Eur. J. Biochem. 205, 163-172 (1992)

0 FEBS 1992

The C-terminal region of subunit 4 (subunit b) is essential for assembly of the Fo portion of yeast mitochondria1 ATP synthase Marie-Franqoise PAUL, Bernard GUERIN and Jean VELOURS Institut de Biochimie Cellulaire du Centre National de la Recherche Scientifique, Universitk de Bordeaux 11, France (Received September 30, 1991) - EJB 91 1304

The role of the C-terminal part of yeast ATP synthase subunit 4 (subunit b) in the assembly of the whole enzyme was studied by using nonsense mutants generated by site-directed mutagenesis. The removal of at least the last 10 amino-acid residues promoted mutants which were unable to grow with glycerol or lactate as carbon source. These mutants were devoid of subunit 4 and of another Fo subunit, the mitochondrially encoded subunit 6. The removal of the last eight amino-acid residues promoted a temperature-sensitive mutant (PVY161). At 37 "C this strain showed the same phenotype as above. When grown at permissive temperature (30°C) with lactate as carbon source, PVY161 and the wild-type strain both displayed the same generation time and growth yield. Furthermore, the two strains showed identical cellular respiration rates at 30°C and 37°C. However, in vitro the ATP hydrolysis of PVY161 mitochondria exhibited a low sensitivity to Fo inhibitors, while ATP synthesis displayed the same oligomycin sensitivity as wild-type mitochondria. It is concluded that, in this mutant, the assembly of the truncated subunit 4 in PVY161 ATP synthase is thermosensitive and that, once a functional Fo is formed, it is stable. On the other hand, the removal of the last eight amino-acid residues promoted in vitro a proton leak between the site of action of oligomycin and F1.

FIFOATP synthase is responsible for the synthesis of ATP from ADP and Pi at the expense of the proton chemical gradient generated by the respiratory chain. In mitochondria the complex is located in the inner membrane and is composed of two domains. F, (peripheral portion of ATP synthase) is the catalytic sector and Fo (integral membrane portion of ATP synthase) is involved in proton translocation. The Fo domain of eucaryotes is rather complex, since it contains not only the three subunits of Escherichia coli Fo (a, b, c), designated subunits 6, b (or F01 or 4) and 9 (dicyclohexylcarbodiimidebinding protein, respectively, but in addition, subunits d, OSCP (oligomycin-sensitivity-conferringprotein), A6L (or 8) and F6 (for review see Senior, 1988). Owing to thiscomplexity, the Fo sector of eucaryotes has received less attention than the procaryotic enzyme. Since yeast is a microorganism with which genetic and molecular biology is easily applied, this eucaryote now appears as a model for understanding the assembly and function of the subunits composing the complex. The Fo part of Saccharomyces cerevisiae contains at least six different subunits whose primary structure is known. Three of them are mitochondrially encoded and are hydrophobic proteins (subunits 6, 8 and 9) (Nagley, 1988). The other three are nuclear-encoded and termed subunits 4 (Velours, et al., 3988), OSCP (Uh et al., 1990) and subunit d (Norais et al., 1991). Subunit 4 is homologous to the E. coli b subunit

(Velours et al., 1988) and to the b subunit of beef heart mitochondria (Walker et al., 1987). From proteolysis experiments of F1-depleted inverted membranes of E. coli (Hoppe et al., 1983) and labeling with the hydrophobic probe trifluorophenyldiazirine (Hoppe et al., 1984), it was suggested that this protein is embedded in the membrane by the N-terminus and that the hydrophilic C-terminus is in contact with F,. In mammalian mitochondria, Houstek et al. (1988) and Zanotti et al. (1988) have shown by proteolytic cleavage and reconstitution experiments that subunit b is involved in proton conduction through Fo and in the sensitivity of oligomycin. The counterpart of the E. coli b subunit in yeast, subunit 4, is involved at least in the assembly of the complex, since disruption of the ATP4 gene promotes a mutant with an F1 part loosely linked to the Fosector and whose mitochondrially encoded subunit 6 is not integrated in the complex (Paul et a]., 1989). Since subunit 4 is apparently heavily involved in the assembly of the complex, we have introduced stop codons towards the 3' end of the ATP4 gene by using site-directed mutagenesis, with a view to determining the role of the Cterminal part of the subunit in assembly. The consequences of the mutations in the assembly and function of ATP synthase are reported here.

Correspondence to J. Velours, Institut de Biochimie Cellulairc du CNRS, 1, rue Camille Saint Saens, F-33077 Bordeaux Cedex, France Ahhreviafions. (cHxN)*C, dicyclohexylcarbodiimide;CCCP, carbony1 cyanidc m-chlorophenylhydrazone;Fo and F1, integral membrane and peripheral portions of ATP synthase; OSCP, oligomycinsensitivity-conferring protein; Et3SnCI, triethyl tin chloride. E n z ~ mATP ~ . synthase or ATP phosphohydrolase (EC 3.6.1.34).

