Current Genetics
Current Genetics (1981) 4:197-204
© Springer-Verlag 1981
Genetic and Biochemical Characterization of Antisuppressor Mutants in the Yeast Saccharomyces cerevisiae Junpei Ishiguro Department of Biology, Faculty of Science, Konan University,Okamoto, Kobe 658, Japan
Summary. Five mutants carrying anti.suppressors which reduce the efficiency of SUP46 suppressor of the yeast, Saccharomyces cerevisiae, were isolated. One of them was found to have an altered protein, $27, of 40S ribosomal subunit by using two-dimensional gel electrophoresis. A single recessive gene, designated asull, was responsible for the mutation, and the heterodiploid (asu11/+) strain was shown to contain both altered and normal $27 proteins, suggesting that the asull locus codes for the $27 protein. The mutant was resista_nt to an aminoglycoside antibiotic, paromomycin which has been known to induce translational errors. These results together with the previous finding that SUP46 suppressor mutant contains an altered S l l ribosomal protein (Ishiguro et al. 1981) suggest that the secondary mutation in the ribosomal protein, $27, renders the restrictive effect to increased translational ambiguity caused by the alteration of S11 protein. Key words: Yeast-Ribosomal proteins-Antisuppressor
Introduction
Investigation of ribosomal mutations which affect translational fidelity has provided a lot of genetical and biochemical information about ribosomal components in Escherichia coli (Nomura et al. 1977, see review). In an eukaryotic organism, Saccharomyces cerevisiae, several omnipotent suppressors which act on a wide range of mutations including ochre (UAA), amber (UAG) and umber (UGA) mutations have been found and thought to be analogous to ram suppressors of t?. coll. Two loci responsible for the recessive omnipotent suppressors have been mapped, namely supl (sup45 or supQ) on the right arm of chromosome II, and sup2 (sup35 or supP)
on the right arm of chromosome IV (Inge-Vechtomov and Andrianova 1970; Hawthorne and Leupold 1974; Gerlach 1975; Surguchov et al. 1980a). As expected, genes supl and sup2 were suggested to code for proteins involved in termination of protein synthesis (Smimov et al. 1974, 1976; Surguchov et al. 1980b). Furthermore, Surguchov et al. (1980a) showed that the supl mutant ribosome led to mistranslation at a higher level than the wild type ribosome in a cell-free system. However, the mutational alteration of the ribosomal components has been obscure, although the sup2 mutant ribosome was found to contain an increased amount of L30 protein as compared with that of the parental ribosome (Smirnov et al. 1978). Recently, Ono et al. (1981) have found a novel dominant omnipotent suppressor, SUP46, which is located on the right arm of chromosome II, but distinct in the locus from the recessive suppressors described above. The wide range of suppression of SUP46 has been shown to be due to an abnormally high rate of mistranslation for which 40S ribosomal subunit is responsible Masurekar et al. 1981). Finally, the SUP46 ribosome was found to contain an altered protein S11 of 40S ribosomal subunit, the protein participating in the ribosomal ambiguity (Ishiguro et al. 1981). On the other hand, Liebman and Cavenagh (1980) and Liebman et al. (1980}have isolated two mutants carrying antisuppressors which reduce the efficiency of the recessive omnipotent suppressors. They were referred to as asu9 and ASUIO, which were recessive and dominant, respectively when scored on the basis of suppression of leu2-1 (ochre). Molecular mechanism of action of the antisuppressors has not yet been clarified. The author isolated five mutants carrying antisuppressors which act on the dominant omnipotent suppressor, SUP46. In the present paper, the author describes isolation and some genetic and biochemical characterization of the mutants. The results will suggest that an antisup0172-8083/81/0004/0197/$01.60
198 pressor, designated asull, locus c o d e s for $27 p r o t e i n o f the 40S ribosomal s u b u n i t and the m u t a t i o n renders the restrictive e f f e c t to the increased m i s t r a n s l a t i o n caused b y the SUP46 suppressor.
Materials and M e t h o d s
Chemicals. Yeast-Nitrogen Base W/O Amino Acids and Bacto Peptone were obtained from Difco Laboratories. Ampholines (pH 5 - 7 and pH 3.5-10) were obtained from LKB. Aminoglycoside antibiotics, Hygromycin B and Paromomycin were kindly supplied by Takeda Chemical Industries, Osaka, Japan.
Strains, Culture Conditions and Genetic Procedures. Strains used in this work are listed in Table 1. Strains, BO133-3B and 0-9003 are described by Ono etal. (Ono etal. 1979a, b; Ono et al. 1981). Strains, OK29-1C and IS14-9D, and strain, OG7-2C were kindly supplied by Dr. B-I. Ono and Dr. K. Matsumoto, respectively. For scoring of genetic markers, minimal medium (0.67% bactoyeast nitrogen base w/o amino acids, 2% glucose, 2% agar) containing the appropriate supplements was used (Sherman and Lawrence 1974; Ono et al. 1979a). Susceptibility to aminoglycoside antibiotic was scored by using YEPD medium (1% yeast extract, 2% bacto peptone, 2% glucose, 2% agar) containing paromomycin (500 t~g/ml). For ribosome preparation, ceils were grown aerobically in liquid YEPD medium at 27 °C, and harvested at late logarithmic growth phase. Standard methods for crossing, sporulation and dissection were used as described by Sherman and Lawrence (1974).
Isolation of Antisuppressor Mutants. A strain BO133-3B was able to grow on YEPD medium containing 100 ~g/ml ofhygromycin B, while growth of a 0-9003 strain was completely inhibited at the drug concentration. About 107 cells of 0-9003 (SUP46 strain) were spread on YEPD plate and irradiated by UV light with a dose of about 50% survival. After overnight incubation in the dark, the plate was replica plated on YEPD medium containing hygromycin B (200 ~g/ml). Resistant revertants to hygromycin B were isolated, and their genetic markers were scored by using the appropriate synthetic medium. In order to select the mutants carrying antisuppressors, revertants which lost the ability of suppression of leu2-1 gene were crossed with tester strain, OK29-1C. Resultant diploid strains were isolated and sporulated. Each of the spore suspensions obtained from the respective diploid strains was spotted on synthetic medium lacking leucine. When a certain group of the spore suspensions was observed to grow up on the medium, the corresponding parental revertant was used as an antisuppressor mutant.
