potlion of ~ ' ~ ¢ ~ / ~ ~'a/i H'-ATPa~, Carboxyl-terminal ~egi~ of the b sub~aaa is ~.~nlial f~" a,~mhly of functi~al F,., J, Biol. Chore. 2f~ (11~) 161t~-lff112.
[24] Spannag¢l C., Vaillier J., Chaigncpain S.. Velours L. Topography of the yeast ATP synthase FO sector by using cysteine substitution mutants, Cross-linking between subunits 4, 6 and f, Biochemistry 37 (19981 615-62 !. [25] Fields S,, Song O., A novel genetic system to detect proteinprotein interactions. Nature 340 (19891 245-246. [26] Miller L, Assays of 13.galactosidase, in: Experiments in biolecular Genetics, Cold Spring Harbor Laboratory, Cold Sprin g Harbor, NY, 1972, pp, 352-355, [27] Arselin D.E Chateaubodeau G.. Gu6rin M., Gu6rin B., Permeabilit6 de la membrane interne des mitochondries de levure: 6rude des relations entre structure el activitY, Biochimie 58 (19761601-610, 1281 L~g B., Burger G., Doxiadis |., Thomas D.Y,, Bundler W., Kaudewiw. E, A simple n~elhod for the Ire'go-scale preparation of mitochondda from microorganisms Anal. Biochem. 77 (1977) 110-121. 129I Gudrin B.. Labbe P.. Somlo M,, Preparation of yeast mitochondria (Sacchanm~yces cerevi.~iae) with g ~ P10 and n,~spiratory control ratios. Methods En~ymol, 55 (1~}791 14~159, 130l Lowry O.H.. Rosebrough NJ., Farr A,Lo, Randall R J., P~x~lei!~ measurement with the folin phenol reagenL J, Biol. Chem. 193 (19511 265-275, 13! ] Todd R.D., Grie,~nbeck T.A., Douglas M.G., The yeast mitochondrial adenosine triphosphatasc complex. Subunit stoichiomctry and physical charactedza|ion. L Biol. Chem. 255 (19801 5461-5467. [32] Tabor S,, Richardson C.C., A bacteriophage T7 RNA polymerase/ promoter system for controlled exclusive expression of specific genes, Prec, Natl, Acad. Sci. USA 82 (19851 1074--!078. [33] Mukhopadhyay A., Zhou X.Q., Uh M., Mueller D.M., Heterologoas expression, purification, and biochemistry of the oligomycin ~nsitivity confen'ing protein (OSCP) from yeast. J. Biol. Chem. 267 (!g92) 25690-25696. 1341 Ansorge W., Fast visualisation of protein hands by impregnation in potassium permanganate and silver nitrate, in : Stathakos D (Ed.L E!ectrophoresis, 82, Walter de Gruyter. Berlin, New York, 1983, PR 235.24Z 135I Velours l., Durmns E. Aigle M.. Gu6rin B,. ATP4. tile structural gone tot yeast FoFj ATPase sut~unit 4. Cur. J, !~iochem0 170 (1988) 637-642. I36l Schneider E.. Aitendort" g.. Mad)Ileal)on of suhunit b of Ihe F. c~tnplcs f~tJ~ !;..whe~ichi, coli ATP ~ynthasc by a hydrophol~ic m~d¢imide and i|s effects on FO filn¢ik~ns, Cur. J. I]ioehemo 153 1985) 1055-.~:111~}. 137] Midloa %, Galant¢ M,, Velours L, NH~.terminal sequence of the i~laled yeast ATP synlhase subunit 6 ~vea!s posbtranslational cleavage, Ear. J. B i ~ m , 172 (1988) 621~25. 1381 Macino G,, Tzagoloff A., Assembly of the mitochondriat membrane system: Sequence analysis of a yeast mitochondrial/WPase gone containing the oli-2 and oli.4 loci, Cell 20 (1980) 507-517. 1391 Hermolin J., Gallant J., Fillingara¢ R.H., Topology, organization. and function of the psi subunit in the F0 ~ctor of the H'-ATPase of Escherichia co!i. J. Biol. Chem. 258 (19831 14550-14555. 1401 Ads LE, Simon) R,D., Cross-linking and labeling of the Escherichin colt F~Fo-ATP synthase reveal a compact hydrophilic portion of FO clo~ to an FI catalytic subunit, L Biol. Chem. 258 ( 19831 1459%14609. 1411 Yamada 11., Moriyama Y., Maeda M., Futai M., Transmembrane topology of 6~'cherichia coli it (+)-ATPase (ATP syntha~) subunit a, FEBS Lett. 39t.t ( 1 ~ ) 34~38. 1421 Skakoon E.N., Dunn S.D., Location of conserved residue histidinc-38 of the epsilon subunit of b,~'cherich;a colt ATP synthase, Arch. Biochem. Biophys. 302 (19931 272-278. 143[ Bdogmdov G.I., Ibm)oh J,M., Hateli Y., Membrane topography and near-neighbor relationships of the mitochondrial ATP synthase subunils e, f, and g, J. Biol. Chem. 271 (19961 20340-20345. 1441 Takoda M., Chert W J., Saltzgaber J., Douglas M.G.. Nuclear genes encoding the yeast mitochondrial ATPase complex. Analysis of M'PI coding the FI-ATPase alpha-subunit and its assembly, J. Biol. Chem. 261 (19861 15126-15133.
