Molecular Basis for Resistance to Myxothiazol, Mucidin (Strobilurin A)

THE JOURNAL OF BIOLOGICAL CHEMISTRY 8 1989 by The American Society for Biochemistry and Molecular Biology. Inc.

Val. 264, No 24 , Issue of August 25, pp. 14543-14548,1989 Printed In US.,‘.

Molecular Basis for Resistance to Myxothiazol, Mucidin (Strobilurin A), and Stigmatellin CYTOCHROME b INHIBITORS ACTING AT THE CENTER o OF THE MITOCHONDRIAL UBIQUINOL-CYTOCHROME c REDUCTASE IN SACCHAROMYCES CEREVZSIAE*

(Received for publication, January 23, 1989)

Jean-Paul di RagoS, Jean-Yves Coppee, and Anne-Marie Colsons From the University of Louuain, Laboratoire de Genetique Microbienne, Place Croix du Sud, 4, B-1348 Louuain-la-Neuue, Belgium

The respiratory bcl complex transfers the electrons from ubiquinol to cytochrome c oxidase. Myxothiazol, strobilurin A (mucidin), and stigmatellin are center o inhibitors preventing electron transfer at the ubiqui- none redox site Qo, which is located closer to the outer side of the inner mitochondrial membrane. The cytochrome b gene is carried by the organelle DNA. Yeast mutants resistant to myxothiazol and mucidin have been previously isolated and mapped to specific loci of the cytochrome b gene.

In the present work, stigmatellin-resistant mutants were isolated and genetically analyzed. The mutated amino acid residues from seven myxothiazol-, four mucidin-, and six stigmatellin-resistant mutants have been identified by sequencing the relevant segments of the resistant cytochrome b gene. A third myxothiazol- resistant locus and the first stigmatellin-resistant locus were identified. The mutated codons were found to be clustered in two regions of the cytochrome b protein which appeared to be responsible for the resistance to Qo site inhibitors. The first region is within the end of the first, the second, and the beginning of the third exon whereas the second region is within exon five and the beginning of the sixth exon.

The mitochondrial respiratory chain transfers electrons to oxygen and drives proton uptake and proton release across the inner mitochondrial membrane. In the bcl complex, a transmembrane electron circuit is thought to be responsible for this proton displacement (1). Cytochrome b, a transmem- branous protein which carries two hemes, bS6* and b566, plays a central role in this electron circuit (2). Hemes b562 and b566 are presumably located nearer the inner or outer sides of the membrane, respectively (3-5). Two classes of bc, inhibitors can be distinguished on the basis of their site of action in this electron cycle. Inhibitors acting downstream of b562 (inner side) are called center i inhibitors and include antimycin, funiculosin, and diuron whereas inhibitors acting upstream of b566 (outer side) are called center o inhibitors including myxo-

* This work was supported in part by IC1 Agrochemicals (Imperial Chemical Industries, Great Britain) and by the National Fund for Scientific Research (Belgium). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ Supported by the Belgian Institut pour 1’Encouragement de la Recherche Scientifique dans 1’Industrie et YAgriculture fellowship.

§ Research Associate to the National Fund for Scientific Research, Belgium.

thiazol, strobilurin A, and stigmatellin (6-8). Among center o inhibitors, strobilurin A was found to have an identical structure to mucidin (4). Center i and center o are formed by the cytochrome b itself or by its interaction with other proteins of the bc, complex such as the Rieske iron-sulfur protein and the cytochrome c1 (1, 2, 9).

Numerous center i and center o inhibitor-resistant mutants have been isolated during the past decade, and their mutations have been mapped on the cytochrome b gene (10-19). The genetic map of the inhibitor-resistant mutations revealed the probable location of each mutation within the split cytochrome b gene. Taking into account the mosaic organization of the yeast cytochrome b gene, four exons (exon 1, exon 4, exon 5, and exon 6) were found to carry the inhibitor-resistant mutations. The first exon carries both center i and center o inhibitor-resistant mutations. Exon four seems to carry center i inhibitor-resistant mutations only whereas exons 5 and 6 carry center o inhibitor-resistant mutations.

By sequencing mutant cytochrome bs, the mutated codons which express a resistance phenotype to center i inhibitors were found to be clustered in two regions of the gene (22,23). The first region was located near the NHz-terminal end of the cytochrome b protein; the second region was situated at about 30 amino acids distant from the conserved His-197, a presumed heme attachment site (24). An eight-transmembrane helices model has been recently proposed for cytochrome b (25-27). In this model, the two center i inhibitor- resistant regions appeared to belong to the presumed inner side (negative side) of the membrane.

In the present work, the sequencing of mutant cytochrome bs leading to center o inhibitor resistance has also been performed. The DNA base substitutions in seven myxothiazol-resistant mutants, four mucidin (strobilurin A)-resistant mutants, and six stigmatellin-resistant mutants are presented. Selection genetic and phenotypic properties of new stigmatellin-resistant mutants are also shown. Two center o inhibitor-resistant regions were also identified. The first region was found to belong to the end of the first exon, the second exon, and the beginning of the third exon whereas the second region was found located in the fifth exon and the beginning of the sixth exon.

In the eight helices model (25-27), the resulting location of the two center o regions is the presumed outer side (positive side) of the membrane.