Effects of Aminoglycoside Antibiotics and High Temperature {38 °C) on Cell Growth. After cells were grown in YEPD medium at 27 °C for 2 h, either paromomycin (final conc. of 500 #g/ml) or hygromycin B (final eonc. of 200 ~g/ml) was added and cultivation was continued. Cells were also grown in YEPD medium at 38 °C. Cell growth was measured by liglat absorbance at 660 nm.
Preparation of Ribosomal Proteins. Purified 80S ribosomes and ribosomal subunits were prepared according to essentially the same method described by Torafio et al. (1974). Cells werewashed and disrupted with glass beads in medium A (10 mM Tris-HC1 pH 7.5. 5 mM magnesium acetate, 10 mM potassium chloride, 10 mM 2mercapto ethanol). The cell homogenate was centrifuged at
J. Ishiguro: Characterization of Yeast Antisuppressor 8,000 Xg for 10 min and then, 30,000 Xg for 20 min twice. The supernatant was centrifuged at 105,000 Xg for 2 h. The pellet (crude ribosomes) was suspended in medium B (30 mM Tris-HC1 pH 7.2, 0.5 M ammonium chloride, 0.1 M magnesium acetate, 0.25 M sucrose, 5 mM 2-mercaptoethanol), and left in ice for overnight. The suspension was mixed with medium A (1 : 1 volume), and centrifuged at 30,000 Xg for 20 min and then, 105,000 Xg for 90 min. The pellet obtained was suspended again in medium A, and centrifuged at 30,000 Xg for 20 rain. Purified ribosomes were obtained from the final supernatant by centrifugation. For the preparation of ribosomal subunits, crude ribosomes were suspended in medium C (50 mM Tris-HC1 pH 7.7, 0.8 M potassium chloride, 12 mM magnesium acetate, 20 mM 2-mercaptoethanol), and layered on 15-30% sucrose gradient made in medium C. The centrifugation was performed at 20,000 rpm for 15 h with Hitachi RPS25-2 rotor. Fractions corresponding to the 40S and 60S subunits were collected separately, and each of the suspensions was diluted with medium D (30 mM magnesium acetate, 20 mM 2-mercaptoethanol) and sedimented by centrifugation at 190,000 Xg for 5 h. Extraction of ribosomal proteins was carried out as described by Sherton and Wool (1974). The ribosomes and ribosomal subunits were suspended in medium E (10 mM Tris-HC1 pH 7.7, 33 mM magnesium acetate) and two volumes'of glacial acetic acid were added. The mixture was left in ice for 60 min with shaking, and rRNA was sedimented by centrifugation. The protein solution was dialyzed against 1 N acetic acid followed by lyophilization.
Two-Dimensional Polyacrylamide Gel Electrophoresis. Two-dimensional polyacrylamide gel electrophoresis was performed as described by Ishiguro and McLaughlin (unpublished data). Nonequilibrium pH gradient gel based on the method of O'Farrell et al. (1977) and acidic gel based on the method of Kaltschmidt and Wittmann (1970) were used in the first (l-D) and the second (2-D) dimensions, respectively. The solutions needed are as follows. Sample solution: 9 M urea, 5% 2-mercaptoethanol, 0.2% Ampholines (pH 3.5-10). Overlay solution: 8 M urea, 0.2% Ampholines (pH 3.5-10), 0.8% Ampholines (pH 5-7). Separation gel for l-D: 4% acrylamide, 0.2% N,N'-methlenebisacrylamide, 9 M urea 2% Ampholines (pH 3.5-10). For polymerization 20 pl of 10% ammonium persulfate and 20 ~1 of N,N,N',N'-tetramethylethylenediamine (TEMED) were added to 10 ml of gel mixture. Electrode buffer for l-D: 0.016 M phosphoric acid (upper) and 0.019 M sodium hydroxide (lower). Dialysis buffer for 1-D gel: 8.3 M urea, 0.013 M acetic acid, 0.012 M potassium hydroxide (final pH 6.4). Separation gel for 2-D: 18% acrylamide, 0.5% N,N'-methylenebisacrylamide, 6 M urea, 0.91 M acetic acid, 0.048 M potassium hydroxide, 0.58 TEMED (final pH 4.6). For polymerization ammonium persulfate was added to make final 0.06%. Electrode buffer for 2-D: 0.186 M glycine, 0.026 M acetic acid. Sample proteins (approx. 300 ug) were dissolved by the sample solution (50 ~1), and the protein solution was applied on the surface of a gel. The overlay solution was carefully layered over the sample solution and then, a tube was filled with the upper electrode buffer solution. Electrophoresis for 1-D was carried out at 400 constant voltage for 2-2.5 h with the anode on the top and the cathode on the bottom. After the electrophoresis, the gel was dialyzed against the dialysis buffer at room temperature for 90 min and then, embedded in the 2-D gel. Electrophoresis for 2-D was carried out at 80 constant voltage for 15 h with the anode on the top and the cathode on the bottom. The small size gel plate '(120 mm wide - 100 mm long - 2 mm thick) was used in the experiment.
199
J. Ishiguro: Characterization of Yeast Antisuppressor Table 1. Genotype of Strains used in this Investigation Strain
Genotype
BO133-3B 0-9003 IS14-9D OK29-1C OG7-2C
a cycl-72 canl-lO0 met8-1 leu2-1 ura4-1 his5-2 lysl-1 trp5-48 ~ + a SUP46cycl-72canl-100 met8-1 leu2-1 ura4-1his5-2 lysl-1 trp5-48 ~+ SUP46 met8-1 leu2-1 his5-2 ura4-1 ade2-1 ~ + a met8-1 leu2-1 ~ gal4-62 met8-1 leu2-1 t) +
Results
ura4-1, his5-2 and amber (UAG) mutation, metS-1 were scored. None of the revertants was able to suppress any markers, while 0-9003 strain was able to suppress all of them except his5-2 (Table 2). In the following genetic study, leu2-1 was mainly used as a UAA suppressible marker, since UAG marker, metS-1, was sometimes weakly suppressed in the presence of cytoplasmic factor [psi+]. The revertants were crossed with tester strain, OK29-1C, and the resultant diploids which were homozygous for leu2-1 and heterozygous for SUP46, were sporulated. Each of the spore suspensions obtained from the respective crosses was spread on appropriate synthetic medium lacking leucine (SC-leu) in order to distinguish between antisuppressor carrying-revertants and intragenic revertants at SUP46 locus. Five groups of the spore suspensions grew up on the medium, indicating that the corresponding five revertants still have SUP46 suppressor, and the suppression activity could be weakened by the secondary mutation unlinked to SUP46. The suppression patterns and other properties of these antisuppressor mutants are shown in Table 2.
