J. Mol. Biol.
(1977) 117, 369-386
Yeast Mutants Defective in Iso-2-cytochrome c ,J. ALLAN DOWNIE~,JOHN
W. STEWART AND YRED SHERMAN
of Radiation Biology and Biophysics of Rochester School of Medicine and Dentistry Rochester, N.Y. 14642, U.S.A.
Department
Univemity
(Received 9 May 1.977) The structural gene CYC7 for yeast iso-2-cytochrome c was previously identified by isolating a mutant, ~~~7-1-1, totally lacking iso-2-cytochrome c and demonstrating that revertants of this mutant contained iso-2-cytochrome c with an altered primary structure (Downie et al., 1977). In this paper we describe a variety of different types of mutants that completely or partially lack iso-2-cytochrome c due to mutations in either the structural gene, CYC7, or unlinked “regulatory” genes. The iso-2-cytochrome c-deficient mutants were isolated by benzidine staining of over 3 X 105 colonies from p- strains (cytoplasmic petites) that lacked iso-1-cytochrome c due to the deletion cycl-1 and that contain abnormally high levels of iso-2-cytochrome c due to a chromosomal translocation, CYC7-I. adjacent to the normal structural gene CYC7f. The cytochrome c content of mutants not staining with the benzidine reagents was estimated by low temperature spectroscopy, and 139 mutants containing significantly decreased levels of iso-2-cytochrome c were analyzed genetically by complement&ion with previously identified cyc mutants. In this way 50 mutants at the cyc2 and cyc3 loci wercs identified along with a group of 62 mutant,s of the structural gene cyc7. Tht) different types of mutants of the structural gene which were uncovered and which were more or less anticipated included those that completely lacked iso-2-cytochrome c, those that were suppressible by UAA or UAG suppressors, those that lacked iso-2.cytochrome c but had increased levels after growth at lower temperatures, and those that exhibited visibly altered c= absorption bands of iso-2-cytochrome c. Iso-2-cytochrome c mutants with altered primary structures were obtained from intragenic revert,ants of several of these mutants, confirming our earlier conclusion that cyc7 is the structural gene. In addition we observed an unexpected class of mutants that lacked iso-2-cytochrome c when in the p- state but contained approximately the CYC7-1 parental level when in the p+ state. Two of these mutants, ~~~7-1-47 and cyc7-l-49, were shown to contain altered iso-2-cytochromes c. The different contents of the abnormal iso-2. cytochromes c suggest that cytochrome c has different environments in p+ and p- mitochondria and that the p + condition may stabilize certain alterod proteins.
1. Introduction ~I;ormal
wild-type
cytochromes (Slonimski about
bakers’
yeast
(Xacchuromyce
scerevisiae)
contains
two
distinct
c which are referred to as iso-1-cytochrome c and iso-2-cytochrome c et al., 1965; Sherman et aE., 1965). Generally iso-1-cytochrome c constitut*es
950/l, and iso-2-cytochrome
c about
5% of the total
t Present address: Department of Biochemistry, John Curtin Australian National University, Canberra, A. C. T. 2601, Australia.
369
amount
of cytochrome
School of Medical
c in
RRsP&rrh.
370
J. A. DOWNIE,
J. W.
STEWART
AND
F. SHERMAN
aerobically grown yeast. The iso-cytochromes c have different amino acid sequences (Stewart & Putterman, manuscript in preparation) and both function in mitochondriel oxidative phosphorylation (Mattoon & Sherman, 1966). Although the reasons for two distinct cytochromes c in yeast are unknown, their absolute amounts and relative proportions varies with different physiological states (Sels et al., 1965; Sherman $ Stewart, 1971). Mutation of six loci, cycl through cyc6, can result in reduced levels of total cytochrome c (Sherman, 1964). The cycl locus is the structural gene for iso-1-cytochrome c and it appears to specifically affect only iso-1-cytochrome c while the other loci, especially cyc2 and cycd, appear to affect the levels of both iso-cytochromes c. Such mutants were first identified by low temperature spectroscopic screening (Sherman, 1964), later by staining colonies with benzidine reagents (Sherman et al., 1968) and more recently by selecting strains resistant to chlorolactate, an inhibitor which preferentially affects lactate-utilizing strains (Sherman et al., 1974). This latter technique selects mutants which contain certain low levels of cytochrome c that permit growth on glycerol but not on lactate media. Although large numbers of mutants containing reduced levels of cytochrome c were isolated by these techniques, no mutants specifically lacking iso-2-cytochrome c were obtained (Sherman et al., 1965,1974). This could be anticipated since normally the change in total amount of cytochrome c due to a deficiency of iso-2-cytochrome c would be undetectable by the screening methods used. However, we have recently reported the isolation of a mutant of the structural gene, CYC7, of iso-2-cytochrome c (Downie et al., 1977). This mutant was isolated by using as a starting strain a mutant totally lacking iso-1-cytochrome c due to the deletion cycl-1 and containing an abnormally high level of iso-2-cytochrome c due to a chromosomal translocation, CYC’I-1, adjacent to the normal structural gene CYC7+ (Sherman t Helms, 1978). It proved possible to isolate mutants containing reduced levels of iso-2-cytochrome c by selecting colonies which had a reduced benzidine staining reaction. Benzidine reagents stain all haem proteins, but since iso-2-cytochrome c is the major haem protein present in the strain described above, most of the unstained colonies had diminished amounts of iso-2-cytochrome c. A mutant of the structural gene ~~~7-1-1 has already been described and was shown to contain a UAG nonsense codon corresponding to position 24 in iso-2cytochrome c (Downie et al., 1977). In this paper we describe over 100 other mutants having reduced amounts of iso-2-cytochrome c. The largest group was the result of mutations of the structural gene for iso-2-cytochrome c, cyc7, while others were the result of mutations of the cyc2 and cyc3 loci. The types of cyc7 mutants included those that completely lacked iso-2-cytochrome c, those that were suppressible by UAA and UAG suppressors, those that contained low levels of non-functional iso-2-cytochromes c, those having amounts of iso-2-cytochrome c that increased after lower incubation temperatures and, unexpectedly, those having iso-2-cytochrome c in p+ strains but not in corresponding p- strains.