MATERIALS AND METHODS

Strains-Parental strains: D6, 01 arg met; GM50-3C, a trp2 hisl; D225-5A, 01 adel lysZ KL14-4A, a hisl trp&; D273-10B/A21, LY ne t . Myxothiazol-resistant mutants (14): D6/10 myxl-10; GM50-3C/103

14543

14544 Mynothiatol, Mucidin, and Stigmatellin Resistance S. cerevisiae myxl-103; GM50-3C/118 myx1-118; D6/31 myx2-31; GM50-3C/119 myx3-11.9; GM50-3C/124 myx2-124; D6/184 myx2-184. Mucidin- resistant mutants (16,17): D225-5A/3D mucl-771; K50 a ural muc3- K.50; D225-5A/101 muc2-772; K16 muc2-Kl6. Stigmatellin-resistant mutants (this work): KL14-4A/ stil-1; KL14-4A/ stil-2; KL14-4A/ stil-3; KL14-4A/ stil-4; KL14-4A/ stil-5; KL14-4A/ stil-6. Rho- mutants: D273-10B/A21/Rll; D273-10B/A21/FR91; D273-10B/ A21/F101. The retained segments of the COB-BOXgene in each rho- mutant are approximately from codon 1 to 384 for R11, from codon 31 to codon 282 for F101, and from codon 252 to codon 282 for FR91.’ Tester strains: D273-10B/A21 diul-724 (22); D225-5A/3A mucl- 771 (this paper); D225-5A/101 muc2-772 (this paper); K50 muc3- K50 (this paper).

Inhibitors-Stigmatellin and mucidin were kindly provided to us by Dr. G. Hofle (28) (Gesellschaft fur Biotechnologische Forschung, Braunschweig, Germany) and Dr. J. Subik (Food Research Institute, Bratislava, Tchecoslovaquia), respectively.

Isolation and Genetic Analyses of the Stigmatellin-resistant Mu- tants-Freshly grown single colonies derived from the sensitive parental strain KL14-4A were plated individually on glycerol (yeast extract 1%, glycerol 3%, agar 1.5%) containing2.5 pg/ml stigmatellin. After 10-15 days of incubation a t 28 “C, few resistant clones per plated colony did appear on the selective medium. Only one resistant clone per plated colony was retained. Six independent stigmatellin- resistant mutants were further analyzed.

Genetic analyses were performed as described in Ref. 18. Prepa- ration of the mitochondrial membranes and respiratory measure- ments was performed as previously described (29).

Mitochondrial RNA isolation and RNA sequencing were performed as previously described (23).

Primers-The primers were synthesized by Dr. Xavier Perea Centre de Genetique Moleculaire du Centre National de la Recherche Scientifique, Gif-sur-Yvette, France. They were numbered from P1 to P6. The primers P1-P5 were utilized in Refs. 22 and 23. Primers P1-P3 were used for the sequencing of exon 1, exon 2, and the beginning of exon 3. Primer P6 was used for the sequencing of exon 5 and the beginning of exon 6. The positions of the primers are given on the basis of their first and last base pair numbers on the cytochrome b split gene.

P1,109-128: 3”CCAAATACAAATCAATAAGT-5’ P2,279-298: 3”CAAATACGTATACCCGATTTC-5’ P3,2636-2655: 3’-GGTAAACATCCATTGCTATA-5’ P6, 6886-6905: 3’-CTCAATAAGATTACAAACGT-5’

RESULTS

Myxothiazol- and mucidin-resistant mutations have been previously mapped within the split cytochrome b gene (13- 19). Two myxothiazol-resistant loci ( M Y X I , M Y X 2 ) were previously allocated to exons 1 and 6 of the gene (Fig. 1). Three mucidin-resistant loci (MUCI, MUC2, MUC3) were found in exons 1,6, and 5, respectively.

Stigmatellin-resistant mutants were selected on glycerol medium in the presence of 2.5 pg/ml stigmatellin, and six independently isolated mutants were further analyzed. Stig- matellin resistance was found to be increased by a factor of 2 in. vivo and in vitro. The Iso values (concentration in pg/ml stigmatellin necessary to decrease by 50% the growth yield or the rate of NADH oxidation) were found to be 0.038 in these mutants and 0.018 in the wild type strain). All mutants presented an impaired growth on glycerol. The generation time on different substrates was measured for mutant sti-5 and its parental strain KL14-4A. Although the two strains had an identical generation time when grown on glucose, the mutant growth rate was, however, lowered relative to that of the wild type by a factor of 2 on lactate, of 2.5 on ethanol, and of 3 on glycerol.

The rate of oxygen consumption was measured on isolated mitochondrial membrane preparations from the sti-5 mutant and its parental strain in the presence of increasing concen-

D. Meunier, P. P. Slonimski, and J.-P. di Rago, unpublished results.

41 5 15 75 2 5 2 54 3 5 1

4

E l E2 E3 E4 E 5 E6 - n - n ” n m MUCI rnucl-771

MUC3 MUCZ muc3-K50 rnuc2-77.2

rnuc.2-K16

M Y H l MYH3 MYHZ

myK! - ! (?3 myK2-184

rnyxr-10

rnyx1-!!8 rnyx.2-31

m y x 3 - i 19 rnyx2-I24

ST1 i

stil-2 s t i l - I

s t i l -3 s l i l -4 s t i i -5 si i l -6

FIG. 1. Genetic location in the cytochrome b gene of Q. inhibitor-resistant mutations. The exons are numbered from El to E6. The length of each exon is given in base pairs. The genetic loci for myxothiazol resistance are M Y X I , M Y X 2 , and M Y X 3 (Ref. 14 and this work), those for mucidin resistance are MUCl, MUC2, and MUC3 (15-17), and that for stigmatellin resistance is STZl (this work). The names of the corresponding sequenced individual mutations are listed under each inhibitor-resistant locus.