Isolation o f A ntisuppressors
Aminoglycoside antibiotics, paromomycin and hygromycin B have been shown to cause mistranslation in vivo and in vitro in the yeast Saccharomyces cerevisiae (Singh et al. 1979; Palmer et al. 1979). The SUP46 mutant is hypersensitive to paromomycin because of its additive effect on mistranslation (Masurekar et al. 1981). The SUP46 m u t a n t was also found to be hypersensitive to hygromycin B. The antibiotic of 200/~g/ml concentration inhibited the growth of SUP46 m u t a n t completely, while the parental strain was not affected to grow at the drug concentration. Mutants carrying antisuppressors which reduce the efficiency of SUP46 suppressor were isolated from the hygromycin B-resistant revertants of 0-9003 strain carrying SUP46 gene. Sixteen resistant revertants were isolated and their genetic markers including ochre (UAA) mutation canl-lO0, leu2-1, lysl-1,
Table 2. Properties of Antisuppressor Mutants Strain
BO133-3B O-9003a J200 le J2002e J2004e J2006e J2022e
Gro'wth on YEPD + paromo a
SC -met
SC +canb
SC -leu
+ + + + + +
± + + + + + ±
+ ± + + + + +
+ . . .
Ribosomal Protein c SC -lys
SC -ura
SC -his
S11
$27
+
-+
-
Normal Altered Altered Altered Altered Altered Altered
Normal Normal Normal Normal Normal Altered Normal
.
.
.
. .
. .
. .
a Paromomycin (500 #g/ml) b Canavartine Sulfate (60 t~g/ml) c Ribosomal proteins were prepared from each of the strains and analyzed by two-dimensional gel electrophoresis (see Material& Methods) d Parental strain (SUP46 strain) e Antisuppressor mutants obtained from the strain, 0-9003 Scoring was performed as follows: + good growth by 5 days; ± poor growth by 7 days; - no sign of growth by 7 days
200
J. Ishiguro: Characterization of Yeast Antisuppressor
Table 3. Segregation of Antisuppressor (asu11) from SUP46 suppressor Strain
Genotypea
Number of Tetrads b
SUP46~+asu11/+
D 5 (J2006 x OK29-1C)
P
NP
T
4
7
20
D5 diploid strain is homozygous for leu2-1 T-he type of segregation of the tetrads was determined by scoring the degree of growth on SC-leu plates as follows. P (SUP46/asu11 X 2, +/+ X 2): 0+ and 4 - ; NP (SUP46~+ X 2, +/asu11 X 2): 2+ and 2-; T (SUP46/asu11, SUP46~+,+/asu11, +/+): 1+ and 3 . + means good or poor growth by 5 days and - means no sigh of growth by 5 days
a
b
c
B
o
I.B" A
g
/_ O.EJ
5
1'0
rl m4
5
10
UlR4
8
10
1114
Hours Fig. l a - c . The effects of aminoglycoside antibiotics on cell growth. The arrows indicate dosage of Higromycin B (final conc. of 200 pg/ml) or Paramomycin (finn conc. of 500 pg/ml). Control (o-o), Hygromycin B (zx-z~)and Paramomycin (D--D). BO133-3B (a), 0-9003 (SUP46) strain (b) and J2006 (asu11) strain (c)
Genetic Properties o f A ntisuppressors Five antisuppressor mutants isolated independently were crossed with tester strain, OK29-1C. The resultant diploids which were homozygous for the suppressible marker, leu2-1, and heterozygous for the SUP46 suppressor and each of the five antisuppressors, respectively. All of the five diploid strains grew up on the SC-leu plate, indicating that the corresponding mutant strains, namely J2001, J2002, J2004, J2006 and J2022 contained recessive antisuppressors. An antisuppressor that was carried in the strain, J2006, was denoted asull, and the mutant strain was studied further (see Table 2). In order to examine the segregation of asu11, a diploid strain homozygous for leu2-1 and SUP46 and heterozygous for asull was constructed (J2006 x IS14-9D) and sporulated. How-
ever, most of the asci gave rise to two or three spores. This observation suggests that in most cells meiosis was abnormally performed because of the presence of SUP46 homo-allele in the diploid. The SUP46 and other omnipotent suppressors generally cause retardation of mating, sporulation, spore germination and cell growth (Ono et al. in preparation). Only two sets of complete tetrads were obtained and they segregated 2+ and 2 - for the suppression of leu2-1. The segregation of asul 1 was also examined by using a diploid strain which was homozygous for leu2-1 and heterozygous for SUP46 and asull, respectively. The meiotic progenies obtained from the diploid strain were scored by the suppression of leu2-1, and the types of the segregation were determined in each of the tetrads as follows. Parental ditype (P): SUP46/asull X2, +/+ X2; Nonparental ditype (NP):
J. Ishiguro: Characterization of Yeast Antisuppressor
a
201
C
b
2,r"
E o ¢.D 1.5
e-. ~;~ 1 . 0
//
o5 / 8 /
I
B
I 1.,1 2 4
10
'
5
' I IR-
10
I
5
*
I n
"'
I#E4
Hours Fig. 2a-c. The effects of high temperature (38 °C) on cell growth. Cells were incubated at 27 °C ( o - o ) or 38 °C (*-*). BO133-3B strain (a), 0-9003 (SUP46) strain (b) and J2006 (asu11) strain (c)
SUP46~+ X2, +/asul 1 X2; Tetratype (T): SUP46/asul 1, SUP46~+, +/asull, +/+. Since only the meiotic progeny with the genotype of SUP46/+ can grow up on Sc-leu plate, P, NP and T types of tetrads might reveal 0 + : 4 - , 2+ : 2 and 1+ : 3 - segregations for supprssion of leu2-1, respectively. As seen in Table 3, the antisuppressor, asull, segregated independently from SUP46 suppressor. The ratio of P : NP : T was nearly 1 : 1:4. These results demonstrate that a single recessive gene might be responsible for the asull mutation.