Topography of the yeasl ATP syntimse F(} sector [45] Saltzgaber-Muller ,I,, Kunapuli S.P,, Douglas M,G,, Nuclear genes coding lhe yeast milochol~d~kd adeuosine triphosphatase complex, |solafion of ATP2 coding ~he FI-ATPase beta subunit, J. Biol. Chem. 258 (1983) i1465-I 1470. 1461 Paul M.E, Ackerman S., Yuc L, Arselin G., Velours J., Tzagolol'f, A., Clt~ning of lhe yeast ATP3 gene coding for lhe gamma subunit of F~ and chavacteri~.ation of alp3 mutants, J. Biol. Chem. 269 (1994) 26158-26164. 147] Giraud M,E, Velours J,, ATP synthase of yeast mitochondria: Isolation of the F1 delta subunit, sequence and disruption of the struclur,! gene, Eur. J. Bioehem. 222 (1994) 851-859. 1481 Gu~lin E., Chevallier J., Rigoulet M., Gu6rin B., Velours ]., ATP synthase of yeast mitochondria. Isolation and disruption of the ATP¢) gene, J. Biol. Chem. 268 (1993) 161-167. [49[ Macreadie I.G., Novitski C.E,, Maxwell R.J., John U., Ooi B.G., McMullen G.I,,, Lukins H.B., Linnane A.W., Nagley E, Biogenesis of mitochondria: the mitochondrial gene (aapl) coding for mit~chondria! ATPase subunit 8 in Saccharomyces cerevisiae, Nucleic Acids Rcs. 11 (1983) 4435-4451. 150l Macino G., Tzagoloff A., Assembly of the mitochondrial membrane system:Partial sequence of a mitochondriai ATPase gene in Saccharomyces cerevisiae, Proc. Natl. Acad. Sci. USA 76 (1979) 131-135.
80t 151R Hensgens, L.A.M., Gri~cll, I,.A., Borst P., Bos LL.. Nucteo~ide sequence of the mitochondriai structural gone ft~r subunit 9 of yeast ATPase complex, Pr~c. Natl. Acad. Sci. USA 76 (1979} 1663-1667. [52] lchikawa N., Yoshida Y., Hasbimoto T., Ogasawara N.. Yoshika~a H., [mamoto E, Tagawa K., Activation of ATP hydrolysis by an uncoupler in mutant mitochondria lacking an intrinsic ATPase inhibitor in yeast, J. Biol. Chem. 265 (1990) 6274-6278. [53] Akashi A., Yoshida Y., Nakagoshi H., Kuroki K., Hashimoto T., Tagawa K,, Imamoto E, Molecular cloning and expression of a gene for a factor which stabilizes formation of inhibitormitochondrial ATPase complex from Saccharomyces cerevisiae, J. Biochem. (Tokyo) 104 (1988) 526-530. 154] Yoshida Y., Sato T., Hashimoto T., Ichikawa N., Nakai S., Yoshikawa H., Imamoto E, Tagawa K., Isolation of a gene for a regulatory 15-kDa subunit of mitochondrial FjFo-ATPase and construction of mutant yeast lacking the protein, Eur, J. Biochem. 192 ( ! 990) 49-53. [551 Tokatlidis K., Junne T., Moes S., Schatz G., Glick B.S., Kronidou N., Translocalion arrest of an intramitochondrial sorting signal next to Tim ll at the inner-membrane import site, Nature 384 (1996) 585-588. 156] Arnold I., Bauer M.F., Brunner M., Neupert W., Stuart R.A.. Yeast mitochondrial FIF,-ATPase: the novel subunit e is identical to Timll, FEBS l,ell. 411 (1997) 195-20.
Topography of the yeast ATP synthase F o sector Jean Velours*, Christelle Spannagel, St6phane Chaignepaim Jacques Vaillier, Genevieve Arselin, Pierre Vincent Graves, Gis le Velours, Nadine Camougrand Institut de Biochimie et G~n~tique Cellulaires du CNRS, Universit6 de Bordeaux IL !, rue Camille-Saint-SaChs, 33077 Bordeaux cedex, France
(Received 9 March 1998: accepted 3 June 1998) Abstract ~ The interaction between the hydrophilic C-terminal part of subunit 4 (subunit b) and OSCE which are two cornponents of the connecting stalk of the yeast ATP synthase, was shown alter reconstitution of the two over-expressed proteins and by the two-hybrid method. The organization of a part of the Fo sector was studied by the use of mutants containing cysteine residues in a loop connecting the two N-terminal postulated membrane-spanning segments. Labelling of the mutated subunits 4 by a maleimide fluorescent probe revealed that the sulfhydryi groups were modified upon incubation of intact mitochondria. In addition, non-permeant maleimide reagents labeled .,,ubunit 4D54C, thus showing a location of this residue in the intermembrane space. Cross-linking experiments revealed the proximity of subunits 4 and f. In addition, a disulfide bridge between subunit 4D54C and subunit 6 was evidenced, thus demonstrating near-neighbor relationships of the two subunits and a location of the N-terminal part of the mitochondrially-encoded subunit 6 in the intermembrane space. © Soci6t6 franqaise de biochimie et biologic mol6culaire / Elsevier, Paris ATP synthase I cross-linking / subunit 4 / subunit 6 1 subunit f I. Introduction
The mitochondrial ATP synthase is the major enzyme responsible tbr aerobic synthesis of ATP. ATP synthase exhibits a tripartite structure consisting of a headpiece (catalytic sector), basepiece (membrane sector) and a connecting stalk. However, the enzyme resolves into only two parts. The catalytic sector F~, with subunits tt, [], y, 5 and t:, is a water-soluble unit retaining the ability to hydro!yse ATP when in a soluble form, and a detergent soluble unit, the Fo sector, which is embedded in the membrane and is composed of hydrophobic subunits forming a specific proton pathway. The connecting stalk is composed of components of both F~ and Fo. When the two sectors ate coupled, the enzyme functions as a reversible * Correspondence and reprints Abbreviations: AMDA, 4-acetamido-4'-maleimidylstilbene-2-
2'disulfonic acid; APA-Br, p-azidophenacyl bromide; APDP, N-I4. p-azidosalicylamido)butyi]-3'(pyridyldithio) propionamide; ASIB, i-(p-azidosalicylamido)-4-(iodoacetamido) butane; C-ter4, C-terminal part of subunit 4; DACM, N-(7dimethylamino-4-methyl-3-coumarinyl)maleimide; DSP, dithiobis(succinimidyl propionate); EDTA, ethylenediamine tetraacetic acid; Fo and F~, integral membrane and peripheral portions of ATP synthase; GnCI, guanidinium chloride; HEPES, N-(2hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid); MPB, 3-(N-maleimidylpropionyl) biocytin; OSCP, oligomycinsensitivity-conferring protein; SDS, sodium dodecyi sulfate; su, subunit.