Bacterial strains and plasmids

MATERIALS AND METHODS The bacterial strain JMlOl was grown in DYT medium (1.6% tryptone, 1% yeast extract, 0.5% NaC1) containing, when appropriate, 50 pg ampicillin/ml; it was transformed by the CaClz procedure (Maniatis et al., 1982). The yeast vector shuttle YRp7-14 was the ATP4 gene source (Velours et al.,

164 Table 1. Yeast strains used in this study.

HP E

I

Name D273-10B/A/U/H PVY 10 PVY162 PVY 154 PVY 174 PVY 168 PVY 160 PVY161

Genotype and other information MATu, ntet, uru3, his3 MATa, ntet, ura3, his3, A T P I : :U R A 3 PVYlO [pRS313-ATP4] PVY 10 [pRS313-atp4-183end] PVY 10 [pRS313-atp4-196end] PVY 10 [pRS313-atp4-200end] PVY 10 [pRS313-atp4-202end] MATa, met, ura3, h i d , utp4-202end

B

E

I

X

H

X

H

1 X &IH

E0 B

Yeast strains and procedures

The S. cerevisiur strain D273-10B/A/U/H (MATa, met, ura3, lris3) was derived from S. cerevisiae D273-10B/A (MATu, met) by ultraviolet mutagenesis, nystatin selection (Shaffer Littlewood, 1975) and crossing with the rho- isogenic strain F98-3C [MATa, his, (Ery‘, Oligo‘, Diuron l‘)] (a gift from Dr F. Foury). Rapid screening of rho- colonies was done on agar plates according to McEwen et al. (1985). The yeast strain PVYlO (MATa, met, his3, uru3, utp4: : URA3) was transformed either with pRS313 bearing the ATP4 gene or with the linear fragment containing the whole ATP4 gene. Table 1 summarizes the yeast strains used in this study. Nucleic acid techniques

Published procedures were used for transforming cells of yeast (Ito et al., 1983) or E. coli JM 101 (Maniatis et al., 1982), for isolating yeast DNA (Hoffman and Winston, 1987), and for small-scale isolations of plasmids from E. coli (Crouse et al., 1983). Commercially available restriction enzymes and other DNA-modifying enzymes were used as advised by the suppliers. The strain PVYlO was constructed by integrative transforination (Orr-Weaver et al., 1981) with a linear piece of DNA containing the U R A 3 gene flanked on one side by the 5‘ region 28 bp upstream of the initiating ATG, and on the other side by the 3’ region of the ATP4 gene. In a previous paper, we have constructed the plasmid pS4-13 which contained a truncated version of the ATP4 gene (Paul et al., 1989). The 3’ missing end of the ATP4 gene was isolated from the double-stranded form of M13 tg 130 containing a Hind111 - BumHI fragment 1660 bp long (Fig. 1) which was used previously for sequencing the 3’ region of the gene (Velours et al., 1988). This fragment was ligated to pS4-13 cleaved by HindlII. After transformation of E. coli JM101, plasmids were isolated and cut with BnmH1, which released a fragment 2400 bp long. The

E

B

S

ATP4

Baa

2520

1988). pFL38 (a gift from Dr Francois Lacroute) provided the URA3 gene. This version of the URA3 gene is flanked by &/I1 sites instead of initial Hind111 sites, and its PstI site was removed by the exonuclease activity of the DNA polymerase. Constructions were performed in a pUC8 whose EcoRI site was removed as above. Construction of plasmid pS4-13 containing 80% of the ATP4 gene was described by Paul et al. (1989). The shuttle vector pRS313 (Sikorski and Hieter, 1989) was used for transformation of the yeast strain PVY10. The E. coli strain BMH 71-18 mutS [ A (lac-proAB), thi, supE; FlucP, Z A M15,p r o A + B + ,mutS215: :TnlO] was provided by Boehringer Mannheim and used for mutagenesis experiments.

SBg

I

ATP4

~Jv3

S

bp

Fig. 1. Maps of the ATP4 gene region and of inserts borne by plasmids pJV3 and pJV4. pJV3 contained a 1770-bp insert. The restriction site B is part of the multi-clonIng site of the plasmid. pJV4 contained a 2520-bp insert. The 1170-bp U R A J fragment was ligated to pJV3 after deletion of the 420-bp EcoRI-XhoI fragment. The resulting 2520-bp BurnHI-Sol1 fragment was used to disrupt the yeast nuclear gene ATP4 of the strain D273-10 B/A/U/H. E” and Bg” correspond to EcoRI and &/I1 restriction sites destroyed after ligation of blunt ends. H p = Hpall, E = EcoRI, X = XhoI, H = HindIII, S = SuIl, Bg = &/[I, B = BUMHI.