Effects of Aminoglycoside Antibiotics and High Temperature on Cell Growth Effects of aminoglycoside antibiotics on cell growth are shown in Fig. 1. Cell growth of SUP46 strain (0-9003) was completely inhibited by paromomycin (500 #g/ml) or hygromycin B (200/lg/ml), while that of the parental wild strain (BO133-3B) was not significantly affected at the drag concentrations. The asull strain (J2006) which was isolated as hygromycin B-resistant revertant appeared to have cross-resistance to paromomycin. It also can be seen that in the J2006 strain, retardation of cell growth caused by SUP46 mutation was restored to almost the normal level. Effects of high temperature on cell growth are shown in Fig. 2. The growth of the 0-9003 strain was significantly inhibited at 38 °C, while that of the BO133-3B strain was not affected. The J2006 strain also appeared to gain the resistance to the high temperature.
Ribosomal Proteins from A n tisuppressor Mutants Ribosomal proteins obtained from five antisuppressor mutants were analyzed by two-dimensional gel electrophoresis. The results are summarized in Table 2. The ribosomes of all mutants had an altered S11 protein, which had been shown to be responsible for SUP46 mutation (Ishiguro et al. 1981, and see Fig. 3a, b in this paper). This result consists with the genetic evidences that SUP46 suppressor is still present in the five revertants. Added to this, another altered ribosomal protein, $27, was found in the strain, J2006, as shown in Fig. 3c. This finding was also confirmed in the 40S ribosomal subunits (Fig. 4). The mutated protein moved less towards the cathode in the pH gradient gel as compared with the corresponding wild protein, indicating that the mutant protein has a decreased pH of its isoelectric point. A diploid strain heterozygous for asull and homozygous for SUP46 was constructed (J2006 x IS14-9D) and the ribosomal proteins were examined. As seen in Fig. 3d, both altered and normal $27 proteins and only altered S11 protein were clearly observed in the gel. This result suggests that the structural gene coding for the $27 protein might be altered but not for the modifying enzyme acting on the protein.
Correlation between A ntisuppressor (asul 1) and Alteration of Ribosomal Protein ($27) A diploid strain was constructed to be homozygous for the suppressible marker, leu2-1, and heterozygohs for
202
J. Ishiguro: Characterization of Yeast Antisuppressor
Fig. 3a-d. Sections of two-dimensional gel electrophoretograms of 80S ribosomal proteins. Nonequilibrium pH gradient gel electrophoresis (pH 3.5-10) in 1-D and acidic gel electrophoresis (pH 4.6) in 2-D were carried out as described in the Materials and Methods. Typical examples obtained from BO133-3B strain (a) 0-9003 (SUP46), J2001, J2002, J2004 and J2022 strains (b) J 2006 (asu11) strain (c) and Heterodiploid (asu11/+) strain (d). The S l l and $27 proteins are indicated by the arrows
the SUP46, asull and other markers, respectively, in order to ascertain correlation between asull mutation and alteration of $27 protein. The meiotic segregants from the diploid were scored by the appropriate markers, and their ribosomal proteins were analyzed above. The result is shown in Table 4. None of the segregants grew up on the SC-leu plate, indicating that the tetrad has a presumable genotype of P type (SUP46/asu11 X2, +/+ X2). Segregation of other markers indicated that meiosis was normal. In this case, it was shown that two out of four segregants contained altered S l l and altered $27 proteins, and the remaining two segregants contained normal S l l and normal $27 proteins. In the case o f a T type-tetrad, it was also observed that only one segregant was able to grow up on the SC-leu plate of which ribosomes contained altered S11 and normal $27 proteins (data not shown). In certain progenies with a presumable genotype of SUP46/asull, normal $27 protein was found to be somewhat leaky (Table 4).
, F i g . 4a and b. Two-dimensional gel electrophoretograms of proteins of 40S ribosomal subunits obtained from 0-9003 (SUP46) strain (a) and J2006 (asu11) strain (b). The $27 proteins are indicated by the arrows
J. Ishiguro: Characterization of Yeast Antisuppressor
203
Table 4. Correlation between Antisuppressor (asu11) and Alteration of Ribosomal Protein ($27) Strain
Growth on SC-
presumable genotype
leu
lys
ura
his
+
-
+ -
+ +
Ribosomal Protein S 11
$27
Altered Altered Normal Normal
Altered Altered & Normal Normal Normai
D6 (J2006 x OG7-2C)a Segregant
a
11-A -B -C -D
.
-
.
. + +
.