trations of stigmatellin. The oxygen consumption of sti-5 was found to be only 77% of that observed for its parental strain. These results explain the observation of the slower growth rate of sti-5 mutant on nonfermentable substrate. In vivo cross-resistance of the stigmatellin-resistant mutant sti-5 to myxothiazol mucidin, antimycin, and diuron was tested. The sti-5 mutant did not show cross-resistance to myxothiazol or mucidin nor to diuron or antimycin.

The genetic mapping of six stigmatellin-resistant mutants using discriminating rho- mutants in the cytochrome b region of the mitochondrial DNA has been performed as described in Ref. 18. It revealed that all six mutations were within the cytochrome b gene. Indeed, in crosses between the six sti mutants and a rho mutant R11 (see “Materials and Methods”) carrying the wild type cytochrome b gene, sensitive recombinants were found at a frequency fluctuating between 30 and 45%, indicating the presence of the wild type allele of the sti mutations in the cytochrome b gene. In a second set of crosses, four sti mutants were tested with a rho- mutant FR91 (see “Materials and Methods”) carrying only a small segment of the cytochrome b gene, mainly the fourth intron, the fifth exon, and a portion of the sixth exon. No stigmatellin-sensitive recombinants were obtained indicating that the sensitive alleles of the sti mutants were not within the cytochrome b gene region which includes MYX2,MYX3 and MUC2,MUC3. The rho- mutant FlOl which lacks introns 1, 2, and 3 is known to carry a segment of the cytochrome b gene which includes the end of exon 1 from codon 131 carrying the MYXl locus, exons 2, 3, 4, 5 , and a portion of the sixth exon. In crosses between sti mutants and rho- F101, the frequency of sensitive recombinants was significantly lower suggesting that the mutated allele might be located close to the end of the retained segment. The 3’ end of the retained segment, corresponding to exons 5 and 6, does not seem to be involved since it has already been shown that the sti mutations do not belong to that region. Allelism tests between sti mutants and diul- 724 (codon 225 in exon 4), mucf-771 (codon 137 in exon 11, muc2-772 (codon 275 in exon 6), and muc3-K50 (codon 256 in exon 5 ) showed a reduced frequency of recombinants only in crosses with muc1-771 indicating an absence of linkage

Myxothiazol, Mucidin, and Stigmatellin Resistance S. cerevisiae 14545

with MUC2 and MUC3 and suggesting that the sti mutations are close to the MUCl locus. Thus, the mutation seems to be located in the vicinity of the end of the first exon around codon 131 and near MYXl.

The alterations in the mutant cytochrome b genes leading to Qo inhibitor resistance are shown in Table I. The sequencing gels are shown in Fig. 2. Mutational alterations were identified as single or double point mutations leading to missense codons. Both transitions and transversions were observed. The sequences exhibited almost no polymorphisms. By identifying the mutated codons, it was possible to deduce according to the mitochondrial genetic code (30) the nature and position of the amino acids which are involved in the resistance mechanism.

Four amino acid positions are implicated in the myxothiazol resistance. (i) Phenylalanine 129 which is changed into a leucine and (ii) glycine 137 becoming an arginine both belong to exon I. Because of their close proximity, they were not distinguished in the recombination analysis. (iii) At position leucine 275, myxothiazol resistance can be caused by three different amino acid substitutions (to serine, phenylalanine, or threonine) and (iv) in mutant myx-119 an asparagine 256, in the fifth exon, becomes a tyrosine. I t is, therefore, consid-

TABLE I Mutations in cytochrome b gene responsible

for resistance to center o inhibitors The mutations have been allocated to specific exons (El, exon 1;

E3, exon 3; E5, exon 5; and E6, exon 6) of the cytochrome b split gene (Fig. 1). The positions of the deduced amino acid changes in a cvtochrome b 8-a-helices model are Dresented in Fie. 4.

Exons Mutations Mutated codons A ~ ~ ~ ~ ~ i d Positions

E l myxl-10 UUU UUA Phe Leu 129 E l myxl-103 UUU UUA Phe Leu 129 E l myxl-118 GGA AGA Gly Arg 137 E l mucl-771 GGA AGA Gly Arg 137 E3 stil-1 AUU UUU Ile Phe 147 E3 stil-2 AUU UUU Ile Phe 147 E3 stil-3 AUU UUU Ile Phe 147 E3 stil-4 AUU UUU Ile Phe 147 E3 stil-5 AUU UUU Ile Phe 147 E3 stil-6 AUU UUU Ile Phe 147 E5 myx3-119 AAC UAC Asn Tyr 256 E5 muc3-K50 AAC UAC Asn Tyr 256 E6 myx2-124 UUA UUU Leu Phe 275 E6 myx2-184 UUA CUA Leu Thr 275 E6 myx2-31 UUA UCA Leu Ser 275 E6 muc2-772 UUA UCA Leu Ser 275 E6 muc2-Kl6 UAC AAC Tyr Asn 214