2. Materials and Methods (a) Nonaenclature The current nomenclature of mutants affecting cytochrome c has been described recently by Sherman et al. (1974,1975) and Downie et al. (1977). Briefly, mutants of different loci having reduced amounts or activities of cytochrome c are designated cycl, cyc2,
YEAST
MUTANTS
OF ISO-2.CYTOCH.ROME
c
371
etc., while the corresponding wild-type alleles are designated, respectively, C YCI + , G YC2 f etc. The cycl symbol refers to mutants of the structural gene of iso- 1 -cytochrome c and independently derived mutants at this locus are designated cycl-1, cycl-2, etc. CYCl-2-A, CYCl-2-B. etc. A Intragenic revertants of 9~1-2, for example, are designated similar nomenclature has been adopted for mutants of the structural gene of iso-2. cytochrome c whose wild-type allele has been denoted CYC7+. The symbol, CYC7-1. denotes a mutant that produces a high level of iso-2-cytochrome c due to a chromosomal translocation adjacent to the normal gene CYC7+-. Subsequent mutations at this gene causing a loss of iso-2-cytochrome c are designated ~~~7-1-1, ~~~7-1-2, etc. Intragenic rcvertants of the deficient mutants are designated by letters following the allele numbers, e.g. CYC7-l-l-A, CYC7-l-l-B, etc. Examples of mutational pathways affecting iso-2. cytochrome c are outlined in Fig. 1. ‘Ihe standard genetic symbols, p+ and p- denote, respectively, wild-type and mitochondrial mutants deficient in cytochromes u*a3 and b.
Rare mutant
on lactate
medium
cycl
CYC7-I cycz-IO (15%)
cyc7 mutants 050%)
cyc3 mutants (O-15%)
cycZmutants (15.ZO%l cycl
cycl
CYC7-I cycJ-I8 (0%) Revertant
on lactate
medw cycl
FIG. c. The amount due to specific
cyc7-l-2 (0%)
1 CYC7+z?A (150%)
1. The mutational pathways of mutants having high snd low amounts of iso-2eytochrome approximate amounts of iso-2.cytochrome o are given as the percentages of the total of cytochrome c in normal CYCI+ CYC7+ strains. All strains lack iso-1-cytochrome ( cycl mutations. The range of deficiencies refer to mutants grown at 22°C. Examples of deficient mutants and an intragenic revertant are presented at the bottom of the Figure. (b) Strains
The mutant B-3770 (~~~1-345 CYC7-I) having an approximate 30.fold increase of iso-2-cytochrome c wa.s isolated on lactate medium as previously described (Downie et al., 1977). It was subsequently demonstrated that the CYC7-I mutation was a chromosomal translocation adjacent to the normal structural gene CYC7+ but outside the translated region (Sherman & Helms, 1978). Presumably the translocation caused the formation of an abnormal region in front of the structural gene, giving rise to properties expected for strains having mutations in the promotor region. Iso-2-cytochrome c-deficient mutants CH33-lD-1 (p- CL were isolated from strains CH33-IB-1 (p- LXcycl-1 CYC7-1 &@-I), cycl-1 CYC7-1 trpl-1 arg4-17) and WlOO-23A-1 (p- a cycl-1 CYC7-1 1~82-1 canl-100 ural-I). These strains were constructed by conventional yeast genetic procedures involving crosses with strain B-3770 and converted to the p- strains by growth in the presence of ethidium bromide. Other strains having other cyc mutations (cycl, cyc2 or cyc3) or containing UAA or UAG suppressors (SUP-o or SUP-a, respectively) were also constructed as required or were chosen from our stock collection. (0) Cfenetic
methods
Yeast genetic procedures (Sherman & Lawrence, 1974) and the types of media (Sherman et al., 1974) were as described previously. Lactate and glycerol media do not permit growth 25
372
J. A. DOWNIE,
J. W.
STEWART
AND
F. SHERMAN
of strains lacking both iso-cytochromes c. In contrast to glycerol medium, lactate medium does not permit the growth of cycl CYC7+ strains which lack iso-1-cytochrome c but which contain the normal low amount of iso-2-cytochrome c. Mutants lacking iso-2-cytochrome c were isolated from p- cycl-1 CYC7-I strains with the benzidine staining procedure (Sherman et al., 1968; Downie et aZ., 1977), after either treatments with 30 Jm-2 of U.V. irradiation (Downie et al., 1977) or 3 h treatment with 2% (v/v) ethyl methanesulfonate in 50 mM-phosphate buffer (pH 7-O). Revertants containing iso-2-cytochrome c at high levels typical of CYC7-I strains were selected from using glycerol or lactate media and after irradiation with P+ cycl-1 ~~~7-1-2 segregants either 30 JmM2 of U.V. irradiation or 10 krad of X-rays. The levels of total cytochrome c in intact cells of mutants and segregants were estimated by low temperature (- 190°C) spectroscopic examinations (Sherman & Slonimski, 1964) or spectroscopic recordings (Sherman et al., 1968). Derepressed cells for spectral examination were prepared by growth at 22°C or 30°C for 2 or 3 days on nutrient plates as previously described (Sherman et al., 1978). Tests of suppression were performed either by examining diploid strains that were and heterozygous for the cyc7 heterozygous for the suppressors (e.g. SUP7-a/sup7+) mutant (e.g. cyc7-I-I/CYC7+) (Downie et al., 1977) or by examining meiotic haploid segregants that contained the suppressor and the cyc7 mutant (e.g. XUP4-o ~~~7-1-2). The suppressed haploid strains were derived either from heterozygous translocation crosses or homozygous translocation crosses using the procedures described by Sherman & Helms (1978). (d) Identijcation
of altered iso-2-cytochromes c
Structural changes in iso-2-cytochromes c were identified from amino acid analyses, tryptic and chymotryptic peptide maps, and sequential Edman degradations as described earlier (Stewart et al., 1971). In most’ instances iso-2-cytochromes c were prepared by the procedure used to prepare iso-I-cytochrome c (Sherman et al., 1968; Stewart et al., 1971). p+ strains were purified as Labile iso-2-cytochromes c from the ~~~7-1-47 p+ and cyc7-I-49 described by Sherman et al. (1968) except that only the center portion of cytochrome c eluting from the first column of Amberlite CG-50 cation exchange resin (100 to 200 mesh) (Mallinckrodt) was collected. This peak fraction of cytochrome c WAS diluted 4-fold in 0.1 M-sodium phosphate buffer (pH 7*2), adsorbed onto a column (1 cm x 20 cm) of Amberlite CG-50 cation exchange resin (200 tjo 400 mesh) and eluted with a solution of 0.8 M-NaCl in O-1 M-sodium phospha,te buffer (pH 7.2). The peak fraction of cytochrome c was collected, dialyzed against 0.01 M-(NH,)2C0, and freeze-dried. All steps in this purification procedure were carried out at 5 to 10°C.