H+-transporting ATPase or ATP synthase [ 1,2]. The model lbr energy coupling by F~Fo ATP synthase that has gained the most general support is the binding change 131. This concept has been strengthened by the crystal structure of the major part of the mitochondrial Fj 141. The affinity change of substrates and products at catalytic sites is coupled to proton transport by the rotation of st, bunits which belong to F~ and Fu (15-81 for reviews). As a result, the ATP synthase could be a rotary motor with a rotor consisting of subunits y, s and the DCCD binding protein oligomer in E. colt, all other subunits being parts of the stator Whereas the E. colt ATP synthase contains eight different subunits, the bovine enzyme is composed of 16 different subunits 191. The Saccharomyces cerevisiae AI"P synthase is composed of at least 13 different subunits that are involved in the structure of the enzyme (table I). The Fo part contains at least eight different subunits. Thtve of them are mitochondrially encoded (subunits 6, 8 and 9) 1101. The other five are subunit 41111, OSCPII21 subtmit d 1131, subunit h 1141 and subunit f l151. Subunit 4 is a component of the stator and is homologous to the b-subunit of beef heart mitochondria 1161. It contains 209 amino acid residues and has a molecular mass of 23 250 Da. The structure of the eukaryotic b-subunit is composed of two domains: the N-terminal part which is predominantly hydrophobic, and the C-terminal part which is charged and hydrophilic. This subunit is considered to traverse the membrane twice via two hydrophobic
Velours el al.
7~ Tal14eI, Yeast ATP s~,ntha.~ subunits. Subunit Ft
Fo
Associat¢~ proteins
Amino acids
Mass (Da)
get~e
ct I]
5 !2 490
55 394 52 800
ATP1 ATP2
3 3
[441 [45l
y
278 138 61 249 48 76 209 !95 173 95 88 63 63 83 96
30 649 14 555 6612 27 943 5850 7761 23 253 20 874 19 722 I0 567 9953 7384 7287 9455 10 876
ATP3 ATPI6 ATPI5 OLI2 (ATP6) AAP! (ATP8) OLli (ATPg) ATP4 ATP5 ATP7 ATP 17 ATP!4 INH I STFi STF2 TIM I I
I i i
1461 [471 [48~ 1381 I491 150, 511 l! II I 12I [I 31 ! 15] [ 14I 1521 [53 ! 1541 155, 561
su.6 su.8 su.9 su.4 oscp su.d su.f su.h IF1 9 kDa 15 kDa su.e
Stoichiomets3'
I 9-12 I I ! n.d. n.d. I n.d. n.d. n.d.
Ref~,re~z~'e
n.d,, not determined.
stretches of amino acids, with N- and C.terminal parts emerging from the membrane on the Ft side 1171. Like its c o u n t e ~ r t subunit b in E, coll, the euka~),otic b-subunit appears necessary for a tight coupling between proton flux and ATP synthesis 118~231. This study was designed: i) to examine the relationships between OSCP and the hydrophilic Cqerminal part of subunit 4; and ii) m examine the accessibility and the environment of the N-termina! part of the e ukaryotic subunit.
2, M a l ¢ ~
aml methods
APA-Br, DACM and oligomycin we~ p u ~ h a ~ d from Sisma, APDP and ASIB we~ from Pierce, AMDA and MPB were obtained from Molecular Probes. All other chemicals were of reagent grade quality,
2,1, Yeast strains and nucleic acid techniques "rb~ Saccht~romyces ce~visiae strain D273- !0B/A/H/U (MATot met6, ur~3, his3)1181 was the wild type strain. Wild type ~ d mutant strains were obtained after complementation of the deleted-disrupted yeast strain PVYI0 (MATer, met6, urn3, his3, ATP4::URA3) by the low copy shuttle ~ t o r pDRI containing the wild tyt~ ATP4 gene ~ mutated ve~ions of ATP4 gene, respectively 1241, M u ~ t s were n ~ as (wild type residue)(residue num~r)(mutant residue) where the residues are given a single-letter code, The two-hybrid system of Fields and Song | ~ 1 was u ~ , The coding sequences were introd~ into the unique Smal or BamHl sites in plasmids
pGBT9 and pGAD424 which were transformed into the yeast strain SFY526. The I~-galactosidase assay was used to determine the interaction 1261.
2.2. Biochemical procedures Cells were grown aerobically at 28 °C in a complete liquid medium containing 2% lactate as carbon source [27l and harves!ed in logarithmic g~x~wth phase. Mitochondria were prepared according to Lang et al. 1281 and Gu~rin et al. 1291. Protein amounts were determined ~tccording to Lowry et al. 130,1in the presence of 5% SDS. Bovine serum albumin was used as standard protein, The s~eific ATPase activity of the purified F~Fo ATP synthase was measured in the presence el" exogenous phospholipids [141. DACM-labelling, MPB-labelling, AMDA protection and cross-linking experiments were performed as descried in [241, !mmunoprecipitation of the yeast ATP synthase was done according to 1311.