Sun site on the 3’ side of the fragment allowed reduction of the fragment up to 1770 bp. This construction, which contained the whole ATP4 gene, was confirmed by sequencing. The resulting plasmid pJV3 was cut with EcoRI ( - 34) and XlzoI ( +387). The blunt-ended Bgfll U R A 3 fragment 1170 bp long was ligated to the dephosphorylated blunt-ended plasmid. The resulting plasmid pJV4 (Fig. 1) was cut with BamHI and SulI and the 2520-bp fragment was used to transform the strain D273-3OB/A/U/H. Transformants (PVY 10 strain) were selected on minimal medium supplemented with methionine and histidine and 2% glucose as carbon source. Site-directed mutagenesis was performed by using the double-primer method (Zoller and Smith, 1987). The 1770-bp BumHI - SulI fragment containing the wild-type ATP4 gene was inserted into the polylinker of the bacteriophage MI 3 tg 131. Single-stranded template DNA was prepared and used for directed mutagenesis by using the mutagenic oligonucleotide and the ‘universal’ 15-mer MI 3 sequencing primer. Repair of the mutation was prevented by the use of the E. coli strain BMH 71-18 mutS. Mutants were detected by hybridization with the radiolabeled mutagenic oligonucleotide. Mutation was confirmed by DNA sequencing of the region containing the target sequence. Sequencing of the entire gene was also performed with the use of four primers: 5‘AAAGACTGACGAGAATT-3’ (- 46 to - 30), S’XTCTATCATCAATGCCA-3’ (153 to 169), 5’-GCCGATGCAAGAATGAA-3’ (346 to 362), 5’-AATTAGCTCACGAAGCA3’ (539 to 555). The replicative form DNA carrying the mutation was cut with BumHI and SulI and the 1770-bp BurnHI- Sull fragment was inserted into the shuttle vector pRS313. The resulting plasmid bearing the yeast marker HIS3 was used to transform the PVY 10 ycast strain. Transformants were selected on minimal medium containing methionine and glucose as carbon source. They were assayed in a complete medium containing 2% lactate or 2% glycerol.

Biochemical preparations and analyses

Yeast growth was described previously (Arselin de Chateaubodeau et al., 1976). Mitochondria were prepared

165 Table 2. Oligonucleotide-directed mutagencsis of ATP4 gene and growth of mutants with lactate as carbon course. (+) Indicates growth, (-) no growth. Parcntheses in the oligonucleotides indicate mupation. Glu383-1end (etc.) indicates protein ends at Glu183. Mutation

Synthetic oligonucleotides

None Lys73 +Glu

5'-AGTGGCA(G)AGTATTT-3'

Growth at

____

17°C

30°C

37°C

+ +

+

+

+ +

+

+

+

Asp82-1 Gly

5'-TTTTGCCG(G)TGCAAG-3'

Argl59+Lys Argl59-1Thr Argl59+Ilc

5'-GTGGGTTA C ATATGAAG-3'

+ + +

+ + +

+ + +

Argl65+His Argl65 +Pro Arg165-1 Leu

5'-CTTCCTTGC C TCAATTGG-3'

+ + +

+ + +

+ + +

Glul83-1Lys Glul83m1d

5'-GTTCAGTCA A AATTGGGT-3' (T)

+-

+-

+

Lysl92+Arg Lysl92+Ile

5'-CCAAGAGA G AGTTTTGC-3' (TI

+ +

+ +

+ +

Glnl96-Glu Glnl96+end

5'-GTTTTGCAA G AGTCTATA-3' (T)

+-

-

-

(%I I:(

+

-

+

Glu200 +Ly s Glu200+Gln Glu200+end

5'-AGTCTATATCT C AAATTGAACAA-3'

+ +-

+ +-

+ +-

Glu202 -1Gln Glu202+end

5'-CTGAAATT C AACAATTG-3' (T)

+ +

+ +

+

Lys209 Aend

5'-CTAAATTG(T)AGTAATCA-3'

+

+

+

0

from protoplasts according to GuCrin et al. (1979). The ATP synthase was immunoprecipitated with F1 antiserum as described by Todd et al. (1980). Extraction of Fo subunits was performed as in Paul et al. (1989). Protein amounts were determined with the method of Lowry et al. (1951) in the presence of SDS. Bovine serum albumin was used as standard protein. SDSjPAGE was performed as previously described (Velours et al., 1987). The silver-staining method of Ansorge (1983) was used. ATPase activity was measured at basic pH (8.4) according to Somlo and Krupa (1974). ATP synthesis and oxygen consumption rates were measured as described by Rigoulet and Guerin (1979).

-

-

RESULTS Previous results have shown that disruption of ATP4 gene causes the absence of an oligomycin-sensitive ATPase activity (Paul et al., 1989). It was postulated that subunit 4 is involved in the biogenesis of the whole complex since the absence of subunit 4 led to the lack of integration of the mitochondrially encoded subunit 6. The mutant PVYlO was constructed by the method of deletion-disruption in order to produce a true null mutant (see Methods). The PVYlO strain had exactly the same phenotype as the disrupted PVY6 strain containing a selective marker in the middle of the ATP4 gene (Paul et al., 1989). Moreover, Southern analysis of the genomic DNA of the PVYlO strain confirmed the integration of the URA3 gene

166

Fig. 2. SDS/PAGE of ATP synthase immunoprecipitates and crude extracts of Fo subunits. ATP synthase immunoprecipitates were obtained from 2 mg mitochondrial proteins (see Methods): wild type (lane 1). PVYlO(1ane 2), PVY160 (lane 3 ) , PVY154(lane4), PVY174 (lane 5). Crude extracts of subunits 6 , 8 and 9 (from 0.2 mg mitochondrial protein): wild type (lane 6) PVYlO (lane 7), PVY160 (lane 8), PVY 154 (lane 9), PVY 174 (lane 10). Crude extracts of subunit 4 (from 0.2 mg mitochondrial protein): PVY162 (lane l l ) , PVYlO (lane 12), PVY 160 (lane 13). PVY 154 (lane 14), PVY 174 (lanc 15). The stars indicate thc location of the truncated subunit 4 (lanes 3 and 13); su = subunit.