UUA UUU Leu Phe 215

A T

FIG. 2. Sequencing gels from wild type and inhibitor-resistant cytochrome b genes. G, guanine; A, adenine; T, thymine; C, cytosine; *, mutated base. Myxothiazol resistance results in a change from T to C in myx2-31 and from A to T in myx2-124.

ered as a third myxothiazol-resistant locus MYX3 (Fig. 1). Four amino acid positions were identified as responsible for

mucidin resistance. One mutation affects, as in myxothiazol resistance, glycine 137, which is converted into an arginine. Similarly, asparagine 256 is changed into a tyrosine in the mucidin-resistant mutant, muc3-K50, as well as in the myxothiazol-resistant mutant myx.3-119. Leucine 275 is also changed into a serine in the mucidin-resistant mutant, muc2- 772, as had been found in the myxothiazol-resistant mutant, myx2-31. So far, no mutation in codon 129 was found among the mucidin-resistant mutants. The mutant myxl-10 (codon 129), however, presents a cross-resistance to mucidin.

Thus, in three identical positions (137, 256, and 275), myxothiazol and mucidin resistances result from the same base change leading to the same amino acid change. Most mutants carry single base pair change except muc2-K16, which exhibits a double change in two adjacent codons corresponding to two distinct amino acids, tyrosine 274 and leucine 275. Myxothiazol and mucidin resistances result pri- marily from the same changes a t identical amino acid positions, and myxothiazol resistance can be expressed in leucine 275 by a single change into phenylalanine. Both inhibitors belong to the methoxyacrylate family of inhibitors known to carry a methoxy group (47). This partial structural similarity between the two inhibitors and the identity of the amino acid changes in the resistant mutants might be considered as arguments in favor of a partially common inhibitory site for myxothiazol and mucidin (strobilurin A) on the cytochrome b.

It is most likely that mucidin resistance in the double mutant muc2-Kl6 is due to the change from leucine 275 into phenylalanine. The single change from the adjacent tyrosine 274 into asparagine has not yet been observed in a mucidin- resistant mutant. Tyrosine 274 is a 100% conserved amino acid in a highly conserved region of the gene. It is rather striking, therefore, that this double mutational change did not lead to respiratory deficiency. Moreover, the double mutational change found in two diuron-resistant mutants (22, 23) and one mucidin-resistant mutant may have a distinctive effect on the spectral properties of the bcl complex compared with that resulting from single mutational changes.

The six sti mutants carry an alteration which changes isoleucine 147 to phenylalanine. Isoleucine 147 (exon 3) is close to glycine 137 (exon 1) which is changed into myxothiazol- and mucidin-resistant mutants. The amino acid segment between phenylalanine 129, glycine 137, and isoleucine 147 seems to be representative of a center o inhibitor-resistant region of the cytochrome b protein. Isoleucine 147 belongs to the third exon of the gene. A loss (at phenylalanine 129) or an acquisition (at isoleucine 147 and asparagine 256) of an aromatic amino acid seems to cause the resistance; no charge modification is observed since the mutated amino acids re- main nonpolar or hydrophobic or with uncharged polar groups. Structural and charge modifications, however, were found in position 137 coincident with myxothiazol or mucidin resistance where the polar uncharged glycine is changed into a positively charged arginine.

Myxothiazol-resistant mutations do not express a cross- resistance to stigmatellin (14). Stigmatellin-resistant mutations at isoleucine 147 do not express a cross-resistance to myxothiazol. This presumably reflects structural differences in stigmatellin, which, by contrast with myxothiazol, interacts both with cytochrome b and the Rieske iron-sulfur protein (48).

Changes affect mostly preferentially used codons in yeast mitochondria (30). In all positions the occurrence of alterna-

14546 Myxothiazol, Mucidin, and Stigmatellin Resistance S. cerevisiae

tive changes would have produced amino acids with other structural or charge properties or should be derived from codons, absent or rarely used in the mitochondria. The selection of resistant mutants seems to favor particular structural or charge modifications in relation to the amino acid position.

Prokaryotic and eukaryotic cytochrome bs derived from respiratory and photosynthetic organisms have been compared. The amino acid alignments of the regions carrying myxothiazol-, mucidin-, and stigmatellin-resistant mutations are presented in Fig. 3. Phenylalanine 129, glycine 137, isoleucine 147, and asparagine 256 are conserved among 15 respiratory organisms whereas leucine 275 is found only in yeast, Neurospora crassa, and Aspergillus. It is striking that myxothiazol resistance at leucine 275 results in a change to phenylalanine which is already present in most aligned cytochrome bs including chloroplast cytochrome b6 (Fig. 3). The mutated amino acids belong also to two regions of the protein which are composed of highly conserved amino acids. The conserved nature of the mutated amino acids leading to resistance to center o inhibitors as well as the highly conserved nature of the two center o inhibitor-resistant regions suggest that these regions are essential for cytochrome b activity. A partial impaired growth on nonfermentable substrate of all mutants of this class provides experimental evidence in favor of this hypothesis.