3. Results (a) Isolation of iso-2-cytochrome c-dej%ent mutants Mutants completely or partially lacking iso-2-cytochrome c were isolated from p- cycl-I CYC7-1 strains CH33-lB-1, CH33-lD-1 and WlOO-26A-1. All three of these strains contain a high level of iso-2qtochrome c due to the CYC’I-1 translocation, completely lack iso-1-cytochrome c due to the cycl-I deletion and completely lack cytochrome a-a3 and b due to the mitochondrial mutation p-. Cultures were mutagenized with either ethyl methanesulfonate or ultraviolet irradiation, were plated on nutrient medium, and the resulting colonies stained with benzidine reagents. Unstained colonies were subcloned and the cytochrome c levels of the subclones were estimated by spectroscopic examination of intact cells. Only those mutants which had less than 40% of the cytochrome c content further analysis. In this way 139 mutants obtained after examining approximately
procedure.
of the parent strains were retained for deficient in iso-2-cytochrome c were
322,000 colonies with
the benzidine
YEBST
MUTANTS
OF ISO-2-CYTOCHROME
r
373
(b) IdentiJication of mutant loci It was anticipated that mutations of a variety of distinct loci could cause deficiencies of iso-2qtochrome c. In our previous publication (Downie et al., 1977) one iso-2-cytochrome c-deficient mutant, Bz39, was shown to contain a mutation, cyc7-l- 7, in the structural gene of iso-2-cytochrome c. Also cyc2 and cyc3 mutants were previously shown to cause marked deficiencies of iso-1-cytochrome c and presumably of iso-2qtochrome c (Sherman et al., 1965). In addition it was expected that mutations of other loci, such as those involved in porphyrin synthesis, could result in benzidinrnegative mutants. Genetic analyses were carried out in order to determine if a known cyc2 mutant’ gene could produce deficiencies of iso-2-cytochrome c in the presence of CYC7-2 branslocation that normally causes the formation of a high level of iso-2-cytochromc c. Meiotic segregants of the genotype cycl-1 CYC7-1 cyc2-8 were obtained from a cross cycl-1 CYC7-+ cyc2-8 x cycl-1 CYC7-1 CYC2+. Spectroscopic examinations indicated that the cyc2-8 allele reduced the level of iso-2-cytochrome c in CYC7- 1 strains by about 60%. The action of a cyc3 mutation on the C’YCT-1 gene was revealed with the new mutant Bz84 that contained only approximately 5% of iso-2-cytochrome c of the cycl-1 CYC7-1 parental strains. The Bz84 mutant was crossed to a cycl-1 CYCT+ CYC3+ strain and to a cycl-1 CYC7+ cyc3-3 strain and the cytochrome c contents of the resulting two diploids were estimated by spectroscopy. The diploid from the former cross contained a level of cytochrome c characteristic of cycl-1 CYC7-llcycl-7 CYC7-t- strains while the latter diploid contained less than 5% of the normal level. These results indicate that the Bz84 strain contains the CYC’I-I gene and a recessive gene which is allelic to the known cyc3-3 gene and which was designated ~~~3-18. Ttms a cyc3 mutant gene can diminish the level of iso-2-cytochrome c in CYC7-I st!rains by as much as 950/,. If the iso-2-cytochrome c deficiencies were caused by either the cyc2, cyc3 or cyc7 recessive mutations, the mutant loci could be conveniently identified by crossing the mutants to known cyc tester strains, and examining the diploids for complementation of their cytochrome c deficiencies. Each of the 139 mutants were first crossed to &rain AD176-2D (cycl-1 CYC’I-1) and the cytochrome c level of the diploid crosses were estimated by spectroscopic examinations; all such diploids contained high levels of cytochrome c characteristic of cycl-1 CYC7-llcycl-1 CYC7-1 or cycl-1 CYC7-11 cycl-1 GYC7+ crosses, demonstrating that all of the mutants were recessive. Further tests were carried out by determining the cytochrome c content from spectroscopic examinat,ions of diploids that were constructed by crossing the series of iso-2cvtochrome c-deficient mutants to several tester strains and determining whether or not complementation occurred. Table 1 illustrates how such complementation tests were used to identify mutations of two different genes, e.g. Bz39 having the ~~~7-1-1 mubation in the structural gene and Bz118 having the cyc3-19 mutation in the cyc3 gene. The mutation in the structural gene was detected by crossing Bz39 to the tester strain D672-5D (cycl-1 CYC7+) and examining the cytochrome c content of the diploid: the low level of iso-2-cytochrome c of the diploid cross is typical of cycl-1 CJ’C7-I- strains. Similarly the low level of iso-2-cytochrome c in the cross Bz118 (cycl-1 CYC7-I cyc3-19) x AD179-9D (cycl-I CYC7-1 ~~~3-18) indicates that t,he iso-2cytochrome c deficiency in Bz118 is due to cyc3 mutation.
374
J. A. DOWNIE,
J. W.
STEWART
AND
F. SHERMAN
TABLE 1
Examples of complementation tests with the parental strain (cycl-1 CYC’I-1 CYC3+) and with mutants dejicient in iso-Z-cytochrome c
Strain
Genotype
Parental strain Bz39 Bz118
cycl-1 GYC7-1 cyc3+ cycl-1 cyc7-l-l cyc3+ cycl-1 CYC7-1 cyc3-19
Approximate oh of iso-2.cytochrome c Crossed to taster strains: cycl-1 CYC7+ CYC3+ cycl-1 CYC7-1 cyc3-18 (D672-6D) (AD179-9D)
Haploid
160 1 1
100 6 100
160 100 1
All strains lack iso-1-cytochrome c due to the deletion cycl-1. The content of iso-2-cytoohrome c is presented as the percentage of the amount of total cytochrome c found in normal strains (cYcl+ cyc7+ cYc3+).