at the ATP4 locus. On the other hand, the PVYlO strain did not complement with the disrupted PVY6 strain of the opposite mating type (not shown). Phenotypic analyses of mutants bearing mutated ATP4 gene on a plasmid The PVYlO strain was used as a recipient strain for complementation studies with plasmids bearing mutated forms of the ATP4 gene. Table 2 shows oligonucleotides used for construction of mutations. The effect of mutations on cell growth with lactate as carbon source and at three different temperatures is reported. Modification of conserved amino acids such as Lys71, Argl59, Arg165 and Lys192 were without any effect on growth with an oxidative substrate, showing that the complex was not modified and that these amino acids were not involved in a structural relationship. Another strategy was to define the role of the C-terminal part of the protein in the assembly of the complex. Introduction of stop codons in the 3' region of the ATP4 gene revealed

that mutants containing a subunit 4 deleted by at least the last 10 amino-acid residues (PVY154, PVYl74 and PVY 168) were unable to grow on lactate (Table 2). The mutation removing residues C-terminal to Glu202 (Glu2024end) resulted in the temperature-sensitive strain PVYl60 which was unable to grow on lactate at 37"C, but this mutant, bearing a deletion of the last eight amino-acid residues of subunit 4, was still able to grow on lactate at the permissive temperature of 30'C. ATP synthase was isolated from a mitochondrial Triton X-100 extract by immunoprecipitation with a rabbit antiserum directed against the F1 sector. Provided that the complex is stable, this polyclonal antibody, although directed against F1, is able to immunoprecipitate the whole ATP synthase. ATP synthase immunoprecipitates and extracts of Fo subunits were performed from mitochondria isolated from cells grown on 2% galactose. SDSjPAGE revealed the presence of Fl subunits in each ATP synthase immunoprecipitate, but Fo subunits were visible for the wild-type (Fig. 2, lane 1) and only slightly visible for the mutant Glu2024end (PVY 160) (Fig. 2, lane 3). Immunoglobulins were also detected under the p subunit and between they subunit and subunit 4. The presence of the small hydrophobic proteins of the Fo sector were not detected in ATP synthase immunoprecipitates, since subunit 8 was very poorly stained and subunit 9 migrated as an oligomer in the high-molecular-mass range (Velours et al., 1987). However these two subunits were detected easily after either labeling of proteins in uivo in the presence of cycloheximide or after extraction of mitochondria with organic solvents (Paul et al., 1989). Thus, analysis of mitochondrial organic extracts revealed the lack of subunit 6 in PVY154, PVY174 strains (Fig. 2, lanes 9 and 10) as in PVYlO strain (lane 7). Analysis of crude subunit 4 extracts revealed the presence of subunit 4 in strain PVY162 (a PVYlO strain complemented by pRS313 bearing the wild copy of the ATP4 gene) (Fig. 2, lane 11). The crude PVY160 mitochondrial extract contained a protein migrating at 22 kDa (lane 13). This protein was also found in the PVY160 ATP synthase immunoprecipitate (lane 3). Western blotting analysis confirmed that this band was the truncated form of subunit 4 (not shown). PVY 10, PVYl54 and PVY174 mitochondrial extracts did not contain any truncated form of subunit 4 (lanes 12, 14 and 15). The low amount of Fo subunits associated to F1 in immunoprecipitates of PVY 160 mitochondrial extracts suggested that this mutant could display a weak ATP synthase. In order to obviate problems of expression of the mutated ATP4 gene borne by the shuttle vector, the altered ATP4 gene was integrated at its locus by using the homologous transformation method, giving rise to strain PVY 161. Integration was confirmed by Southern blot analysis of genomic DNA of PVY161 strain and by the recovery of uracil auxotrophy (not shown). Comparative study of wild-type and PVY 161 strains Table 3 shows similar generation times for the two strains at 30'C. At 37'C, only the wild-type strain grew with lactate as carbon source. With galactose as substrate the PVY161 strain grew, but the final growth yield was half that of the wild type at 37'C, indicating a defect in the oxidative metabolism of PVY161 strain at this temperature. The effect of temperature was studied in uitro. To preclude any effect due to catabolic repression, lactate was used as carbon source for growth of cells at 30 C. Table 4 shows the same inhibition of the cellular respiration rate upon addition of the Fo inhibitor Et,SnCl (Cain and Griffith, 1977), and the

167 Table 3. Growth of wild type and PVY161 strains at 30'C and 37'C. Cells ofthe wild-type (D273-10B/A/U/H) and PVYl61 strains were grown on complete medium containing 2% lactate or 2% galactose as carbon sources. Turbidimetry was measured at 600 nm for estimation of cell concentrations after appropriate dilutions. The ratio dry mass/A600nm was the same whatever the strain studied. Strain

Lactate

Wild type PVY 161

Galactose

gencration time -__ 30-c 31°C

stationary phase

min 160 160

Asoo

_

_

_

30°C

_

_

37°C

~

generation time

stationary phase

30 "C

30°C

37°C

min

370 -

15 -

150 150

31 C

A 600

150 150

20 20

20 10

Table 4. Respiration of yeast cells at either 3OoC or 37'C. Cells were grown at 30 "C on a complete medium containing 2% lactate as carbon source, harvested during exponential growth, washed and assayed as described in Materials and Methods. Et,SnCI concentration was 80 pM. CCCP concentration was 5 pM. ~