2.3. F~xpression and purification of C-ter4 and OSCP The DNA sequence encoding the C-terminal part of subunit 4 (residues 76 to 209) was cloned into the expression vector pTT-7. One L of liquid medium containing 0. ! mglmL of ampicillin and 0. I mg/mL of kanamycin wcrc inoculated by an overnight culture of the E. coil strain JMI01 containing the pT7-7C-ter4 and the pGPl-2 plasmids 1321. Cell growth was monitored at 30 °C to an A6oo of I. The cells were placed for 3 h at 42 °C and then centrifuged for 5 min at 10 000 g. The cellular pellet was suspended in 6 mL per g wet weight of
Topography of the yeast ATP synthase Fo sector lysis buffer conlaining 50 mM Tris-HCI, 100 mM NaCi, 2 mM EGTA, 2 mM EDTA, |0 mM p-aminobenzamidine, 10 mM e-aminocaproic acid, 0,5 mM PMSE pH 7.5. Cell lysis was performed with lyzozyme ~1 mg/mL, 30 rain at 4 °C) and four sonications of 30 s. DNase was added (10 mg/mL) and incubated for 40 min at room temperature. The inclusion bodies containing C-ter4 were recovered by centfifugation (15 min at 750 g) and washed with lysis buffer. After centrifugation, the pellet was dissolved in the lysis buffer containing 8 M GnCi. The extract was separated by gel permeation chromatography on a Sephadex-G75 column (95 x 2.5 cm) that was eluted in the same buffer at a flow rate of 12 mL/h, and C-ter4 containing fractions were chromatographed by reverse phase HPLC on a Vydak C40.5 mm (0.46 × 25 cm) column. Proteins were eluted with an acetonitrilc gradient in the presence of 0.1% triflu0roacetic acid. OSCP was over-expressed by using the pET3a RV vector in BL21(DE3) E. coil strain. Purilication of the recombinant protein was described previously 1331.22 rng of pure OSCP were obtained from 1 L of culture medium.
2.4. Solubilisation of C-ter4 and interaction of OSCP and C-ter4 Four l.tL of 20 mM Tris-HCl, 1 mM EDTA, 0.001% PMSE pH 7.5, were added to 200 lag of purified C-ter4. Six laL of the same buffet" containing 6 M GnCi were then added to solubilise the sarnple. After 10 rain at room temperature, 10 lxL of the interaction buffet" containing 100 mM Tris-HCI, l m M EDTA, 20% glycerol, 2 mM MgCI,, pH 7.5 were added. After centrifugation (15 rain, 100 000 g), the supernatant was added to 165 l.tL of the interaction buffer containing OSCP or not. The mixture was incubated Ibr I h at room temperature lbr !0 rain at 100 000 g. For cross-linking experiments of C-tcr4 and OSCP by DSP, the Tris bu!'!¢r was replaced by 100 mM HEPES buffer, pH 7.5. Aliquots of 10 M, were incubated for 30 rain and analysed by Western blot.
795 lbllowed by successive dilutions was tested. It resulted in a soluble fraction which chromatographed by molecul~x sieving in the range of 20-10 kDa like subunit OSCP. Incubation of C-ter4 and OSCP led to a new peak located ahead of each subunit. SDS-PAGE analysis of this peak revealed the presence of the OSCP and C-ter4 in a molecular mass range corresponding to the sum of the two partners, thus showing a likely interaction (not shown). Confirmation of the interaction was provided by a crosslinking between OSCP and C-ter4 (figure 1). The bifunctional reagent DSP was used to make a bridge between the two partners. The cationale of this experiment is to maintain the interaction during analysis. Incubation of DSP with C-ter4 probably aggregated or oligomerized the protein which disappeared from the slab gel. OSCP spontaneously dimerized in our experimental conditions by the unique cysteine group. This was reversed under reducing conditions (not shown). DSP incubation of the mixture containing OSCP and C-ter4 led to a new band corresponding to the heterodimer of the two proteins, as shown by the relative molecular mass and by twodimensional electrophoresis. This band was absent when GnC! or SDS were added before cross-linking. The fact that a sharp band was obtained upon cross-linking is in favor of a unique structure of the partners with a limited number of cross-links, which otherwise could lead to a
12
34
5 6 78
9
10
kDa 94-
67-
p ~.~, ~.
4330-
~
o -oscpdimer .- o s c p + C - t e r 4
~
2.5. SDS-PAGE and Western blot analyses SDS-PAGE was perlbrmed as described previously i lll. The slab gel was washed with methanol/ water/acetic acid (5/5/1) for 15 min. Fluorescent bands were photographed under ultraviolet light, and the slab gel was silver-stained1341. Two-dimensional gel electrophoresis was described in 1241. Western blot analyses were described previously 1141. Antibodies raised against subunits 4 and f were used with a i: 10 000 dilution. 3. Results and discussion
3. !. Interaction of OSCP and the C-terminal part of subunit 4 in a preliminary experiment a renaturating method consisting in the solubilization ot" C-ter4 by 6 M GnCI
211--
-- ---
- C-ter4
14"
Figure 1. Cross-linking of subunits OSCP and C-ter4. SDSPAGE of over-expressed OSCP and the C terminal part of subunit 4 incubated with 0.38 mM (lanes 3-5) and 0.9 mM (lanes 6-10) of DSP (see Materials and methods). Lane !, standards; lane 2, OSCP + C-ter4; lanes 3 and 6, C-ter4; lanes 4 and 7, OSCP; lanes 5 and 8, OSCP + C-ter4; lane 9, OSCP + C-ter4 + SDS; lane 10, OSCP + C-ter4 + GnCI. Standard~ were ct-lactalbumin, soybean trypsin inhibitor, carbonic anhydrase, ovalbumin, bovine serum albumin and phosphorylase b.