TABLE 2
Iso-2-cytochrome c-dejcient mutants obtained with the benzidine staining method Strain no.
CH33-lB-1 CH33-lB-1 CH33-lD-1 CH33-lD-1 WlOO-23A-1 WlOO-23A-1
Mutagen
EMS &ii &ii . . &:a1
EMS, ethyl methanesulfonate;
Colonies examined
cyc7
56,000 103,000 10,000 33,000 40,000 80,000 322,000
7 30 1 4 6 14 62
u.v., ultraviolet
Number of mutants cyc2 cyc3 Total 4 16 0 0 4 b
29
6 6 1 0 8 0 21
17 62 2 4 18 19 112
irradiation.
The series of 139 iso-2-cytochrome c-deficient mutants were crossed to the tester strains D672-5D and AD179-9D listed in Table 1 and the cyc7 and cyc3 mutations were identified from the iso-2-cytochrome c deficiencies in the resulting diploids. In addition it was necessary to examine p- as well as p+ diploids from the crosses with D672-5D in order to identify certain cyc7 mutants which have deficiencies only in pstrains (see below). These mutants that complemented the cyc7 and cyc3 tester strains were crossed to AD260-3A (cycl-1 GYC7+ cyc2-8) in order to identify cyc2 mutations. The number of mutants of each of the cyc7, cyc3 and cyc2 loci are summarized in Table 2, along with the parent strains from which they were isolated and the mutagen used. In general, mutant cyc2 alleles reduced the level of iso-2-cytochrome c by about 60 to SO%, whereas cyc3 mutant alleles had a greater effect causing an 80% to nearly 100% reduction in the amount of iso-2-cytochrome c. As mentioned above, only mutants having a decrease in the amount of iso-2-cytochrome c of more than 60% were retained for genetic analysis. Thus mutations of the cyc7, cyc2, cyc3 and other loci producing only slight deficiencies have been excluded. In summary, 139 mutants containing less than 40% of CYC7-1 level of iso-2cytochrome c were isolated after examining approximately 322,000 colonies with the
YEAST
MUTANTS
OF ISO-“-CYTOCHROME
c
375
benzidine procedure; 112 of these mutants were identified as having mutations in slither the cyc7, cyc2 and cyc3 loci. Of the remaining mutants, only four had less tha.n 30/ of the parental level of iso-2-cytochrome c. These four mutants are presumably blocked in the synthesis of porphyrin or haem, since they totally lack not only iso-2cytochrome c but also cytochrome c1 which is at least partially present in p- mutants (Ross & Schatz, 1976). One of these mutants, Bz146, was crossed to strain D672-3B (a cycl-1 lysl-1 trp5-48 leul-12 his5-2) and the cytochrome content of meiotic progen! was determined. The complete tetrads gave rise to a 2 : 2 segregation of a pet phenotype which completely lacked all cytochromes, indicating that Bz146 contained a singk mutant gene preventing synthesis of all cytochromes. The 23 mutants not identified were not studied further. None of these were completelg deficient in iso-2qtochrome c, and only had a 60 to 80% decrease in amount of iso-2-cytochrome c. These mutants partially deficient in iso-2-cytochrome c could have nmtations at the cyc4, cyc5, cyc6 or cydl loci which have been primarily shown to only slightly affect the total amount of cytochrome c (see Sherman et al.. 1965 : Sanders et al.. 1973). Alternatively these 23 mutants may contain mutations nt other loc*i which specifically or generally control cytochrome c synthesis. (c) The cyc7 locus is distinct from the cyc2 and cyc3 loci Genetic tests were undertaken to determine whether the cyc7 locus is linked bo either the cyc2 or cyc3 loci. Poor spore viability from crosses that were heterozygous for the translocation, CYC7-ljCYC7+, made the analysis of the meiotic segregation difficult. The cross between strains AD260-3C (a cycl-1 cycZ-8 C YC7+) and WlOS- 1D (a cycl-1 CYCZ+ CYC7-I) yielded only two complete tetrads, one being tetratype and the other a, parental ditype. Of the other 20 surviving segregants from ten tetrads. ten were recombinant type and ten were parental type. These data clearly demonstrated that the cyc2 and cyc7 genes are distinct and probably unlinked. Similarly it was concluded that the cyc3 and cyc7 genes are distinct and probahl) unlinked since the parental ditype/non-parental ditype/t,etratype rntio was 1 :O : 8 in trtrads having four viable segregants that were derived from several crosses which contained the heterozygous genes, CYC3flcyc3, and either ofthe hetorozygous translocations CYC7+/CYC7-1 or CYC7+/cyc7-l-l. The chromosomal location of these three distinct loci will be reported elsewhere. (d) Characteristics
of mutants of the structural
geyze CYC7
A total of 62 mutants were identified as being at the structural gene of jso-2cytochrome c. We have attempted to group these 62 mutants into classes based on the amount and characteristics of iso-2-cytochrome c formed under different, growt,h conditions and formed by suppression. It has been shown (Sherman et al., 1968,1974) that the level of iso-l-cytochrome c present in many cycl mutants can be affected by various growth conditions. When mutants are grown at the lower temperature of 22°C compared with 3O”C? some produce increased amounts of iso-1-cytochrome c. These iso-1-cytochromes c may 01 may not be functional (Sherman et al., 1974) and some have altered absorption spectra, (Schweingruber et al., 1977). Also various levels of iso-1-cytochrome c appear when certain cycl mutants are coupled to UAG or UAA suppressors (Gilmore et al.. 1971; Liebman et al., 1976). We have used these and other criteria to classify mutants of iso-2-cytochrome c outlined in Table 3.