Inhibitor

~~

30' c

37°C

wild type

PVY161

wild type

PVY161

nmol 0 . min-' . mg protein-' None +htJSnCl + Et $SnCI CCCP

+

122 I 1 3 69 +_ 5

111 1 1 69 .t 5

224 f 13 130 +_ 15

214 f 5 140 & 15

174

153 .t 15

251

256 f

9

25

9

Same stimulation of the Et,SnCl-inhibited respiration rate by the uncoupler CCCP (carbonyl cyanide m-chlorophenylhydrazone) when cells were assayed at 30 "C or 37 "C.This result indicates the samc behaviour of the Fo sectors of the two strains at either 30°C or 31°C towards the Fo inhibitor in our experimental conditions. Mitochondria were prepared from cells grown at permissive temperature with lactate as carbon source. ATP synthase immunoprecipitates isolated from fresh PVY 162 mitochondria clearly displayed the following Fo subunits: subunit 6, truncated subunit 4, OSCP and subunit d (Fig. 3, lanes 2 and 3). We observed that frozen and thawed PVY161 mitochondria gave a very low yield of coprecipitating Fo subunits (lane 4). This result reflects instability of the PVY163 ATP synthase in vitro since this observation was not made from ATP synthase immunoprecipitate isolated from wild-type frozen and thawed mitochondria (Fig. 3, lane 1). The oligomycinsensitive ATPase activities of the two strains were measured from frozen and thawed mitochondria. A 2 - 3-fold increase in the specific activity of the wild-type and PVYl61 mitochondria was observed, but only the mutant showed a threefold increase in oligomycin-insentive ATPase activity (Table 5). The 2 - 3-fold increase in the specific ATPase activity for the two strains could correspond to the release of the F1 inhibitor, which may be equivalent to the heat-activation step of the ATPase activity described by Ryrie (1975). The ATPase stability of the two strains was measured after either hypotonic treatment or hypotonic treatment followed by sonication. Table 6 shows a slight difference (7%) in the release of mutant and wild-type ATPase activities. It was shown previously that, in these latter conditions, PVY6 mito-

Fig. 3. SDSlPAGE of ATP synthase immunoprecipitates and crude extracts of subunit 4. ATP synthase immunoprecipitates prepwed from 2 mg mitochondria1 protein of frozen and thawed mitochondria : wild typc (lane I), PVY161 (lane 4) and from fresh PVYl61 mitochoridria (lanes 2 and 3). Crude extracts of subunit 4 (from 0.2 mg mitochondrial protein): wild type (lanc 5 ) and PVY161 (lane 6). The star indicates thc location of the truncated subunit 4 (lanes 2, 3 , 4 and 6; su = subunit.

chondria which were devoid of subunits 4 and 6 did not contain any membranous ATPase activity (Paul et al., 1989), just like PVYlO mitochondria (not shown). The effect of temperature on ATP synthase assembly was examined in vivo. ATP synthase immunoprecipitates of the wild-type and PVYl61 strains were prepared from cells grown

Table 5. ATPase activity of mitochondria grown on 2% lactate as carbon source. Mitochondria were isolated from cells grown at 3 0 ' C on complete medium containing 2 % lactate as carbon source. Assays were performed at 30'C with addition of 8.5 pg oligomycin/mg protein where indicated. Measurements were performed in triplicatc. Mitochondria strain

ATPase activity no addition

oligomycin

Table 6. ATPase activity after hypotonic treatment and sonication. Mitochondria were isolated from the wild-type and PVYl6l strains grown at 30;C on complete medium containing 2% lactate as carbon source. 3 mg mitochondria1 protein was suspended in 1 ml 10 mM TrislHCl, 0.5 mM EGTA pH 7.5 and sonicated where indicated. The suspension was centrifuged at 100000 x g Tor 15 min. Thc pellet and the supernatant werc recovered and assayed at 30 C for ATPase activity. Measurements were madc in triplicate.

remaining Strain

Fresh wild type PVY161 Frozen and thawed wild typc PVY 161

pmol min-' . mg protein-'

%

2.176 0.027 2.645 k 0.059

0.585 1.148

0.083 0.068

27 43

6.075 k 0.109 5.284 0.140

0.621 f 0.055 3.421 f 0.162

10 65

+

Activity - sonication

+ sonication

pellet

supernatant

pellet

supernatant

0 7+1

70+1 63+4

29k1 37k1

YO

D273-10B/A/'H/U (wild typc) PVY161

100 94+3

Fig. 4. SDS/PAGE of ATP synthase immunoprecipitates and crude extracts of F, subunits prepared from mitochondria isolated from cells grown at either 3OoC or 37OC. ATP synthase immunoprecipitates prepared from the wild-type strain grown at either 30 C (lane 1) or 37 C (lanes 2 and 11) and from PVY 161 strain grown at either 30°C (lanes 3 and 12) or 37°C (lane 4). Crude extracts of subunits 6. 8 and 9 preparcd from the wild-typc strain grown at cither 30°C (lanc 5 ) or 37-C (lanes 6 and 13) and from thc PVY161 strain grown at either 30 .C (lane 7) or 37 C (lane 14). Crude extracts of subunit 4 prepared from thc wild-type strain grown at either 30'C (lane 8) or 37'C (lanes 9 and 15) and from the PVY161 strain grown at either 30LC (lane 10) or 37°C (lane 16). The stars indicate thc location of the truncatcd subunit 4 (lanes 3, 10 and 12); su = subunit.