SUP46/asul i SUP46/asul 1 +/+ +/+
diploid strain is homozygous for leu2-1 and heterozygous for other markers. + indicates good growth no sign of growth by 7 days D6
Discussion
The SUP46 suppressor mutant was previously shown to have ribosomes which cause abnormally high rates of mistranslation, and therefore, to be unusually sensitive to paromomycin which hase been known to stimulate translational errors (Masurekar et al. 1981). The SUP46 xibosomes were also found to contain altered S l l protein of 40S subunits, which was responsible for the suppression by misreading (Ishiguro et al. 1981). In the present study, the mutant carrying an antisuppressor, designated asull, which reduces the efficiency of SUP46 suppressor has been shown to have additional alteration in $27 protein of 40S ribosomal subunits. The antisuppressor mutant which was isolated from hygromycin B-resistant revertants displayed cross-resistance to paromomycin, suggesting that the mutation renders restrictive effect to translational errors caused by alteration of the S11 protein. The correspondence between the asull mutation and the alteration-of $27 protein was ascertained by examining meiotic segregants from a heterozygous diploid. In this experiment, however, it was observed that both normal and abnormal $27 protein were observed in certain meiotic segregants carrying SUP46/asull mutations. The cause of this "leakiness" of the asull mutation in certain meiotic progenies is unknown. One possibility is that asull is not the structural gene for $27, but codes for a modifying enzyme acting on $27. However this is inconsistent with the production of both normal and abnormal $27 proteins in a diploid heterozygous asul 1/+ (Fig. 3d). An alternative explanation is that the "leakiness" is caused by misreading of asull mRNA enhanced by the SUP46 mutation. The SUP46 suppressor mutant was found to be temperature sensitive at 38 °C. The asu11 mutation was accompanied with restoration of the temperature sensitivity, suggesting that the alteration of ribosomal components, S l l and $27 proteins, would participate in the temperature sensitivity of cell growth. It has been demonstrated that ts-revertant which carries recessive sup-
by
5 days and - indicates
pressor, sup2, has a defect in termination of protein synthesis and accumulates 80S ribosomes bearing peptidyl tRNA at 36 °C (Smimov et al. 1974, 1976 ; Surguchov et al. 1980). In the SUP46 mutant, however, polysome decay was not observed under the restrictive temperature (results not shown). Of five antisuppressor mutants isolated independently, one was found to have alteration in ribosomal protein, $27, and the remaining four mutants were not. This result suggests the possibility that at least two kinds of antisuppressors which are effective on SUP46 suppressor would be present, although allelism tests were not performed. Recently two antisuppressors have been found and designated asu9 and ASU10 (Liebman and Cavenagh 1980; Liebman et al. 1980). The former acts on recessive omnipotent suppressors, sup35 and sup45, and the latter is specific for only sup35 suppressor. In the present study, it is not known if asull antisuppressor acts on sup35 or sup45. All five antisuppressors including asull were recessive as well as asu9 when scored on the basis of suppression of leu2-1. Thus, the possibility remains that some of them would be the same as asu9 mutation. Liebman and Cavenagh (1981) have showed by using two-dimensional gel electrophoresis that 40S ribosomal subunits from the strains carrying asu9 mutation contain a small amount of extra protein which is absent in the asu9 non-carrying-strains. However, no other significant differences were observed between them, and the extra protein may not be an intrinsic ribosomal protein. Therefore, the asu11 mutation is considered to be different from the asu9 mutation. The SUP46 mutation resembles ram mutations in Escheriehia coli in the sense that both mutations cause alterations of ribosomal proteins (S11 in yeast and $4 or $5 in E. coli) and lead to translational ambiguity (Masurekar et al. 1981; Ishiguro et al. 1981). Amino acids composition of S11 protein (YS11 of Hiroshima Nomenclature, Otaka and Osawa 1981) seems to be highly analogous to that of $4 p r o t d n of E. coli (Kaltschmidt et al. 1970; Higo and Otaka 1979). Indeed, structural ho-
204 mology between them can be assumed by using Difference Index Method (Difference Index = 7.6, Higo and Otaka at Hiroshima Univ., personal communication). The S11 protein hase been known to be a rRNA bindingprotein as well as in the case of $4 protein of E. coli (Zimmermann 1974; Reyes et al. 1978). In contrast, strA mutations in E. coli restrict ribosomal ambiguity caused by the ram mutations, and alter a ribosomal protein, S12 (Gorini 1974 see review). The antisuppressor mutation described in this paper is thought to be analogous to the strA mutations. Interestingly, the $27 protein of yeast has a similar electrophoretic mobility to the S12 protein ofE. coli in two-dimensional gel. Thus, our findings would be useful for expanding not only biochemical but also evolutional knowledges about eukaryotic ribosomal proteins.
Acknowledgement. The author wishes to thank Dr. B.-I. Ono (Okayama Univ., Okayama, Japan) for supplying the strains and for helpful discussions and comments, and the author is also grateful to Dr. Y. Arakatsu (Konan Univ., Kobe, Japan) for supporting this work and for critical reading of the manuscript.
References Gerlach WL (1975) Mol Gen Genet 138:53-63 Gorini L (1974) Streptomycin and Misreading of the Genetic Code. In: Momura M, Tissi~resA, Lengyel P (ed) Ribosomes. Cold Spring Harbor Monograph Series, p 791 Hawthorne DC, Leupold U (1974) Current Topics Microbiol lmmuno164:1-47 Higo K, Otaka E (1979) Biochemistry 18:4191-4196 Inge-Vechtomov SG, Andrianova VM (1970) Genetika 6:103115 lshiguro J, Ono B-I, Masurekar M, McLaughlin CS, Sherman F (1981) J Mol Biol 147:391-397 Kaltschmidt E, Wittmann HG (1970) Anal Biochem 36:401-412 Kaltsehmidt E, Dzionara M, Wittmann HG (1970) Mol Gen Genet 109:292-297 Liebman SW, Cavenagh MM (1980) Genetics 95:49-61