From the present results, it is clear that myxothiazol-, mucidin-(strobilurin A), and stigmatellin-resistant mutations are clustered in only two center o inhibitor-resistant regions

S Mu

1 2 5 My 135 My 140 Y: I A T A F L G Y C C V Y G Q M S H W G A T V I T N L F S Nc: M A T A F L G Y V L P Y G Q M S L W G A T V I T N L I S

PC: M G T A F M G Y V L P W G Q M S F W G A T V I T G L F G A: M A T A F L G Y V L P Y G Q M S L W G A T V I T N L M S

Rc: M G T A F M G Y V L P W G Q M S F W G A T V I T G L F G B: M A T A F M G Y V L P W G Q M S F W G A T V I T N L L S

M: M A T A F M G Y V L P W G Q M S F W G A T V I T N L L S H : M A T A F M G Y V L P W G Q M S F W G A T V I T N L L S

RA: M A T A F M G Y V L P W G Q M S F W G A T V I T N L L S XL: M A T A F V G Y V L P W G Q M S F W G A T V I T N L L S

DR: M G T A F M G Y V L P W G Q M S F W G A T V I T N L L S T: M A T A F V G Y V L P W G Q M S F W G A T V I T N L L S

MA: I V T A F I G Y V P P W G Q M S F W G A T V I T S L A S Wh: I V T A F I G Y V P P W G Q M S F W G A T V I T S L A S O e : I V T A F I G Y V L P W G Q M S F W G A T V I T S L A S Lw: V S F G V T G Y S L P W D Q I G Y W A V K I V T G V P E Tb: A S F G V T G Y S L P W D Q V G Y W A V K I V T G V P D S p : A S F G V T G Y S L P W D Q I G Y W A V K I V T G V P D

150

MU 252 My 260

MU

Y: G Q P D N Y I P G N P L V T P A S I V P E W Y L L P F Y A I 270 MY 280

N c : G D S E N Y I M A N P M Q T P P A I V P E W Y L L P F Y A I A: G D S E N Y V M A N P M Q T P P A I V P E W Y L L P F Y A I P c : G H P D N Y I F A N P L V T P A H I V P E W Y F L P F Y A I R c : G H P D N Y I Q A N P L S T P A H I V P E W Y F L P F Y A I

H : G D P D N Y T L A N P L N T P P H I K P E W Y F L F A Y T I B: G D P D N Y T P A N P L N T P P H I K P E W Y F L F A Y A I

M: G D P D N Y M P A N P L N T P P H I K P E W Y F L F A Y A I R A : G D P D N Y T P A N P L N T P P H I K P E W Y F L F A Y A I X L : G D P D N P T P A N P L I T P P H I K P E W Y F L F A Y A I

D R : G D P D N F I P A N P L V T P A H I Q P E W Y F L F A Y A I T: G D P D N F T P A N P L I T P P H I K P E W Y F L F A Y A I

M A : G H P D N Y I P A N P M P T P P H I V P E W Y F L P I H A I W h : G H P D N Y I P A N P M P T P P H I V P E W Y F L P I H A I 0 e : G H P D N Y I P A N P M S T P P H I V P E W Y F L P I H A I L w : E P S M I G E P A N P F A T P L E I L P E W Y F F P V F Q I T b : E P S M I G E P A D P F A T P L E I L P E W Y F F P V F Q I S p : E P S M I G E P A D P F A T P L E I L P E W Y F F P V F Q I P e a : E P S M I G E P A D P F A T P L E I L P E W Y F F P V F Q I

FIG. 3. Amino acid alignments of two cytochrome b ( b ~ + subunit IV in chloroplasts) regions around positions 129 and 256 from 19 species. Y, yeast (20, 21); B, bovine (20); H, human (20); M , mouse (20); RA, rat (31); XL, Xenopus laeuis (32); T, toad (32); DR, Drosophila (33); Nc, N . crassa (34, 35); A, Aspergillus (36); MA, maize (37); Wh, wheat (38); Oe, Oenothera (39); PC, Paracoccus denitrificans (40); Rc, R. capsulatus (41); Lw, liverwort (42); Tb, tobacco (43); S p , spinach (44); pea (45). The sequences around amino acid 256 from liverwort, tobacco, spinach, and pea chloroplasts belong to subunit IV. Letters in boldface represent mutated amino acid positions in inhibitor-resistant yeast strains (My, myxothiazol resistance; Mu, mucidin resistance; S, stigmatellin resistance).

of the apocytochrome b, from amino acid positions 129 to 147 and from amino acid positions 256 to 275.

DISCUSSION

Seven myxothiazol-, four mucidin- (strobilurin A), and six stigmatellin-resistant mutations in the cytochrome b gene of Saccharomyces cereuisiae have been analyzed. The myxothiazol- and mucidin-resistant mutant cytochrome bs were sequenced whereas the stigmatellin-resistant mutants were selected, characterized, and sequenced.

The advantages of the sequencing method used in this work have been stressed previously (23).

The sequencing results presented here are in perfect agree- ment with the genetic mapping and provide a more precise localization for these mutations. In addition, a new myxothiazol-resistant locus was identified in exon 5, MYX3 , which is allelic to MUC3.

On the basis of the present results, the mutated amino acids responsible for the resistance to center o-type inhibitors (myxothiazol, strobilurin A (mucidin), stigmatellin) are clustered in only two regions of the cytochrome b apoprotein. The first region is located between phenylalanine 129 and isoleucine 147, with only three changed amino acid positions among a total of 10 independent mutants analyzed, and the second region is located between asparagine 256 and leucine 275 with two changed amino acid positions among a total of 8 independent mutants analyzed. The limited number of mutated sites and their clustering provide strong evidence that these identified mutated sites are, indeed, responsible for myxothiazol, mucidin (strobilurin A), and stigmatellin resistances.