Velours et al~
7% ~I!. ~ocL
OSCP-C4er4 interaction by the yeast two-hybrid
F~ion proteinsa Gal4-bd + Gal4,ad Cat + Gal4-nd ~ b d + Oal4-ad-OSCP Gat4-bd + Ga14A-ad42ter4 G~4.bd43SCP ~ad~SCP Oal4.ad.C-lel~
~bd.OSCP
+
Gal4-ad-C-ter4
fl.galactosidase activi~, b 0.53 0.46 0.34 0.42 0.21 0.19 0.21 752
e x ~ s i o n plasmids carrying the genes for the indicated fusionpix~(ns were cotran~fected into yeast strain SFY5261251. *'VM~ f~ [3~galactosidaseactivity are the mean of at least two trans~ each assayed at lea~ three dines. The activity is shown according to Miller [261 with a slight modification:the optical density at 420 nm of o~nitrophenoi released by the ~ y m ¢ was normalizedby the proteins measured by the Lowry procedure 1301,
and AMDA labelled su4D54C and to a lesser extent su6C23 (not shown), whereas su4N47C and su4E55C were not accessible (L49C mutant was not tested). This was shown by an increase in the relative molecular mass of subunit 4 (fgure 2(7). AMDA protected su4D54C from MPB-binding (figure 2C, D), thus showing that the two maleimides were competitive towards the sulfhydryi group of Cys54. From these data, we propose that the hydrophilic loop containing the amino acid residues 46 to 56 is directed toward the mitochondrial intermembr~ae space. The highly hydrophilic maleimide AMDA efficiently protected su4D54C from MPB-labelling, thus showing that Cys54 is located in a hydrophilic environment. On the contrary, the absence of MPB-labeling of su4N47C and su4E55C (data not shown) is in favor of a location of these residues in a more hydrophobic environment. As a result, the predicted loop connecting the two membrane-spanning segments should expose only a few
A
broad band. ~o-hybrid experiments with a combination of OSCP fused to Gal4-bd and C-let4 fused to Gal4-ad r e s u l ~ in a strong expression of the reporter gene, thus showing that interaction between OSCP and C-ter4 also o e c u ~ d u n d e r in vivo conditions (tab& !1).
B
wt 47 49 54
55
wt47
49 54 55
3,2, Accessibi!ity of subunit 4 oscl
The location of a loop region (residues 46 to 56) connoting the two Notermina! ~stulated membrane~ ~ n a t n g ~ g m e n t s !16, 351 was investigated by using four eysleine sub~tituti,~n mu~nts, each containing a sole cysteine refidue in subunit 4 at locations 47, 49, 54 and 55 (lhe wild type ~,~nit 4 is devoid of ¢ysteiae ~sidue), !~e m u ~ t s t t a i m 8 f e w wi~ l a c ~ as c a ~ n souse, thus ~owin$ that ~ phosphotylation process was not altered in the cysteine m a ~ t s , Furthermore, the mitochondfial oligomycin-~nsitiv¢ ATPase ~tivifies were not significandy modified (not shown), • :cessibility of the eysteine residues of mutated subun~ 4 was examin~ by t cu~uon of intact mito¢hondfia with the fluorescent maleimide hydrophobic t ~ n t DACM, which forms a stable, s~ngly fluore~ent adduct with thiol g ~ p s [361, ATP syntha~s were extracted, perifi~ by immunoptccipitalion and analysed by $DS-PAGE, Of all the putative DACM-labelled subunits, only subunit 6, su4N47C, su4L49C, su4D54C and ~a4ESGC we~ fluorescent, Figure 2 shows the migration of subunits 4, 6, OSCP and d, As DACM is a hyd~obic ~nt, more ~finit¢ evidence for the Iotalion of ~ hydrophilic loop was p m v i ~ by b i l k i n g sulfhydryi ~ with water-soluble maleimides, Freshly isolated ~ int~t D S ~ mitocholldria were reacted with M P B ~ AMDA, The water-soluble maleimides MPB
MPB AMDA
°°
C * ~
-
*
-
+
+
-
D + -
-
+
+
+
Figure 2, Labellingof subunit4. A, B. SDS-PAGEof wild type control and mutant immunoprecipitatedATP synthases isolated from intact mit~hondfia labelledby DACM. A. Silver-staining. B, Analysis for fluorescenceunder ultravioletlight, wt, wild type control (no mutation).The numbersat the top of the figurerefer to the I~ation of the mutations in subunit 4. C, D. Western blot analyses of D54C immunoprecipitatedATP synthases isolated from intact D54C mitochondria labelled by MPB and AMDA (see Materials and methtxh'). Blots were revealed either with antibody raised against subunit 4 (C) or with streptavidin horse radi~ peroxidase (D).
Topography of the yeast ATP synthase Fo sector
797
residues (at least Cys54) to the aqueous phase. In addition, the fluorescent labelling of tile sole cysteine (Cys23) of mature subunit 6 is also in |avour of an intermembrane space location of the N-terminal part of subunit 6, as proposed previously [37].