J. Ishiguro: Characterization of Yeast Antisuppressor Liebman SW, Cavenagh MM, Bennett LN (1980) J Bacteriol 143:1527-1529 Licbman SW, Cavenagh MM (1981) Current Genet 3:27-29 Masurekar M, Palmer E, Ono B-I, Wilhelm JM, Sherman F (1981) J Mol Biol 147:381-390 Nomura M, Morgan EA (1977) Ann Rev Genet 11:297-347 O'Farrell PZ, Goodman HM, O'Farrell PH (1977) Cell 12:11331141 Ono B-I, Stewart JW, Sherman F (1979a) J Mol Biol 128:81 - 100 Ono B-I, Stewart JW, Sherman F (1979b) J Mol Biol 132:507520 Ono B-I, Stewart JW, Sherman F (1981) J Mol Biol 147:373379 Otaka E, Osawa S (1981) Mol Gen Genet 181:176-182 Palmer E, Wilhelm JM, Sherman F (1979) Nature 277:148-150 Reyes R, Vazquez D, Ballesta JPG (1978) Biochim Biophys Aeta 521:229-234 Sherman F, Lawrence CW (1974) In: King RC (ed) Handbook of Genetics. Plenum Press, New York, p 359 Sherton CC, Wool IG (1974) Mol Gen Genet 135:97-112 Singh A, Ursic D, Davies J (1979) Nature 277:146-148 Smknov VN, K_reier VG, Lizlova LV, Andrianova VM, IngeVechtomov SG (1974) Mol Gen Genet 129:105-121 Smirnov VN, Surguchov AP, Fominykch ES, Lizlova LV, Saprygina TV, Inge-Vechtomov SG (1976) FEBS Lett 66:12-15 Smirnov VN, Surguchov AP, Smirnov VV, Berestetskaya YuV, Inge-Vechtomov SG (1978) Mol Gen Genet 163:87-90 Surguchov AP, Berestetskaya YuV, Fominykch ES, Pospelova EM, Smirnov VN, Ter-Avanesyan MD, Inge-Vechtomov SG (1980a) FEBS Letters 111:175-178 Surguchov AP, Fominykch ES, Berestetskaya YuV, Smirnov VN, Inge-Vechtomov SG (1980b) Mol Gen Genet 177:675-680 Torago A, Sandoval A, Heredia CF (1974) Soluble Protein Factors and Ribosomal Subunits from Yeast. Interaction with Aminoacyl-tRNA. In: Moldave K, Grossman L (ed) Methods in Enzymotogy 30. Academic Press, New York London, p 254 Zimmermann RA (1974) RNA-Protein Interactions in the Ribosomes. In: Nomura M, Tissi6resA, Lengyel P (ed) Ribosomes. Cold Spring Harbor Monograph Series, p 225
Communicated b y B. Cox Received July 3, 1981
Current Genetics (1981) 4:197-204
© Springer-Verlag 1981
Genetic and Biochemical Characterization of Antisuppressor Mutants in the Yeast Saccharomyces cerevisiae Junpei Ishiguro Department of Biology, Faculty of Science, Konan University,Okamoto, Kobe 658, Japan
Summary. Five mutants carrying anti.suppressors which reduce the efficiency of SUP46 suppressor of the yeast, Saccharomyces cerevisiae, were isolated. One of them was found to have an altered protein, $27, of 40S ribosomal subunit by using two-dimensional gel electrophoresis. A single recessive gene, designated asull, was responsible for the mutation, and the heterodiploid (asu11/+) strain was shown to contain both altered and normal $27 proteins, suggesting that the asull locus codes for the $27 protein. The mutant was resista_nt to an aminoglycoside antibiotic, paromomycin which has been known to induce translational errors. These results together with the previous finding that SUP46 suppressor mutant contains an altered S l l ribosomal protein (Ishiguro et al. 1981) suggest that the secondary mutation in the ribosomal protein, $27, renders the restrictive effect to increased translational ambiguity caused by the alteration of S11 protein. Key words: Yeast-Ribosomal proteins-Antisuppressor
Introduction
Investigation of ribosomal mutations which affect translational fidelity has provided a lot of genetical and biochemical information about ribosomal components in Escherichia coli (Nomura et al. 1977, see review). In an eukaryotic organism, Saccharomyces cerevisiae, several omnipotent suppressors which act on a wide range of mutations including ochre (UAA), amber (UAG) and umber (UGA) mutations have been found and thought to be analogous to ram suppressors of t?. coll. Two loci responsible for the recessive omnipotent suppressors have been mapped, namely supl (sup45 or supQ) on the right arm of chromosome II, and sup2 (sup35 or supP)
on the right arm of chromosome IV (Inge-Vechtomov and Andrianova 1970; Hawthorne and Leupold 1974; Gerlach 1975; Surguchov et al. 1980a). As expected, genes supl and sup2 were suggested to code for proteins involved in termination of protein synthesis (Smimov et al. 1974, 1976; Surguchov et al. 1980b). Furthermore, Surguchov et al. (1980a) showed that the supl mutant ribosome led to mistranslation at a higher level than the wild type ribosome in a cell-free system. However, the mutational alteration of the ribosomal components has been obscure, although the sup2 mutant ribosome was found to contain an increased amount of L30 protein as compared with that of the parental ribosome (Smirnov et al. 1978). Recently, Ono et al. (1981) have found a novel dominant omnipotent suppressor, SUP46, which is located on the right arm of chromosome II, but distinct in the locus from the recessive suppressors described above. The wide range of suppression of SUP46 has been shown to be due to an abnormally high rate of mistranslation for which 40S ribosomal subunit is responsible Masurekar et al. 1981). Finally, the SUP46 ribosome was found to contain an altered protein S11 of 40S ribosomal subunit, the protein participating in the ribosomal ambiguity (Ishiguro et al. 1981). On the other hand, Liebman and Cavenagh (1980) and Liebman et al. (1980}have isolated two mutants carrying antisuppressors which reduce the efficiency of the recessive omnipotent suppressors. They were referred to as asu9 and ASUIO, which were recessive and dominant, respectively when scored on the basis of suppression of leu2-1 (ochre). Molecular mechanism of action of the antisuppressors has not yet been clarified. The author isolated five mutants carrying antisuppressors which act on the dominant omnipotent suppressor, SUP46. In the present paper, the author describes isolation and some genetic and biochemical characterization of the mutants. The results will suggest that an antisup0172-8083/81/0004/0197/$01.60
198 pressor, designated asull, locus c o d e s for $27 p r o t e i n o f the 40S ribosomal s u b u n i t and the m u t a t i o n renders the restrictive e f f e c t to the increased m i s t r a n s l a t i o n caused b y the SUP46 suppressor.
Materials and M e t h o d s
Chemicals. Yeast-Nitrogen Base W/O Amino Acids and Bacto Peptone were obtained from Difco Laboratories. Ampholines (pH 5 - 7 and pH 3.5-10) were obtained from LKB. Aminoglycoside antibiotics, Hygromycin B and Paromomycin were kindly supplied by Takeda Chemical Industries, Osaka, Japan.