These regions, defined as center o inhibitor-resistant regions, are clearly distinct from the two regions previously identified as the center i inhibitor-resistant regions (22, 23).

In the absence of these sequencing data, the genetic results could only provide approximate locations for the resistant mutations. On the basis of the genetic data, the allocation of any mutation to a particular side of the membrane was not possible without speculations. The MUC3 locus, however, is located within exon 5 (50), a small exon 54 base pairs long, which codes partially for a protruding loop at the presumed outer side (49) of the membrane according to the Widger- Saraste model (20, 21).

A model with eight a-helices has been proposed on the basis of different studies on probability parameters for the occurrence of each amino acid in the buried membrane helices resulting in the withdrawal of helix IV from the membrane (25-27) (Fig. 4).

In this eight-a-helices model, each pair of inhibitor-resistant regions, defined by the location of the mutated codons identified by sequencing experiments (Refs. 22 and 23 and this work) seems to be located on the right side of the membrane. The two center inhibitor-resistant regions seemed to belong to the presumed inner (negative) side of the membrane, and the two center o inhibitor-resistant regions seemed to belong to the presumed outer (positive) side of the membrane. Using protein modeling, the experimental sequencing data fit nicely with the Q-cycle hypothesis. This line of reasoning, however, is based on the assumptions that resistant mutations are included, overlap, or interact partially with the center and center o domains.

In an analogous system, such overlapping has already been demonstrated. A photosynthetic plastoquinone/herbicide binding domain which includes the plastoquinone and herbicide binding sites as well as the resistance mutational sites has been proposed (51-53) for the first membrane protein complex ever x-ray analyzed, the bacterial reaction center of

Myxothiazol, Mucidin, and Stigmatellin Resistance S. cerevisiae 14547

DIU17

FIG. 4. Cytochrome b folding model in the mitochondrial inner membrane. This represents an eight-transmembrane helices model after the withdrawal of the fourth helix from the membrane.

split cytochrome b gene. Amino acids involved in the inhibitor resistance are shown in white surrounded by a black circle (ANA, antimycin; DIU, diuron; M Y X , myxothiazol; MUC, mucidin; STI, stigmatellin). Numbers in subscript are the amino acid positions. p and n represent, respectively, positively and negatively charged sides of the mitochondrial inner membrane according to Cramer and co-workers (49).

- , indicates the positions of the corresponding exon limits of the

Rhodobacter viridis (54, 55). Its eukaryotic analog is the photosystem 11. Moreover, the photosynthetic reaction center-photosystem I1 complexes and the respiratory bcl complex present, indeed, some similarities. Stigmatellin interferes with a plastoquinone binding site on photosystem I1 and chloroplast bd complex and inhibits the ubiquinol oxidation by the mitochondrial bcl complex. The double site of action of stigmatellin on both complexes suggests the existence of similar binding sites at the level of photosystem I1 and bcl complexes (56). Similarly, 3-(3,4-dichlorophenyl)-l,l-dimethylurea (diuron) interferes with the plastoquinone binding site and inhibits the bcl complex.

Therefore, two quinonelinhibitor binding domains, one for the center i region and the other for the center o region, which include or partially overlap the resistant mutations regions can be postulated for the bcl complex.

The comparison with inhibitor-resistant cytochrome bs from other organisms provides an additional argument in favor of this hypothesis. Center i inhibitor-resistant mutations were sequenced in the yeast Kluyueromyces lactis (six mutations),2 in the yeast Schizosaccharomyces pombe (two mutations) (57), and in mouse (two mutations) (58, 59). Center o inhibitor-resistant mutations were found in bacteria Rhodobacter capsuluta (three mutation^)^ and in mouse (four mutations) (46, 59). I t is remarkable that all these mutations were found to be included and in some cases affecting the same codons as in the corresponding S. cerevisiae center i and center o inhibitor-resistant regions.

In addition, amino acid alignments and properties of the resistant mutants seem to reveal differential characteristics

A. Brunner, personal communication. F. Daldal, personal communication.

according to the type of inhibitor-resistant region. First, on the basis of amino acid sequence alignments of 22 cytochrome bs, the center i and center o inhibitor-resistant regions can be differentiated by the primary structure of the cytochrome b. The two center o inhibitor-resistant regions, indeed, belong to the two most highly conserved regions of the protein, whereas the two inhibitor-resistant center i regions belong to less conserved regions (Refs. 20 and 21 and data not shown). Second, myxothiazol-, mucidin-, and stigmatellin-resistant mutants exhibit impaired growth rate on nonfermentable substrate and decreased oxygen consumption (Refs. 14 and 16 and this work). According to the knowledge of the func- tioning of the Q-cycle, the center o domain might need more structural stringency in the cytochrome b electron circuit than the center i domain because it canalizes the splitting of the electron flow between cytochrome b and the iron-sulfur protein. The high degree of amino acid conservation in the center o inhibitor-resistant regions and the decreased efficiency in respiration of center o-resistant mutants may reflect the possible key role played by these two regions of the protein in the cytochrome b electron circuit.