169 Table 7. ATPase activity of mitochondria isolated from cells grown at either 3OoC or 37OC with 2% galactose as carbon source. Mitochondria were isolated from the wild-type and PVY161 strains grown at either 30 C or 37'C. Assays were performed at 30°C with thc addition of 8 pg oligomycin/mg protein where indicatcd. Mcasurcments were performcd in triplicate. Wild-type and PVY161 strains grown at 37°C with galactose as carbon source contained 64% and 73% of rhocells, rcspectively. Strain

Temper- ATPasc activity aturc _ _ _ _ _ _ ~ _ _ _ _ _ _ no addition oligomycin rcmaining pmol . min - ' . mg protein - '

C

.~

30

Wild typc PVY161 PVYl0 Wild type PVYl61

____

1.832 f 0.049 1.673 0.085 1.703 f 0.143 1.672 0.076 2.102 f 0.141

37

___

0.454 0.057 1.221 f 0.108 1.446 k 0.028 0.971 +_ 0.063 2.018 f 0.102

%

0

24 73 85 58 96

1

2

pg -

4

3

oligomycin

2

5

I

I

K

-*

.-K

I-

E

I 0

J

(

6

mg protein

I

I

1

2

patomg 0 / min / mg protein

02

0

5

pg

.-cQI c 2n

10

oligomycin

15

20

25

mg protein

5,

1

\

Fig. 6. Measurement of ATP synthesis during titration of ATP synthase by oligomycin. ATP synthesis (JATP) was measured at 27°C in the prcsence of increasing amounts of oligomycin (A). Mitochondria (0.1 mg protein) were suspended in 2.5 m10.65 M mannitol, 0.36 mM EGTA. 3 mM T r i ~ / [ ~ ~ P ] p h o s p h(6.18 a t c MBq/mmol), 10 mM Tris/ maleate pH 6.7 supplemented with 4 m M NADH. ATP synthesis was initiated by addition of 1 mM ADP. ATP formation rate was cstimated by 3'Pi incorporation into adeninc nucleotides after acid extraction or 200-pI aliquots withdrawn at different times. Measurcments were performed in triplicatc. Mitochondria1 oxygen consumption was measured simultaneously with a Clark oxygen electrode (Gilson) connected to a microcomputer giving an on-line display of thc ratc value (B). Oxygen consumption rates in non-phosphorylating state were 0.658 f 0.023 and 0.855 +_ 0.097 pmol 0 min- mg protein ' Tor wild-type and PVY161 mitochondria respectively (mean of Iivc experiments). (B-B) Wild type mitochondria; (0- -1) PVY161 mitochondria. ~

-0 E

%

I 0

1 5

pg

10

15

20

25

oligomycin / mg protein

Fig. 5. ATPase activity versus oligomycin concentration. ATPase reaction was performed for 30 s with 5 mM ATP in the presence of incrcnsing amounts of oligomycin; the ratc of production of inorganic

phosphate/mass protcin was measured colorometrically. Measurements werc made in triplicatc. (A) Wild-type mitochondria; (B) PVY161 mitochondria. (M---B) 30°C; (U---n) 37°C.

with galactose as carbon source at either 30°C or 37°C. Antibody anti-F1 was not able to coprecipitate Fo subunits from mitochondria1 Triton X-100 cxtract of PVY161 cells grown at 37 C (Fig. 4, lane 4). Furthermore, extraction of Fo subunits rcvealed the absence of subunits 6 and 4 in the mutant mitochondria (Fig. 4, lanes 14 and 16 rcspectively) only when the

cells were grown at 37°C. These results suggest a deficiency in the assembly of the complex during growth at 37°C; this was confirmed by measurements of oligomycin-sensitive ATPase activities. Mitochondria isolated from the PVY 161 strain grown at 37 "C displayed no oligomycin-sensitive ATPase activity (Table 7). The 58% oligomycin insensitivity of wild-type ATPase was attributed to the high amount of rho- cells which appeared spontaneously in the culture at this temperature. The oligomycin-sensitive ATPase activity of the wild-type and PVY161 mitochondria, isolated from cells grown with lactate at 30"C, was measured at 30°C and 3 7 T as a function of inhibitor concentration. Fig. 5 shows a twofold increase in the specific ATPase activity for the two strains between 30 37°C. The percentage of oligomycin insensitivity was increased in PVY161 mitochondria (49% and 62% at 30°C and

170 37 C, respectively, instead of 15% for the wild-type mitochondria at either 30'C or 37'C). The low oligomycin-sensitive ATPase activity (40- 50%) of strain PVY161 was also found with other Fo inhibitors such as ( C H X N ) ~(dicyclohexylcarC bodiimide), Et3SnC1and venturicidin at concentrations which fully inhibited wild-type ATPase activity (not shown). ATP synthesis was not measured at 37°C since wild-type and PVY 161 mitochondria were uncoupled at this temperature. The rate of ATP synthesis was measured at permissive temperature as a function of oligomycin concentration. Fig. 6 A shows a similar linear relationship with mitochondria isolated either from the wild-type or from PVY161 strains. Extrapolation of the two lines gave a full inhibition of ATP synthesis at 7.3 pg oligomycin/mg protein for both strains. This result favors a similar population of ATP synthase for both strains when considering ATP synthesis. However, plots of the ATP synthesis rate versus the oxygen consumption rate during titration of state 3 by oligomycin, gave two linear relationships whose slopes were an indication of the ATPjO ratios (Fig. 6B). Slopes were 1.92 for the wild-type mitochondria and 1.32 for the mutant mitochondria, indicating a lower yield of the mutant ATP synthase in vitro. The two lines crossed the x axis at a similar value for an oxygen consumption rate of 0.6 pmol 0 min-' mg protein-'. This value is very close to the mean oxygen consumption rate of the wild-type mitochondria (0.658 pmol 0 min- mg protein- ') in the absence of ADP (non-phosphorylating state), while PVY161 mitochondria displayed a mean value of 0.855 p m o l 0 min-' mg protein - '.