Strains, Culture Conditions and Genetic Procedures. Strains used in this work are listed in Table 1. Strains, BO133-3B and 0-9003 are described by Ono etal. (Ono etal. 1979a, b; Ono et al. 1981). Strains, OK29-1C and IS14-9D, and strain, OG7-2C were kindly supplied by Dr. B-I. Ono and Dr. K. Matsumoto, respectively. For scoring of genetic markers, minimal medium (0.67% bactoyeast nitrogen base w/o amino acids, 2% glucose, 2% agar) containing the appropriate supplements was used (Sherman and Lawrence 1974; Ono et al. 1979a). Susceptibility to aminoglycoside antibiotic was scored by using YEPD medium (1% yeast extract, 2% bacto peptone, 2% glucose, 2% agar) containing paromomycin (500 t~g/ml). For ribosome preparation, ceils were grown aerobically in liquid YEPD medium at 27 °C, and harvested at late logarithmic growth phase. Standard methods for crossing, sporulation and dissection were used as described by Sherman and Lawrence (1974).
Isolation of Antisuppressor Mutants. A strain BO133-3B was able to grow on YEPD medium containing 100 ~g/ml ofhygromycin B, while growth of a 0-9003 strain was completely inhibited at the drug concentration. About 107 cells of 0-9003 (SUP46 strain) were spread on YEPD plate and irradiated by UV light with a dose of about 50% survival. After overnight incubation in the dark, the plate was replica plated on YEPD medium containing hygromycin B (200 ~g/ml). Resistant revertants to hygromycin B were isolated, and their genetic markers were scored by using the appropriate synthetic medium. In order to select the mutants carrying antisuppressors, revertants which lost the ability of suppression of leu2-1 gene were crossed with tester strain, OK29-1C. Resultant diploid strains were isolated and sporulated. Each of the spore suspensions obtained from the respective diploid strains was spotted on synthetic medium lacking leucine. When a certain group of the spore suspensions was observed to grow up on the medium, the corresponding parental revertant was used as an antisuppressor mutant.
Effects of Aminoglycoside Antibiotics and High Temperature {38 °C) on Cell Growth. After cells were grown in YEPD medium at 27 °C for 2 h, either paromomycin (final conc. of 500 #g/ml) or hygromycin B (final eonc. of 200 ~g/ml) was added and cultivation was continued. Cells were also grown in YEPD medium at 38 °C. Cell growth was measured by liglat absorbance at 660 nm.
Preparation of Ribosomal Proteins. Purified 80S ribosomes and ribosomal subunits were prepared according to essentially the same method described by Torafio et al. (1974). Cells werewashed and disrupted with glass beads in medium A (10 mM Tris-HC1 pH 7.5. 5 mM magnesium acetate, 10 mM potassium chloride, 10 mM 2mercapto ethanol). The cell homogenate was centrifuged at
J. Ishiguro: Characterization of Yeast Antisuppressor 8,000 Xg for 10 min and then, 30,000 Xg for 20 min twice. The supernatant was centrifuged at 105,000 Xg for 2 h. The pellet (crude ribosomes) was suspended in medium B (30 mM Tris-HC1 pH 7.2, 0.5 M ammonium chloride, 0.1 M magnesium acetate, 0.25 M sucrose, 5 mM 2-mercaptoethanol), and left in ice for overnight. The suspension was mixed with medium A (1 : 1 volume), and centrifuged at 30,000 Xg for 20 min and then, 105,000 Xg for 90 min. The pellet obtained was suspended again in medium A, and centrifuged at 30,000 Xg for 20 rain. Purified ribosomes were obtained from the final supernatant by centrifugation. For the preparation of ribosomal subunits, crude ribosomes were suspended in medium C (50 mM Tris-HC1 pH 7.7, 0.8 M potassium chloride, 12 mM magnesium acetate, 20 mM 2-mercaptoethanol), and layered on 15-30% sucrose gradient made in medium C. The centrifugation was performed at 20,000 rpm for 15 h with Hitachi RPS25-2 rotor. Fractions corresponding to the 40S and 60S subunits were collected separately, and each of the suspensions was diluted with medium D (30 mM magnesium acetate, 20 mM 2-mercaptoethanol) and sedimented by centrifugation at 190,000 Xg for 5 h. Extraction of ribosomal proteins was carried out as described by Sherton and Wool (1974). The ribosomes and ribosomal subunits were suspended in medium E (10 mM Tris-HC1 pH 7.7, 33 mM magnesium acetate) and two volumes'of glacial acetic acid were added. The mixture was left in ice for 60 min with shaking, and rRNA was sedimented by centrifugation. The protein solution was dialyzed against 1 N acetic acid followed by lyophilization.
Two-Dimensional Polyacrylamide Gel Electrophoresis. Two-dimensional polyacrylamide gel electrophoresis was performed as described by Ishiguro and McLaughlin (unpublished data). Nonequilibrium pH gradient gel based on the method of O'Farrell et al. (1977) and acidic gel based on the method of Kaltschmidt and Wittmann (1970) were used in the first (l-D) and the second (2-D) dimensions, respectively. The solutions needed are as follows. Sample solution: 9 M urea, 5% 2-mercaptoethanol, 0.2% Ampholines (pH 3.5-10). Overlay solution: 8 M urea, 0.2% Ampholines (pH 3.5-10), 0.8% Ampholines (pH 5-7). Separation gel for l-D: 4% acrylamide, 0.2% N,N'-methlenebisacrylamide, 9 M urea 2% Ampholines (pH 3.5-10). For polymerization 20 pl of 10% ammonium persulfate and 20 ~1 of N,N,N',N'-tetramethylethylenediamine (TEMED) were added to 10 ml of gel mixture. Electrode buffer for l-D: 0.016 M phosphoric acid (upper) and 0.019 M sodium hydroxide (lower). Dialysis buffer for 1-D gel: 8.3 M urea, 0.013 M acetic acid, 0.012 M potassium hydroxide (final pH 6.4). Separation gel for 2-D: 18% acrylamide, 0.5% N,N'-methylenebisacrylamide, 6 M urea, 0.91 M acetic acid, 0.048 M potassium hydroxide, 0.58 TEMED (final pH 4.6). For polymerization ammonium persulfate was added to make final 0.06%. Electrode buffer for 2-D: 0.186 M glycine, 0.026 M acetic acid. Sample proteins (approx. 300 ug) were dissolved by the sample solution (50 ~1), and the protein solution was applied on the surface of a gel. The overlay solution was carefully layered over the sample solution and then, a tube was filled with the upper electrode buffer solution. Electrophoresis for 1-D was carried out at 400 constant voltage for 2-2.5 h with the anode on the top and the cathode on the bottom. After the electrophoresis, the gel was dialyzed against the dialysis buffer at room temperature for 90 min and then, embedded in the 2-D gel. Electrophoresis for 2-D was carried out at 80 constant voltage for 15 h with the anode on the top and the cathode on the bottom. The small size gel plate '(120 mm wide - 100 mm long - 2 mm thick) was used in the experiment.