376
J. A. DOWNIE,
J. W.
STEWART
AND
F. SHERMAN
TABLE 3
Properties of the cyc’l mutants of the structural
gene of iso-2-cytochrome c Is0 -2 levels (% of cucr-1)
Completely or almost completely UAA-suppressible
deficient
30°C
22°C
(1977) 117, 369-386
Yeast Mutants Defective in Iso-2-cytochrome c ,J. ALLAN DOWNIE~,JOHN
W. STEWART AND YRED SHERMAN
of Radiation Biology and Biophysics of Rochester School of Medicine and Dentistry Rochester, N.Y. 14642, U.S.A.
Department
Univemity
(Received 9 May 1.977) The structural gene CYC7 for yeast iso-2-cytochrome c was previously identified by isolating a mutant, ~~~7-1-1, totally lacking iso-2-cytochrome c and demonstrating that revertants of this mutant contained iso-2-cytochrome c with an altered primary structure (Downie et al., 1977). In this paper we describe a variety of different types of mutants that completely or partially lack iso-2-cytochrome c due to mutations in either the structural gene, CYC7, or unlinked “regulatory” genes. The iso-2-cytochrome c-deficient mutants were isolated by benzidine staining of over 3 X 105 colonies from p- strains (cytoplasmic petites) that lacked iso-1-cytochrome c due to the deletion cycl-1 and that contain abnormally high levels of iso-2-cytochrome c due to a chromosomal translocation, CYC7-I. adjacent to the normal structural gene CYC7f. The cytochrome c content of mutants not staining with the benzidine reagents was estimated by low temperature spectroscopy, and 139 mutants containing significantly decreased levels of iso-2-cytochrome c were analyzed genetically by complement&ion with previously identified cyc mutants. In this way 50 mutants at the cyc2 and cyc3 loci wercs identified along with a group of 62 mutant,s of the structural gene cyc7. Tht) different types of mutants of the structural gene which were uncovered and which were more or less anticipated included those that completely lacked iso-2-cytochrome c, those that were suppressible by UAA or UAG suppressors, those that lacked iso-2.cytochrome c but had increased levels after growth at lower temperatures, and those that exhibited visibly altered c= absorption bands of iso-2-cytochrome c. Iso-2-cytochrome c mutants with altered primary structures were obtained from intragenic revert,ants of several of these mutants, confirming our earlier conclusion that cyc7 is the structural gene. In addition we observed an unexpected class of mutants that lacked iso-2-cytochrome c when in the p- state but contained approximately the CYC7-1 parental level when in the p+ state. Two of these mutants, ~~~7-1-47 and cyc7-l-49, were shown to contain altered iso-2-cytochromes c. The different contents of the abnormal iso-2. cytochromes c suggest that cytochrome c has different environments in p+ and p- mitochondria and that the p + condition may stabilize certain alterod proteins.
1. Introduction ~I;ormal
wild-type
cytochromes (Slonimski about
bakers’
yeast
(Xacchuromyce
scerevisiae)
contains
two
distinct
c which are referred to as iso-1-cytochrome c and iso-2-cytochrome c et al., 1965; Sherman et aE., 1965). Generally iso-1-cytochrome c constitut*es
950/l, and iso-2-cytochrome
c about
5% of the total
t Present address: Department of Biochemistry, John Curtin Australian National University, Canberra, A. C. T. 2601, Australia.
369
amount
of cytochrome
School of Medical
c in
RRsP&rrh.
370
J. A. DOWNIE,
J. W.
STEWART
AND
F. SHERMAN
aerobically grown yeast. The iso-cytochromes c have different amino acid sequences (Stewart & Putterman, manuscript in preparation) and both function in mitochondriel oxidative phosphorylation (Mattoon & Sherman, 1966). Although the reasons for two distinct cytochromes c in yeast are unknown, their absolute amounts and relative proportions varies with different physiological states (Sels et al., 1965; Sherman $ Stewart, 1971). Mutation of six loci, cycl through cyc6, can result in reduced levels of total cytochrome c (Sherman, 1964). The cycl locus is the structural gene for iso-1-cytochrome c and it appears to specifically affect only iso-1-cytochrome c while the other loci, especially cyc2 and cycd, appear to affect the levels of both iso-cytochromes c. Such mutants were first identified by low temperature spectroscopic screening (Sherman, 1964), later by staining colonies with benzidine reagents (Sherman et al., 1968) and more recently by selecting strains resistant to chlorolactate, an inhibitor which preferentially affects lactate-utilizing strains (Sherman et al., 1974). This latter technique selects mutants which contain certain low levels of cytochrome c that permit growth on glycerol but not on lactate media. Although large numbers of mutants containing reduced levels of cytochrome c were isolated by these techniques, no mutants specifically lacking iso-2-cytochrome c were obtained (Sherman et al., 1965,1974). This could be anticipated since normally the change in total amount of cytochrome c due to a deficiency of iso-2-cytochrome c would be undetectable by the screening methods used. However, we have recently reported the isolation of a mutant of the structural gene, CYC7, of iso-2-cytochrome c (Downie et al., 1977). This mutant was isolated by using as a starting strain a mutant totally lacking iso-1-cytochrome c due to the deletion cycl-1 and containing an abnormally high level of iso-2-cytochrome c due to a chromosomal translocation, CYC’I-1, adjacent to the normal structural gene CYC7+ (Sherman t Helms, 1978). It proved possible to isolate mutants containing reduced levels of iso-2-cytochrome c by selecting colonies which had a reduced benzidine staining reaction. Benzidine reagents stain all haem proteins, but since iso-2-cytochrome c is the major haem protein present in the strain described above, most of the unstained colonies had diminished amounts of iso-2-cytochrome c. A mutant of the structural gene ~~~7-1-1 has already been described and was shown to contain a UAG nonsense codon corresponding to position 24 in iso-2cytochrome c (Downie et al., 1977). In this paper we describe over 100 other mutants having reduced amounts of iso-2-cytochrome c. The largest group was the result of mutations of the structural gene for iso-2-cytochrome c, cyc7, while others were the result of mutations of the cyc2 and cyc3 loci. The types of cyc7 mutants included those that completely lacked iso-2-cytochrome c, those that were suppressible by UAA and UAG suppressors, those that contained low levels of non-functional iso-2-cytochromes c, those having amounts of iso-2-cytochrome c that increased after lower incubation temperatures and, unexpectedly, those having iso-2-cytochrome c in p+ strains but not in corresponding p- strains.