3.3. Cro,~'s-linking of sub~nits 4, 6 and f The mutated subunits 4 were used for determining neighboring proteins in Fo. In a preliminary experiment, crude mitochondrial Triton X-lO0 extracts of the four mutants were reacted with ASIB and exposed to UV light. Two cross-link products migrating with relative molecular masses of 33 000 and 46 000 were found in D54C extract, but not in N47C extract. L49C and E55C mitochondrial extracts contained only the 33 kDa product (figure 3A). The identilication of the two neighboring proteins wax undertaken from D54C mitochondria. As subunit 4 has a molecular mass of 23 250 Da, the two neighboring proteins displayed molecular masses of 10 and 23 kDa, respectively. Similar cross-links were obtained by incubation of intact D54C mitochondria with ASIB. Wild type subunit 4 was not modified by the reagent, whereas three additional bands with relative molecular masses of 50 000, 46 000 and 33 000 were recognized by our anti-su4 antibodies in D54C mitochondria (¢igure 3B). A 50 kDa band was detected without incubation with the cross-linking reagent. This band was present only in D54C and E55C mitochondria. It disappeared upon reduction with 2% fl-mercaptoethanol and was insensitive to a 10 mM NEM incubation prior to dissociation (not shown). Experiments are under way to identify this product. Polyclonal antibodies raised against most yeast F, subunits were tested. We found that the 33 kDa product
A v.'" 47
49 54
55
B + + + wT 47 49
+ 54
+ 55
ASIB
was a heterodimer of subunits 4 and f, which is in agreement with the sum of the respective molecular masses. When cross-linking experiments were performed with APA-Br, APDP and AS|B, the band of 46 kDa was obtained with the three reagents whereas subunits 4 and f were not cross-linked by APA-Br which has the shorter arm (9 k,) (not shown). Oxidation of crude D54C mitochondrial Triton X-100 extracts and intact D54C mitochondria by cupric 1-10 phenanthrolinate also gave the immunoreactive band of 46 kDa (figure 4A). This band was present in high amoun!s in the purified D54C F:F o ATP synthase (ligure 4B, lane 1). As anti-OSCP and anti-subunit d antibodies did not react with the 46 kDa cross-linked product, we took the disulfide bond formation between su4D54C and the cysteine residue of another yeast Fo subunit to identify the neighboring subunit. Purified D54C ATP synthase was incubated under oxidative conditions. The intensity of the 46 kDa cross-linked product increased under oxidative conditions and dis~ppeared under reducing conditions (figure 5A). In figure 5B the subunits of a CuCl~-induced cross-linked D54C ATP synthase were separated under non-reducing conditions. The gel was then reduced and submitted to a separating slab gel gradient. Silver-staining of the slab gel clearly revealed that subunits 4 and 6 were linked during the first electrophoresis. Since Cys23 is the sole cysteine residue of subunit 6 [38], the disulfide bridge between su4D54C and su6C23 indicates a close proximity of the hydrophilic loop ol subunit 4 and the N-terminus of subunit 6. Despite the presence of light chain immunoglobulins migrating in the 25-30 kDa range, a 4-6 dimer and a 44 dimer were also evidenced by two-dimensional SDS-PAGE of an ATP synthase immunoprecipitate isolated from a crude D54C
C
-+-+
WTWT5454
wr 5454
- 50 -46 ,~
33-
~'~P"
-" + - -
,..- - 3 3
f
'qi,WmmD
-.. 4"-
-23
kDa
f-
-10
kDa
Figure 3. Crossolinking between sub° units 4 and f. Western blot analysis of mitochondrial proteins (30 l,tg). Yeast mitochondria were incubated with ASIB or not in the dark for 2 h at morn temperature. The reaction was blocked with t~-mercaptoethanol, samples were illuminated at 365 nn~ Ibr 10 rain and dissociated. Blots were reacted with antibodies raised against subunit 4 (A, B) and against subunit f (C). wt, wild type control (no mutation). The number 54 at the top of the figure refers to the location of the mutation in subunit 4,
798
Velours et al.
A 2
B 3
2 3 4
.......
|
A 2
B 3
567
kDa -50
°li9°mer" 6
/'-~ .
I'-~
4÷. t
..
-
~v ~ "|"
W
oso : _ __-
i
°
• ,,,
cp
-23 h~
F ~ r ~ 4 , Cross-links with CuCI~ and APDE A. Western blot analyses of crude D S ~ mitochondrial extracts 130 ~tg) incubated without (lane 1), with 1.5 mM (lane 2) and with 3 mM CuCI: (lane 3), B, Purified D54C ATPa~ syntha~ (3 ~tg) was reacted with APDP (lane 1. control; lane 2, 20 taM; lane 3, 50 ~M; lanes 4 and $, I ~ I~M. Lanes 6 and 7, crude D54C mitochondrial extract wa~ cro~olink~ with 2 ~ t~M APDP. Lanes 5 and 6, 2% ~me~pt~lhanol was added to the samples be!bre electropho~ r~sis, Blots were reacled wilh antise~ raised against suhunit 4,
mil~hondrial exlracl crOssolinked with APDP (//gm~:, 5C). As subunit 6 and su4D54C were cnassolinked when using either intact mit~hondria or 1~iton Xo I ~) c-st.rac'u,,sa. .close ... p~sim~ty of t ~ two subunits in the same comples was likely, ~ i not a cross-link ~ e u ~ n g ~ t w e e n two subunils of two d i f t ~ n t complexes, From these results we conclude that subunits 6 and 4 are in contact, like the c o u n t e ~ subunits a and b of the I~, ¢oli ATP synthase 139, 401, Tbe hydrophobic plot of the yeast subunit 6 predicts a hydrophilic N-terminal part that is longer than that of the t ~ v i ~ counterpart (not shown), and a location of Cys23 at the beginning of the fi~t membraae~spanning segment, As a result, the N-terminal of subunit 6 should be in the intermembrane comparlment which leads to an i n v e ~ location to that of the prokaryotic counterpart subunit a 141 I, We did not find any significant ATPase activity decrease in the D54C purified enzyme having either sponta~sly or induced 6--4 cross, link, Specific ATPase ~tivifies of 49,3 ± 2,2 and 50,5 ± 0,3 pmol of Pi rain ling protein ~ in the ab~nce of oligomycin and 29,5 ± 1,5 and 27,3 ± 2,5 lamol of Pi rain ~ mg protein '~ in the presence of oligomycin for the wild type and D54C purified
A-
.b
~,f
,
C Figure $. Disullide bridge between subunit 6 and su4D54C. A. SDS-PAGE of D54C ATP synthases 18 ltg) incubated without (lane 1) or with !,SmM CuC!2 (lanes 2 and 3). Before dissolution and electmphoresis, 2% [~-mercaptoethanol was added to the sample in lane 3. Tile slab gel was silver-stained. The arrow indicates the 4-6 cmsso!ink product. B, C. qkvodhuenniona! gel elec|ropho~sis of D54C ATP synthasc. B. 18 ttg of puri|icd entyme were incubaled with i.5 mM CuCl~ alld submitted to a 15% SDS~PAGE without i~omercaptoethanol treatment (that dimension from left to righ!). Second dinlension, 13~18% gradient SDSoPAGE° The second dimension was done after incubation of the rod gel with 2% ~-me~apt(~thanol (see M(ller6ds amt metlu,ls). C. A crude D54C mitochondrial Triton X°I00 extract was cross-linked with 200 itM APDP. The ATP synthase was immunoprecipitated and analysed as in (B).
enzymes were measured, res~ctively. As a consequence, subunits 4 and 6 do not display large relative movements during catalysis, which is in agreement with the fact that these two subunits are part of the enzyme stator.