DISCUSSION Subunit 4 of the yeast S . cerevisiae is an amphiphilic protein which contains 209 amino-acid residues and whose predicted molecular mass is 23 250 Da (Velours et al., 1988). Like other b subunits, it displays a predominently hydrophobic Nterminal part and a C-terminal region which is highly charged and hydrophilic. It has been suggested that this protein occupies a central position providing an important structural link between the membrane and F1 (Walker et al., 1982). The fact that the missence mutations reported in this paper were without effect is not surprising since b-subunit sequences are only weakly related when comparing subunits of E. coli, chloroplast, cyanobacteria (Cozens and Walker, 1987), Rodospirillum rubrum (Falk and Walker, 1988), Thermophilic bacterium PS3 (Ohta et al., 1988) and mitochondria (Walker et al., 1987; Velours et al., 1988). Only two missense mutations affecting the E. coli b subunit have been reported so far (Jans et al., 1985; Porter et al., 1985) despite efforts in a number of laboratories. These results suggest that only a few amino-acid rcsidues in the b subunit are essential for ATPase function. Construction of nonsense mutants could provide data on the assembly of yeast ATP synthase. Previously, Paul et al. (1989) showed that subunit 4 is involved in the assembly of the yeast Fo sector, since disruption of the ATP4 gene caused lack of integration of subunit 6, one of the main subunits of the Fo sector. The role of the carboxyl-terminal region of subunit 4 in the assembly of the complex was studied in this paper with mutants having shorter versions of subunit 4. Thermosensitivity of PVY 161 Fo sector assembly Deletion of the last ten amino-acid residues of subunit 4 prevented growth of mutant cells with a carbon source

utilizing the aerobic pathway. Deletion of the last eight aminoacid residues resulted in the mutant strain PVY161 which was able to grow at 17°C and 30"C, but not at 37'C. The temperature-sensitive mutant PVY 161 was a useful tool for determining the role of the carboxyl-terminal region of subunit 4 in assembly and function of the subunit. PVY161 and the wild-type strains were indistinguishable in vivo when grown at 30°C from either lactate or galactose, but the PVY161 strain did not grow with lactate as carbon source at 37 "C. With galactose (a carbon source utilizing the glycolytic pathway but with a low catabolic repression), the PVY161 strain did grow, but the cellular yield was half that of the wild type at 37"C, which favors a defect in the oxidative metabolism ofethanol produced during galactose metabolism. This defect is a consequence of a modification of the ATP synthase caused by the mutation, since the electrophoretic analyses of ATP synthase immunoprecipitates and crude subunit extracts showed the absence of subunits 4 and 6 in mitochondria isolated from PVY161 cells grown at 37 C. As a consequence the temperature-sensitive mutant was unable to build an effective Fo sector at 37 'C. Presumably, the truncated subunit 4 disappeared by proteolysis just like subunit 6 in the absence of subunit 4 (Paul et al., 1989). The amount of rhocells which appeared during the culture at 37 ,C on a fermentable substrate depended on the number of generations for both strains. In these experiments, 73% of PVY 161 cells were double mutant (rho- and atpl Glu202+end) and 64% of the wild-type cells were rho- which affected the oligomycinsensitive ATPase activity for both mitochondria (the ATPase of rho- cells is oligomycin-insensitive). However the 27% remaining rho' PVYl61 cells displayed no oligomycin-sensitive ATPase activity since ATPase activity (measured on the mixture of mutant and double-mutant mitochondria) was fully insensitive to the Fo inhibitor. As a result, at 37 'C the PVY161 strain displayed the same phenotype as the disrupted strains PVYl0 and PVY6 (Paul et al., 1989). Cellular respiration rates of PVY 161 and wild-type strains grown at 30°C on non-fermentable carbon source and measured at either 30'C or 37°C were identical and the responses to the Fo inhibitor Et,SnCI were the same, indicating that once functional Fo is formed at 30'C it is stable at least throughout the experiment at 37°C. All these results are in favor of a temperature-sensitive assembly of the truncated subunit 4 inside the complex.