The methoxyacrylate (MOA)4 inhibitors's binding site, however, was found to be different from that for ubihydroquinone (60). The MOA inhibitor's binding site may be involved in the ubisemiquinone anion radical binding site (60). As pointed out by Brandt and co-workers (60), the interfer- ence of the MOA inhibitors in energy transduction could involve short or long range conformational changes. Argu- ments in favor of the proximity of the MOA inhibitors to the ubihydroquinone binding site are based on the fact that the Qo reaction center is very distinctly affected. The red shift induced by these inhibitors is almost exclusively a red shift of b566 (61), and they do not affect redox reactions at the Qi center (62). Although the MOA inhibitors bind to distinct sites different from those bound by ubihydroquinone, these sites most likely affect the same region of the protein. These results are consistent with a center o quinonelinhibitor domain on the bcl complex, which includes the center o inhibitor-resistant mutations.

Acknowledgments-We thank Professor P. P. Slonimski (Centre de Genktique Mol6culaire du Centre National de la Recherche Scien- tifique (CNRS) Gif-sur-Yvette, France) for his suggestion of the sequencing method, Drs. J. Perea and C. Jacq (CNRS, Gif-sur-Yvette, France) for the synthesis of the primers and their advice in the experimental sequencing procedures, and Drs. G. Michaelis and J. Subik for their gifts of myxothiazol- and mucidin-resistant mutants, respectively.

REFERENCES 1. Mitchell, P. (1976) J . Theor. Biol. 62, 327-367 2. Bowyer, J. R., and Trumpower, B. L. (1981) J. Biol. Chem. 256,

3. Ohnishi, T., and Trumpower, B. L. (1980) J. Biol. Chem. 255, 3278-3284

4. von Jagow, G., Gribble, G., and Trumpower, B. L. (1986) Bio- chemistry 25,775-780

5. Berry, E. A., and Trumpower, B. L. (1985) in Coenzyme Q (Lenaz, G., ed) pp. 365-389, John Wiley and Sons, New York

6. Dutton, P. L., Erecinska, M., Sato, N., Mukai, Y., Pring, M., and Wilson, D. F. (1972) Biochim. Biophys. Acta 267, 15-24

7. Becker, W. F., von Jagow, G., Anke, T., and Steglisch, W. (1981)

8. Convent, B., Briquet, M., and Goffeau, A. (1978) Eur. J. Biochem.

9. von Jagow, G., Ljungdahl, P. O., Graf, P., Ohnishi, T., and Trumpower, B. L. (1984) J. Biol. Chem. 259, 6318-6326

2245-2251

FEBS Lett. 132,329-333

92,137-145

10. Michaelis, G. (1976) Mol. Gen. Genet. 146, 133-137

' The abbreviation used is: MOA, methoxyacrylate.

14548 Myxothiazol, Mucidin, and Stigi

11. Burger, G., Lang, B., Bandlow, W., Schweyen, R.J., Backhaus, B., and Kaudewitz, F. (1976) Biochem. Biophys. Res. Commun. 72 , 1201-1208

12. Pratje, E., and Michaelis, G. (1977) Mol. Gen. Genet. 152 , 167- 174

13. Colson A,"., Luu The Van, Convent, B., Briquet, M., and Goffeau, A. (1977) Eur. J . Biochem. 74,521-526

14. Thierbach, G., and Michaelis, G. (1982) Mol. Gen. Genet. 186,

15. Subik, J. (1975) FEBS Lett. 59, 273-276 16. Subik, J., Kovacova, V., and Takacsova, G. (1977) Eur. J .

17. Subik, J., and Takacsova, G. (1978) Mol. Gen. Genet. 161, 99-

18. Colson, A. M., and Slonimski, P. P. (1979) Mol. Gen. Genet. 167,

19. Colson, A. M., Michaelis, G., Pratje, E., and Slonimski, P. P.

20. Widger, W. R., Cramer, W. A,, Herrmann, R. G., and Trebst, A.

21. Saraste, M. (1984) FEBS Lett. 166,367-372 22. di Rago, J.-P., Perea, X., and Colson, A,". (1986) FEBS Lett.

23. di Rago, J.-P., and Colson A.-M. (1988) J. Biol. Chem. 263,

501-506

Biochem. 73, 275-286

108

287-298

(1979) Mol. Gen. Genet. 167, 299-300

(1984) Proc. Natl. Acad. Sci. U. S. A . 81,674-678

208,208-210

12564-12570 24. Hauska. G.. Hurt. E.. Gabellini. N.. and Lockau. W. (1983)

Biochlm. Biophys. Acta 726, 97-'133 25. Rao, J. K., and Argos, P. (1986) Biochim. Biophys. Acta 896,

26. Crofts, A,, Robinson, H., Andrews, K., van Dorm, S., and Berry, E. (1987) in Cytochrome Systems (Papa, S., Chance, B., and Ernster, L., eds) pp. 617-624, Plenum Publishing Corp., New York

197-205

27. Brasseur, R. (1988) J . Biol. Chem. 263, 12571-12575 28. Thierbach, G., Kunze, B., Reichenbach, H., and Hofle, G. (1984)