2. Materials and Methods (a) Nonaenclature The current nomenclature of mutants affecting cytochrome c has been described recently by Sherman et al. (1974,1975) and Downie et al. (1977). Briefly, mutants of different loci having reduced amounts or activities of cytochrome c are designated cycl, cyc2,
YEAST
MUTANTS
OF ISO-2.CYTOCH.ROME
c
371
etc., while the corresponding wild-type alleles are designated, respectively, C YCI + , G YC2 f etc. The cycl symbol refers to mutants of the structural gene of iso- 1 -cytochrome c and independently derived mutants at this locus are designated cycl-1, cycl-2, etc. CYCl-2-A, CYCl-2-B. etc. A Intragenic revertants of 9~1-2, for example, are designated similar nomenclature has been adopted for mutants of the structural gene of iso-2. cytochrome c whose wild-type allele has been denoted CYC7+. The symbol, CYC7-1. denotes a mutant that produces a high level of iso-2-cytochrome c due to a chromosomal translocation adjacent to the normal gene CYC7+-. Subsequent mutations at this gene causing a loss of iso-2-cytochrome c are designated ~~~7-1-1, ~~~7-1-2, etc. Intragenic rcvertants of the deficient mutants are designated by letters following the allele numbers, e.g. CYC7-l-l-A, CYC7-l-l-B, etc. Examples of mutational pathways affecting iso-2. cytochrome c are outlined in Fig. 1. ‘Ihe standard genetic symbols, p+ and p- denote, respectively, wild-type and mitochondrial mutants deficient in cytochromes u*a3 and b.
Rare mutant
on lactate
medium
cycl
CYC7-I cycz-IO (15%)
cyc7 mutants 050%)
cyc3 mutants (O-15%)
cycZmutants (15.ZO%l cycl
cycl
CYC7-I cycJ-I8 (0%) Revertant
on lactate
medw cycl
FIG. c. The amount due to specific
cyc7-l-2 (0%)
1 CYC7+z?A (150%)
1. The mutational pathways of mutants having high snd low amounts of iso-2eytochrome approximate amounts of iso-2.cytochrome o are given as the percentages of the total of cytochrome c in normal CYCI+ CYC7+ strains. All strains lack iso-1-cytochrome ( cycl mutations. The range of deficiencies refer to mutants grown at 22°C. Examples of deficient mutants and an intragenic revertant are presented at the bottom of the Figure. (b) Strains
The mutant B-3770 (~~~1-345 CYC7-I) having an approximate 30.fold increase of iso-2-cytochrome c wa.s isolated on lactate medium as previously described (Downie et al., 1977). It was subsequently demonstrated that the CYC7-I mutation was a chromosomal translocation adjacent to the normal structural gene CYC7+ but outside the translated region (Sherman & Helms, 1978). Presumably the translocation caused the formation of an abnormal region in front of the structural gene, giving rise to properties expected for strains having mutations in the promotor region. Iso-2-cytochrome c-deficient mutants CH33-lD-1 (p- CL were isolated from strains CH33-IB-1 (p- LXcycl-1 CYC7-1 &@-I), cycl-1 CYC7-1 trpl-1 arg4-17) and WlOO-23A-1 (p- a cycl-1 CYC7-1 1~82-1 canl-100 ural-I). These strains were constructed by conventional yeast genetic procedures involving crosses with strain B-3770 and converted to the p- strains by growth in the presence of ethidium bromide. Other strains having other cyc mutations (cycl, cyc2 or cyc3) or containing UAA or UAG suppressors (SUP-o or SUP-a, respectively) were also constructed as required or were chosen from our stock collection. (0) Cfenetic
methods
Yeast genetic procedures (Sherman & Lawrence, 1974) and the types of media (Sherman et al., 1974) were as described previously. Lactate and glycerol media do not permit growth 25
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J. A. DOWNIE,
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of strains lacking both iso-cytochromes c. In contrast to glycerol medium, lactate medium does not permit the growth of cycl CYC7+ strains which lack iso-1-cytochrome c but which contain the normal low amount of iso-2-cytochrome c. Mutants lacking iso-2-cytochrome c were isolated from p- cycl-1 CYC7-I strains with the benzidine staining procedure (Sherman et al., 1968; Downie et aZ., 1977), after either treatments with 30 Jm-2 of U.V. irradiation (Downie et al., 1977) or 3 h treatment with 2% (v/v) ethyl methanesulfonate in 50 mM-phosphate buffer (pH 7-O). Revertants containing iso-2-cytochrome c at high levels typical of CYC7-I strains were selected from using glycerol or lactate media and after irradiation with P+ cycl-1 ~~~7-1-2 segregants either 30 JmM2 of U.V. irradiation or 10 krad of X-rays. The levels of total cytochrome c in intact cells of mutants and segregants were estimated by low temperature (- 190°C) spectroscopic examinations (Sherman & Slonimski, 1964) or spectroscopic recordings (Sherman et al., 1968). Derepressed cells for spectral examination were prepared by growth at 22°C or 30°C for 2 or 3 days on nutrient plates as previously described (Sherman et al., 1978). Tests of suppression were performed either by examining diploid strains that were and heterozygous for the cyc7 heterozygous for the suppressors (e.g. SUP7-a/sup7+) mutant (e.g. cyc7-I-I/CYC7+) (Downie et al., 1977) or by examining meiotic haploid segregants that contained the suppressor and the cyc7 mutant (e.g. XUP4-o ~~~7-1-2). The suppressed haploid strains were derived either from heterozygous translocation crosses or homozygous translocation crosses using the procedures described by Sherman & Helms (1978). (d) Identijcation
of altered iso-2-cytochromes c
Structural changes in iso-2-cytochromes c were identified from amino acid analyses, tryptic and chymotryptic peptide maps, and sequential Edman degradations as described earlier (Stewart et al., 1971). In most’ instances iso-2-cytochromes c were prepared by the procedure used to prepare iso-I-cytochrome c (Sherman et al., 1968; Stewart et al., 1971). p+ strains were purified as Labile iso-2-cytochromes c from the ~~~7-1-47 p+ and cyc7-I-49 described by Sherman et al. (1968) except that only the center portion of cytochrome c eluting from the first column of Amberlite CG-50 cation exchange resin (100 to 200 mesh) (Mallinckrodt) was collected. This peak fraction of cytochrome c WAS diluted 4-fold in 0.1 M-sodium phosphate buffer (pH 7*2), adsorbed onto a column (1 cm x 20 cm) of Amberlite CG-50 cation exchange resin (200 tjo 400 mesh) and eluted with a solution of 0.8 M-NaCl in O-1 M-sodium phospha,te buffer (pH 7.2). The peak fraction of cytochrome c was collected, dialyzed against 0.01 M-(NH,)2C0, and freeze-dried. All steps in this purification procedure were carried out at 5 to 10°C.