4. C o n c l u s i o n
A representation of a part of Fo is shown in .Figure 6. This scheme derives from the above data. OSCP interacts with the C-terminal part of subunit 4. This result is in agreement with those of Collinson el al. [17] who recently
Topography of the yeast ATP synthase Fo sector reported such an imemction between the hydrophilic part of bovine b°subunit and OSCP In lhe N-terminal part of the subunit, two lransmembrane et-helices are linked by a short hydrophilic segment, The incubation of intact mitochondfia with maleimides having different permeability properties led to the conclusion that subunit 6 and the mutated subunits 4 of the yeast ATP synthase display accessible ta~ets from the inte~xnembrane space. The cross-linking reagents AS1B and APDP with spacer arms of 18,8 and 21 A, respectively [42] linked subunits f and 4 and subunits 4 and 6, but the absence of cross-linking with APA-Br indicates that the sulfhydryl group of su4D54C and subunit f should be at least 9 A apart. Belogmdov et al. [43] reported that dissuccinimidyl mr|rate (cross-linking distance of 6.4 ,/~) produced f-b crosslinks in bovine ATP synlhase preparations but not in submitochondrial particles. We also observed a 1"-4crosslink but only in intact mitochondria and crude mitochondrial Triton X-100 extracts with cysteinyl reagents having arms longer than 9/k. The specificity of the reagents could explain this discrepancy. The orientation of subunit f was taken from [43]. By using the cysteine substitution strategy, recent experiments performed with mutated subunits f were in agreement with the orientation of the bovine subunit f (unpublished data). The three cross-linking reagents bound su4D54C and subunit 6, thus indicating a close proximity of the two subunits. In addition, the disulfide bridge induced by oxidation revealed that Cys54 of subunit 4 and Cys23 of subunit 6 are in contact. As a consequence, the disulfide bridge occurring between su4D54C and su6C23 indicates a similar location of both sullhydryl groups on the outer phase of the inner mitochondrial membrane. The N-terminal part of the eukaryotic subunit 6 is placed in the intermembrane compartment which leads to an inverse location to that ol" the prokaryotic counterparl subunil, We cannol exclude a slight difference between the structure of the cystcineo containing hydrophilic loop and the wild type loop. However, this alteration did not significantly disturb the overall structure of the enzyme, as oxidative phoshoryla° tions were conserved. Thus, a modification of the orien° tation of subunit 4 was excluded, since its C-terminal part is involved in the binding of F~ I181. Therefore, data provided by cysteine residues should reflect the global structure of the wild type hydrophilie loop, and validate the existence of the two postulated membrane-spanning segments linked by a short hydrophilic loop located near the surface of the membrane.
Acknowledgments We are grateful to Dr Guy Lauquin and Dr. David M. Mueller for stimulating discussions. Wc thank Dr. Ray Cooke for his contribution to the editing of the manuscript. This work was supported by the Centre National de la Recherche Scientifique,
799
,ace
coott
i n t e r m e m b r a n e space Figurc6. S¢licmatic represcnlatmn of stibumls 4, {~, I and OSCR
the Ministate de ia Recherche el de l'Enseignement sup~ricur. the Universit6 de Bordeaux II and the Etablissement Public R6gional d'Aquitaine. C.S. holds a research grant from the Minist~re de la Recherche et de la Technologic.
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Velours et al. 141 A ~ J.E, Leslie A.G.W., Lutter R.. Walker J.E.,Structure at 2,8 an~trom re~tuti~ of FoloATPa~ from bovine heart mitt~ c~a. Nmure 37iI (19941 621~628, 151 S ~ D.. Engelbrecht S,. Junge W.. Functional and idling rolatory motion within F- 1-ATPa.~, Prec. Natl. Acad. Sci. USA 94 (19971 440l'-4405. 161 ~ Y,. Dm~a~ T.M., Cross R L Subanit re.ion in Escherichia F O F I - ~ s~ad,~s¢ daring oxidative phosphorylation. Proc. NaIL A-cad. SoL USA 94 (1997) 10583-10587. 17] I ~ i H~, Yasuda R., Yo~hida M., Kino~ita K., Direct observation of tl~ rotation of F l o A ~ Nature 386 (19971 299-30Z Iill ~ P,D~, The ATP s y n ~ - a spkndid molecular machine, Annu. ~ . B i ~ . 66 (1997) 717-749. 