Biochim. Biophys. Acta 765, 227-235 29. Villalobo, A,, Briquet, M., and Goffeau, A. (1981) Biochim. Bio-

phys. Acta 637, 124-129 30. Bonitz, S. G., Berlani, R., Coruzzi, G., Li, M., Macino, G., No-

brega, F. G., Nobrega, M. P., Thalenfeld, B. E. and Tzagoloff, A. (1980) Proc. Nutl. Acad. Sci. U. S. A . 77, 3167-3170

31. Koike, K., Kobayashi, M., Yaginuma, K., Taira, M., Yoshida, E., and Imai, M. (1982) Gene (Amst . ) 20, 177-185

32. Roe, B. A., Ma, D.-P., Wilson, R. K., and Wong, J. F.-H. (1985) J. Biol. Chem. 260, 9759-9774

33. Clary, D. O., Wahleithner, J. A,, and Wolstenholme, D. R. (1984) Nucleic Acids Res. 12, 3747-3762

34. Burke, J. M., Breitenberger, C., Heckman, J. E., Dujon, B., and RajBhandary, U. L. (1984) J. Biol. Chem. 259,504-511

35. Helmer-Citterich. M.. Morelli. G.. and Macino, G. (1983) EMBO J. 2,1235-1242

. .

36. Waring, R. B., Barries, R. W., Lee, S., Grisi, E., McPhail-Berks, M., and Scazzocchio, C. (1981) Cell 27,4-11

matellin Resistance S. cerevisiae 37. Dawson, A. J., Jones, V. P.. and Leaver, C . J. (1984) E M R n -1

3,2107-2113 " -.

38. Boer, P. H., McIntosh, J. E., Gray, M. W., and Bonen, L. (1985) Nucleic Acids Res. 13, 2281-2292

39. Schuster, W., and Brennicke, A. (1985) Curr. Genet. 9, 157-163 40. Kurowski, B., and Ludwig, B. (1987) J. Biol. Chem. 262,13805-

41. Davidson, E., and Daldal, F. (1987) J. Mol. Biol. 195, 13-24 42. Ohyama, K., Fukuzawa, H., Kohchi, T., Shirai, H., Sano, T.,

Sano, S., Umesono, K., Shiki, Y., Takeuchi, M., Chang, Z., Aota, S., Inokuchi, H., and Ozeki, H. (1986) Nature 322, 572- 574

43. Shinozaki, K., Ohme, M., Tanaka, M., Wakasugi, T., Hayashida, N., Matsubayashi, T., Zaita, N., Chunwongse, J., Obokata, J., Yamaguchi-Shinozaki, K., Ohto, C., Torazawa, K., Meng, B. Y., Sugita, M., Deno, H., Kamogashira, T., Yamada, K., Ku- suda, J., Takaiwa, F., Kato, A,, Tohdoh, N., Shimada, H., and Sugiura, M. (1986) EMBO J. 5, 2043-2049

44. Heinemeyer, W., Alt, J., and Herrmann, R. G. (1984) Curr. Genet.

45. Phillips, A. L., and Gray J. C. (1984) Mol. Gen. Genet. 194,477- 484

46. Howell, N., and Gilbert, K. (1987) in Cytochrome Systems (Papa, S., Chance, B., and Ernster, L., eds) pp. 79-86, Plenum Pub- lishing Corp., New York

47. von Jagow, G., and Link, T . A. (1986) Methods Enzymol. 126,

48. von Jagow, G., and Ohnishi, T. (1985) FEBS Lett. 185, 311-315 49. Cramer, W. A,, Black, M. T., Widger, W. R., and Girvin, M. E.

(1987) in The Light Reactions (Barber, J., ed) pp. 447-493, Elsevier Science Publishers, Amsterdam

50. Takacsova, G., Subik, J., and Kotylak, Z. (1981) Mol. Gen. Genet.

51. Trebst, A,, and Draber, W. (1986) Photosynth. Res. 10, 381-392 52. Johanningmeier, U., Bodner, U., and Wildner, G. F. (1987) FEBS

53. Youvan, D. C., Bylina, E. J., Alberti, M., Begusch, H., and Hearst,

54. Deisenhofer, J., Epp, O., Miki, K., Huber, R., and Michel, H.

55. Deisenhofer, J., Epp, O., Miki, K., Huber, R., and Michel, H.

56. Oettmeier, W., Godde, D., Kunze, B., and Hofle, G. (1985)

57. Weber, S., and Wolf, K. (1988) FEBS Lett. 237, 31-34 58. Howell, N., Appel, J., Cook, J. P., Howell, B., and Hauswirth, W.

59. Howell, N., and Gilbert, K. (1988) J. Mol. Bid. 203, 607-618 60. Brandt, U., Schagger, H., and von Jagow, G. (1988) Eur. J .

61. von Jagow, G., Link, T. A., and Onishi, T. (1986) J. Bioenerg.

62. Becker, W. F., von Jagow, G., Anke, T., and Steglich, W. (1981)

13811

8,543-606

253-271

179,141-146

Lett. 21 1, 221-224

J. E. (1984) Cell 37,949-957

(1984) J . Mol. Biol. 180,385-398

(1985) Nature 318, 618-624

Biochim. Biophys. Acta 807, 216-219

W. (1987) J . Biol. Chern. 262,2411-2414

Biochem. 173,499-506

Biomembr. 18,157-179

FEBS Lett. 132,329-333

Documents

Molecular Basis for Resistance to Myxothiazol, Mucidin (Strobilurin A)