3. Results (a) Isolation of iso-2-cytochrome c-dej%ent mutants Mutants completely or partially lacking iso-2-cytochrome c were isolated from p- cycl-I CYC7-1 strains CH33-lB-1, CH33-lD-1 and WlOO-26A-1. All three of these strains contain a high level of iso-2qtochrome c due to the CYC’I-1 translocation, completely lack iso-1-cytochrome c due to the cycl-I deletion and completely lack cytochrome a-a3 and b due to the mitochondrial mutation p-. Cultures were mutagenized with either ethyl methanesulfonate or ultraviolet irradiation, were plated on nutrient medium, and the resulting colonies stained with benzidine reagents. Unstained colonies were subcloned and the cytochrome c levels of the subclones were estimated by spectroscopic examination of intact cells. Only those mutants which had less than 40% of the cytochrome c content further analysis. In this way 139 mutants obtained after examining approximately
procedure.
of the parent strains were retained for deficient in iso-2-cytochrome c were
322,000 colonies with
the benzidine
YEBST
MUTANTS
OF ISO-2-CYTOCHROME
r
373
(b) IdentiJication of mutant loci It was anticipated that mutations of a variety of distinct loci could cause deficiencies of iso-2qtochrome c. In our previous publication (Downie et al., 1977) one iso-2-cytochrome c-deficient mutant, Bz39, was shown to contain a mutation, cyc7-l- 7, in the structural gene of iso-2-cytochrome c. Also cyc2 and cyc3 mutants were previously shown to cause marked deficiencies of iso-1-cytochrome c and presumably of iso-2qtochrome c (Sherman et al., 1965). In addition it was expected that mutations of other loci, such as those involved in porphyrin synthesis, could result in benzidinrnegative mutants. Genetic analyses were carried out in order to determine if a known cyc2 mutant’ gene could produce deficiencies of iso-2-cytochrome c in the presence of CYC7-2 branslocation that normally causes the formation of a high level of iso-2-cytochromc c. Meiotic segregants of the genotype cycl-1 CYC7-1 cyc2-8 were obtained from a cross cycl-1 CYC7-+ cyc2-8 x cycl-1 CYC7-1 CYC2+. Spectroscopic examinations indicated that the cyc2-8 allele reduced the level of iso-2-cytochrome c in CYC7- 1 strains by about 60%. The action of a cyc3 mutation on the C’YCT-1 gene was revealed with the new mutant Bz84 that contained only approximately 5% of iso-2-cytochrome c of the cycl-1 CYC7-1 parental strains. The Bz84 mutant was crossed to a cycl-1 CYCT+ CYC3+ strain and to a cycl-1 CYC7+ cyc3-3 strain and the cytochrome c contents of the resulting two diploids were estimated by spectroscopy. The diploid from the former cross contained a level of cytochrome c characteristic of cycl-1 CYC7-llcycl-7 CYC7-t- strains while the latter diploid contained less than 5% of the normal level. These results indicate that the Bz84 strain contains the CYC’I-I gene and a recessive gene which is allelic to the known cyc3-3 gene and which was designated ~~~3-18. Ttms a cyc3 mutant gene can diminish the level of iso-2-cytochrome c in CYC7-I st!rains by as much as 950/,. If the iso-2-cytochrome c deficiencies were caused by either the cyc2, cyc3 or cyc7 recessive mutations, the mutant loci could be conveniently identified by crossing the mutants to known cyc tester strains, and examining the diploids for complementation of their cytochrome c deficiencies. Each of the 139 mutants were first crossed to &rain AD176-2D (cycl-1 CYC’I-1) and the cytochrome c level of the diploid crosses were estimated by spectroscopic examinations; all such diploids contained high levels of cytochrome c characteristic of cycl-1 CYC7-llcycl-1 CYC7-1 or cycl-1 CYC7-11 cycl-1 GYC7+ crosses, demonstrating that all of the mutants were recessive. Further tests were carried out by determining the cytochrome c content from spectroscopic examinat,ions of diploids that were constructed by crossing the series of iso-2cvtochrome c-deficient mutants to several tester strains and determining whether or not complementation occurred. Table 1 illustrates how such complementation tests were used to identify mutations of two different genes, e.g. Bz39 having the ~~~7-1-1 mubation in the structural gene and Bz118 having the cyc3-19 mutation in the cyc3 gene. The mutation in the structural gene was detected by crossing Bz39 to the tester strain D672-5D (cycl-1 CYC7+) and examining the cytochrome c content of the diploid: the low level of iso-2-cytochrome c of the diploid cross is typical of cycl-1 CJ’C7-I- strains. Similarly the low level of iso-2-cytochrome c in the cross Bz118 (cycl-1 CYC7-I cyc3-19) x AD179-9D (cycl-I CYC7-1 ~~~3-18) indicates that t,he iso-2cytochrome c deficiency in Bz118 is due to cyc3 mutation.
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AND
F. SHERMAN
TABLE 1
Examples of complementation tests with the parental strain (cycl-1 CYC’I-1 CYC3+) and with mutants dejicient in iso-Z-cytochrome c
Strain
Genotype
Parental strain Bz39 Bz118
cycl-1 GYC7-1 cyc3+ cycl-1 cyc7-l-l cyc3+ cycl-1 CYC7-1 cyc3-19
Approximate oh of iso-2.cytochrome c Crossed to taster strains: cycl-1 CYC7+ CYC3+ cycl-1 CYC7-1 cyc3-18 (D672-6D) (AD179-9D)
Haploid
160 1 1
100 6 100
160 100 1
All strains lack iso-1-cytochrome c due to the deletion cycl-1. The content of iso-2-cytoohrome c is presented as the percentage of the amount of total cytochrome c found in normal strains (cYcl+ cyc7+ cYc3+).