ATP synthase of PVY161 strain exhibits a proton leak in vitro In vitro experiments showed an alteration of the Fo sector of PVY 161 mitochondria prepared from cells grown at 30 C with lactate as carbon source. Freezing and thawing increased the oligomycin-insensitive ATPase activity of PVYl61 mitochondria, thus indicating a decrease in the linkage between the two sectors, leading to a leak of H' outside the H' pathway through Fo. Moreover, the link between the two sectors was disrupted upon immunoprecipitation with antiF1 antibodies. The action of polyclonal anti-F, antibodies on the stability of the complex was previously discussed by Hadikusumo et al. (1984). It should be noted that hypotonic treatment of fresh PVY161 mitochondria resulted in a small release of soluble F, ATPase. The difference in stability with the wild-type strain (7%) was not increased upon sonication, which indicates that the complex was rather stable. However, this small difference could explain the low oligomycin-sensitive ATPase activity of the temperature-sensitive mutant. In contrast, ATP syntheses

171 of mitochondria prepared from PVY161 and wild-type strains were inhibited to the same degree by oligomycin. ATP hydrolysis and ATP synthesis rates were not measured in the same conditions, since yeast mitochondria do not hydrolyze ATP at neutral p H (Ezzahid et al., 1986). Thus the low oligomycin-sensitive ATPase activity of PVY161 mitochondria might be due to experimental conditions at basic pH. However, in phosphorylation conditions (at neutral pH) the mutant mitochondria displayed a non-phosphorylating state higher than that of the wild-type mitochondria, which is in favor of an H leakage through the mutant ATP synthase. Consequently, the efficiency of the two ATP synthases were different, since in vitro and in our experimental conditions a higher oxygen consumption rate was measured to build ATP from PVY161 mitochondria. Since the oxygen consumption rate (state 3) of the mutant mitochondria was higher than that of the wild-type mitochondria, and since full inhibition of state 3 by oligomycin led to the same oxygen consumption rate for either mitochondria, a n H + leak is postulated beyond the site of action of oligomycin when considering the H + flux occurring during ATP synthesis (i. e. towards FJ However, such an H + leak might result in vivo in a loss of energy giving rise to a lower cellular yield for the PVY161 strain. Careful studies of growth yields with limiting amounts of lactate were similar in both strains (not shown). Therefore, the alteration of ATP synthase observed in vitro could be the consequence of an amplification of the mutation effect by the experimental conditions of either the analyses o r the preparation of organelles. +

Deletion of the last eight amino-acid residues would remove most of the CI helix, which therefore would contain only four amino-acid residues. We suppose that this structure is important for cohesion either of subunit 4 itself o r of subunit 4 with other subunits of the complex. Experiments will be conducted by site-directed mutagenesis to test this hypothesis. We thank Dr M. Rigoulet for advice and discussion during thc course of this work and Dr Cooke for linguistic help. This work was supported by the Centre National de la Recherche Scientifique, the Universitk de Bordeaux I I and the Etablissement Public Regional d'Aquitaine. M. F. P.holds a research grant from the Ministere de la Recherche et de la Technologie.

REFERENCES Ansorge, W. (1983) in Electrophoresis, 82 (Stathakos, D., ed.) pp. 235 -242, Walter de Gruyter, Berlin. Arselin de Chateaubodeau, G., Gukin, M. & Guerin, 9. (1976) Biochimie (Paris) 58, 601 -610.

Cain, K . & Griffith, D. E. (1977) Biochem. J . 162, 575-580. Cozens, A. L. &Walker, J. E. (1987) J . Mol. Biol. 194, 359-383. Crouse, G. F., Frischauf, A. & Lehrach; H. (1983) Methods Enzymol. 101, 78-89.

Ezzahid, Z . , Rigoulet, M. & Guerin, B. (1986) J . Gen. Microhiol. 132, 1153-1158.

Falk, G. & Walker, J. E. (1988) Biochem. J . 254, 109-122. Gaboriaud, C., Bissery, V., Benchetrit, T. & Mornon, J. P. (1987) FEBS Lett. 224, 149-155.

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Conclusions

GuCrin, B., Labbe, P. & Somlo, M. (1979) Methods Enzymol. 55,

Removal by proteolysis of the C-terminal part of the E. coli b subunit prevents rebinding of F1 without modifying the passive H + pathway (Hoppe et al., 1983). However, Takeyama

et al. (1988) have shown that the loss of the carboxyl residue results in reduction of both F1 binding and H+translocation. Thus, the carboxyl region of the E. coli b subunit is required for the assembly of the amino-terminal region of the b subunit with the a and c subunits. However, once functional Fo is formed, the carboxyl-terminal region of the b subunit is not required for maintaining the H + pathway. The main result in this paper is the role of the carboxyl-terminal region of yeast subunit 4 in the assembly of the subunit into the complex, in the integration of the mitochondrially encoded subunit h and finally in the assembly of the whole complex. The study of the temperature-sensitive mutant devoid of the last eight aminoacid residues showed that, once a functional Fo is formed at 30 "C, it is stable in vivo at 37 "C; as a consequence, temperature interferes in assembly of the truncated subunit 4 with other components of ATP synthase. Although observed only in vitro, the low oligomycin-sensitive ATPase activity of the temperature-sensitive mutant might be considered as a n argument in favor of the role of the carboxyl-terminal region of subunit 4 in maintaining a n effective H + pathway in eucaryotes. This is in agreement with the results of Zanotti et al. (1988) and Guerrieri et al. (1989) who have shown by using proteolytic cleavages that this subunit is essential for H + conductivity in the ATP synthase and for the inhibition by oligomycin of H conduction in mitochondria1 Fo. Prediction methods proposed an CI helix for the region He198 to Lys209 (Gamier et al., 1978; Gaboriaud et al., 1987) of yeast subunit 4. This CI helix is probably amphiphilic. The hydrophobic side of the helix could be shielded from the aqueous phase by contact with other hydrophobic surfaces. +

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