191 C~.liastm I.R, Runswick M J.. Buchanan S.K.. Fearnley I.M., Ske,l~l J.M.. V a n R ~ MJ.. G¢iffiths D.E.. Walker J.E.. F-0 m ~ donna of ATP s y n ~ from bo~'in¢ heart mitochondeia: Purification. sabunit composition, and r¢constitution with F-I oATPa~. B i o c ~ s t r y 33 (1994) 7971-7978. II0t Nagley P., F.,ukaryote ~ b r a n e genetics. The Fo sector of mitochondriai ATP symha~e. Trends Genet. 4 (19881 46-52. fill Vetou~ J,.A~,.elin de Chateaubodeau G., Galante M., Gu6rin B.. Sabunit 4 of ATP syntha~ (POFI) from yeast mitochondria. Puri~caticm, amino-acid ~ i t i o n and partial N-terminal sequance, Ear. J, Bio¢l'~m. 164 (19871 579.-584. !12! Uh M.. Jones D.. MueUer DM.. The gone coding fi~r the oli;gomycin ~nsilivity-conferring prolein, J. Biol, Chore. 265 (1990) 19047-190.52, !131 Norris N.. Prom~ D.. Velours J, ATP syntha~ of yeast mitochono dfla, Characterization of sabunit d and sequence analysis of the structural gone ATPT, J, Biol. Chore, 266 (19911 16541~16549, II,ll A ~ ! i n G,, Vaillier J,. Graves P.V, Velours J,, ATP synth~¢ of y ~ mitoeho~fia, l~datio~ of suhunit h and disruption of the ATPI4 ge~, J, Bio!, Chore, 271 ( ! ~ ) 20284~20290, l~_Sl Sl~nnag¢l C,, Wilier J, Ars~lin G,. Gr~ves Per., Velours J., The sub,nit f of mi!~bondfia! yeas! ATP syntha~, Characterization of the p~o~¢in ~d all,up!ion of the stru¢tura! gone ATPt7, Eur~ L Bioeh¢:m, 247 (19~7) !1!!~1!17, It01 W~!ker J,E,, R.u.n~wick, M.J., Paulter L., ATP synthase from bovine n!lt~hondl'ifl, The eh~teri~.ation and ~.quonset, ~na!ysis of t~,~ n~mhrane~as~i~t~ sah:units and of the corresponding ~DNA,' ~ J, Mol, BIoL 197 11987) 89~1(X). 1171 Cotlinsoa I,R,. V~neaaij M J.. Rullgwtek MJ,, Feamley I M.. Skeh~l J,M,, O~ss G,L,, Mirous B,, W~lker J.E.=/~I'P synthase from bovi~ henri mih~hondfla, In vitro as.~emhly of a stalk ~|¢~ in Ih~ t ~ of F t~ATP~ ~nd in it..~absence, J, Mul0 lliol,242 ( ! ~ ) ~ 2 1 , h a l M,F,, G ~ n B,, Velo~ J,, 1 ~ Cotem ~" fmal region of subuail~ (subunit b) is ¢s\~ntial t°ot as~mbly of lh~ Fo l ~ i o n of yea~ m i i ~ f a l ATP synlha~, I~ur, J, Biod~m, ~5 (1992) 10=172, l lgl th~stek J,, K ~ k y J,, 7 ~ x t i F,, Guerrieri E, Jirillo E,, Capo~za G,, ~ S,~ Topological and fut~tionai ch~teri~ation of the F,,! sul~i~ of Ih~ n~n~rane meaty of the mitoehondria! H'~ATP sy~th~, E~, J, Bioc~m, 173 (1988) 1~8, I~1 ~ f i F,, G ~ t F,, C a ~ . ~ G,, Houstek J,, Roach) S., Papa S,, I ~ liit~ ! lk~ of ~u¢leus~¢at~d i~I pt~ein of bovine heart m i l o c ~ a l H*oATPa~ as ~ functiona~ part of Fo moiety, FEBS Left, 237 (1988) 9~14, t211 Ra~k~4olly D,, Rigoulet M,, Gu~rin B., Vdours J,, Mutation in the, hydtophobie domain of ATP syntha~ subunit 4 (subunit b) of ~¢a~ mit~bo~kia disturbs c~pling betw~n proton transloeation a ~ eala!ysis, Bi~hemistr~ 33 11994) 9684~9691, [22t SI¢ffens K,, Seh~i~¢ E., ~k¢~s-Ilct'~sltcit G,, Alten&~f K.. !:,, ! t ~ i ~ of g,w/~t~ich/a co//ATP syatha,~, Furtlvzr resolution of t1~I~in-~r~rat~d l~1~nts fr~n subunit b, J, Biol, Chem, 262
t231 Take)~a~ M,, N~mi I",, Maoda M,, Fatal M,, Fo potlion of ~ ' ~ ¢ ~ / ~ ~'a/i H'-ATPa~, Carboxyl-terminal ~egi~ of the b sub~aaa is ~.~nlial f~" a,~mhly of functi~al F,., J, Biol. Chore. 2f~ (11~) 161t~-lff112.
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80t 151R Hensgens, L.A.M., Gri~cll, I,.A., Borst P., Bos LL.. Nucteo~ide sequence of the mitochondriai structural gone ft~r subunit 9 of yeast ATPase complex, Pr~c. Natl. Acad. Sci. USA 76 (1979} 1663-1667. [52] lchikawa N., Yoshida Y., Hasbimoto T., Ogasawara N.. Yoshika~a H., [mamoto E, Tagawa K., Activation of ATP hydrolysis by an uncoupler in mutant mitochondria lacking an intrinsic ATPase inhibitor in yeast, J. Biol. Chem. 265 (1990) 6274-6278. [53] Akashi A., Yoshida Y., Nakagoshi H., Kuroki K., Hashimoto T., Tagawa K,, Imamoto E, Molecular cloning and expression of a gene for a factor which stabilizes formation of inhibitormitochondrial ATPase complex from Saccharomyces cerevisiae, J. Biochem. (Tokyo) 104 (1988) 526-530. 154] Yoshida Y., Sato T., Hashimoto T., Ichikawa N., Nakai S., Yoshikawa H., Imamoto E, Tagawa K., Isolation of a gene for a regulatory 15-kDa subunit of mitochondrial FjFo-ATPase and construction of mutant yeast lacking the protein, Eur, J. Biochem. 192 ( ! 990) 49-53. [551 Tokatlidis K., Junne T., Moes S., Schatz G., Glick B.S., Kronidou N., Translocalion arrest of an intramitochondrial sorting signal next to Tim ll at the inner-membrane import site, Nature 384 (1996) 585-588. 156] Arnold I., Bauer M.F., Brunner M., Neupert W., Stuart R.A.. Yeast mitochondrial FIF,-ATPase: the novel subunit e is identical to Timll, FEBS l,ell. 411 (1997) 195-20.