TABLE 2
Iso-2-cytochrome c-dejcient mutants obtained with the benzidine staining method Strain no.
CH33-lB-1 CH33-lB-1 CH33-lD-1 CH33-lD-1 WlOO-23A-1 WlOO-23A-1
Mutagen
EMS &ii &ii . . &:a1
EMS, ethyl methanesulfonate;
Colonies examined
cyc7
56,000 103,000 10,000 33,000 40,000 80,000 322,000
7 30 1 4 6 14 62
u.v., ultraviolet
Number of mutants cyc2 cyc3 Total 4 16 0 0 4 b
29
6 6 1 0 8 0 21
17 62 2 4 18 19 112
irradiation.
The series of 139 iso-2-cytochrome c-deficient mutants were crossed to the tester strains D672-5D and AD179-9D listed in Table 1 and the cyc7 and cyc3 mutations were identified from the iso-2-cytochrome c deficiencies in the resulting diploids. In addition it was necessary to examine p- as well as p+ diploids from the crosses with D672-5D in order to identify certain cyc7 mutants which have deficiencies only in pstrains (see below). These mutants that complemented the cyc7 and cyc3 tester strains were crossed to AD260-3A (cycl-1 GYC7+ cyc2-8) in order to identify cyc2 mutations. The number of mutants of each of the cyc7, cyc3 and cyc2 loci are summarized in Table 2, along with the parent strains from which they were isolated and the mutagen used. In general, mutant cyc2 alleles reduced the level of iso-2-cytochrome c by about 60 to SO%, whereas cyc3 mutant alleles had a greater effect causing an 80% to nearly 100% reduction in the amount of iso-2-cytochrome c. As mentioned above, only mutants having a decrease in the amount of iso-2-cytochrome c of more than 60% were retained for genetic analysis. Thus mutations of the cyc7, cyc2, cyc3 and other loci producing only slight deficiencies have been excluded. In summary, 139 mutants containing less than 40% of CYC7-1 level of iso-2cytochrome c were isolated after examining approximately 322,000 colonies with the
YEAST
MUTANTS
OF ISO-“-CYTOCHROME
c
375
benzidine procedure; 112 of these mutants were identified as having mutations in slither the cyc7, cyc2 and cyc3 loci. Of the remaining mutants, only four had less tha.n 30/ of the parental level of iso-2-cytochrome c. These four mutants are presumably blocked in the synthesis of porphyrin or haem, since they totally lack not only iso-2cytochrome c but also cytochrome c1 which is at least partially present in p- mutants (Ross & Schatz, 1976). One of these mutants, Bz146, was crossed to strain D672-3B (a cycl-1 lysl-1 trp5-48 leul-12 his5-2) and the cytochrome content of meiotic progen! was determined. The complete tetrads gave rise to a 2 : 2 segregation of a pet phenotype which completely lacked all cytochromes, indicating that Bz146 contained a singk mutant gene preventing synthesis of all cytochromes. The 23 mutants not identified were not studied further. None of these were completelg deficient in iso-2qtochrome c, and only had a 60 to 80% decrease in amount of iso-2-cytochrome c. These mutants partially deficient in iso-2-cytochrome c could have nmtations at the cyc4, cyc5, cyc6 or cydl loci which have been primarily shown to only slightly affect the total amount of cytochrome c (see Sherman et al.. 1965 : Sanders et al.. 1973). Alternatively these 23 mutants may contain mutations nt other loc*i which specifically or generally control cytochrome c synthesis. (c) The cyc7 locus is distinct from the cyc2 and cyc3 loci Genetic tests were undertaken to determine whether the cyc7 locus is linked bo either the cyc2 or cyc3 loci. Poor spore viability from crosses that were heterozygous for the translocation, CYC7-ljCYC7+, made the analysis of the meiotic segregation difficult. The cross between strains AD260-3C (a cycl-1 cycZ-8 C YC7+) and WlOS- 1D (a cycl-1 CYCZ+ CYC7-I) yielded only two complete tetrads, one being tetratype and the other a, parental ditype. Of the other 20 surviving segregants from ten tetrads. ten were recombinant type and ten were parental type. These data clearly demonstrated that the cyc2 and cyc7 genes are distinct and probably unlinked. Similarly it was concluded that the cyc3 and cyc7 genes are distinct and probahl) unlinked since the parental ditype/non-parental ditype/t,etratype rntio was 1 :O : 8 in trtrads having four viable segregants that were derived from several crosses which contained the heterozygous genes, CYC3flcyc3, and either ofthe hetorozygous translocations CYC7+/CYC7-1 or CYC7+/cyc7-l-l. The chromosomal location of these three distinct loci will be reported elsewhere. (d) Characteristics
of mutants of the structural
geyze CYC7
A total of 62 mutants were identified as being at the structural gene of jso-2cytochrome c. We have attempted to group these 62 mutants into classes based on the amount and characteristics of iso-2-cytochrome c formed under different, growt,h conditions and formed by suppression. It has been shown (Sherman et al., 1968,1974) that the level of iso-l-cytochrome c present in many cycl mutants can be affected by various growth conditions. When mutants are grown at the lower temperature of 22°C compared with 3O”C? some produce increased amounts of iso-1-cytochrome c. These iso-1-cytochromes c may 01 may not be functional (Sherman et al., 1974) and some have altered absorption spectra, (Schweingruber et al., 1977). Also various levels of iso-1-cytochrome c appear when certain cycl mutants are coupled to UAG or UAA suppressors (Gilmore et al.. 1971; Liebman et al., 1976). We have used these and other criteria to classify mutants of iso-2-cytochrome c outlined in Table 3.
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AND
F. SHERMAN
TABLE 3
Properties of the cyc’l mutants of the structural
gene of iso-2-cytochrome c Is0 -2 levels (% of cucr-1)
Completely or almost completely UAA-suppressible
deficient
30°C
22°C