199
J. Ishiguro: Characterization of Yeast Antisuppressor Table 1. Genotype of Strains used in this Investigation Strain
Genotype
BO133-3B 0-9003 IS14-9D OK29-1C OG7-2C
a cycl-72 canl-lO0 met8-1 leu2-1 ura4-1 his5-2 lysl-1 trp5-48 ~ + a SUP46cycl-72canl-100 met8-1 leu2-1 ura4-1his5-2 lysl-1 trp5-48 ~+ SUP46 met8-1 leu2-1 his5-2 ura4-1 ade2-1 ~ + a met8-1 leu2-1 ~ gal4-62 met8-1 leu2-1 t) +
Results
ura4-1, his5-2 and amber (UAG) mutation, metS-1 were scored. None of the revertants was able to suppress any markers, while 0-9003 strain was able to suppress all of them except his5-2 (Table 2). In the following genetic study, leu2-1 was mainly used as a UAA suppressible marker, since UAG marker, metS-1, was sometimes weakly suppressed in the presence of cytoplasmic factor [psi+]. The revertants were crossed with tester strain, OK29-1C, and the resultant diploids which were homozygous for leu2-1 and heterozygous for SUP46, were sporulated. Each of the spore suspensions obtained from the respective crosses was spread on appropriate synthetic medium lacking leucine (SC-leu) in order to distinguish between antisuppressor carrying-revertants and intragenic revertants at SUP46 locus. Five groups of the spore suspensions grew up on the medium, indicating that the corresponding five revertants still have SUP46 suppressor, and the suppression activity could be weakened by the secondary mutation unlinked to SUP46. The suppression patterns and other properties of these antisuppressor mutants are shown in Table 2.
Isolation o f A ntisuppressors
Aminoglycoside antibiotics, paromomycin and hygromycin B have been shown to cause mistranslation in vivo and in vitro in the yeast Saccharomyces cerevisiae (Singh et al. 1979; Palmer et al. 1979). The SUP46 mutant is hypersensitive to paromomycin because of its additive effect on mistranslation (Masurekar et al. 1981). The SUP46 m u t a n t was also found to be hypersensitive to hygromycin B. The antibiotic of 200/~g/ml concentration inhibited the growth of SUP46 m u t a n t completely, while the parental strain was not affected to grow at the drug concentration. Mutants carrying antisuppressors which reduce the efficiency of SUP46 suppressor were isolated from the hygromycin B-resistant revertants of 0-9003 strain carrying SUP46 gene. Sixteen resistant revertants were isolated and their genetic markers including ochre (UAA) mutation canl-lO0, leu2-1, lysl-1,
Table 2. Properties of Antisuppressor Mutants Strain
BO133-3B O-9003a J200 le J2002e J2004e J2006e J2022e
Gro'wth on YEPD + paromo a
SC -met
SC +canb
SC -leu
+ + + + + +
± + + + + + ±
+ ± + + + + +
+ . . .
Ribosomal Protein c SC -lys
SC -ura
SC -his
S11
$27
+
-+
-
Normal Altered Altered Altered Altered Altered Altered
Normal Normal Normal Normal Normal Altered Normal
.
.
.
. .
. .
. .
a Paromomycin (500 #g/ml) b Canavartine Sulfate (60 t~g/ml) c Ribosomal proteins were prepared from each of the strains and analyzed by two-dimensional gel electrophoresis (see Material& Methods) d Parental strain (SUP46 strain) e Antisuppressor mutants obtained from the strain, 0-9003 Scoring was performed as follows: + good growth by 5 days; ± poor growth by 7 days; - no sign of growth by 7 days
200
J. Ishiguro: Characterization of Yeast Antisuppressor
Table 3. Segregation of Antisuppressor (asu11) from SUP46 suppressor Strain
Genotypea
Number of Tetrads b
SUP46~+asu11/+
D 5 (J2006 x OK29-1C)
P
NP
T
4
7
20
D5 diploid strain is homozygous for leu2-1 T-he type of segregation of the tetrads was determined by scoring the degree of growth on SC-leu plates as follows. P (SUP46/asu11 X 2, +/+ X 2): 0+ and 4 - ; NP (SUP46~+ X 2, +/asu11 X 2): 2+ and 2-; T (SUP46/asu11, SUP46~+,+/asu11, +/+): 1+ and 3 . + means good or poor growth by 5 days and - means no sigh of growth by 5 days
a
b
c
B
o
I.B" A
g
/_ O.EJ
5
1'0
rl m4
5
10
UlR4
8
10
1114
Hours Fig. l a - c . The effects of aminoglycoside antibiotics on cell growth. The arrows indicate dosage of Higromycin B (final conc. of 200 pg/ml) or Paramomycin (finn conc. of 500 pg/ml). Control (o-o), Hygromycin B (zx-z~)and Paramomycin (D--D). BO133-3B (a), 0-9003 (SUP46) strain (b) and J2006 (asu11) strain (c)
Genetic Properties o f A ntisuppressors Five antisuppressor mutants isolated independently were crossed with tester strain, OK29-1C. The resultant diploids which were homozygous for the suppressible marker, leu2-1, and heterozygous for the SUP46 suppressor and each of the five antisuppressors, respectively. All of the five diploid strains grew up on the SC-leu plate, indicating that the corresponding mutant strains, namely J2001, J2002, J2004, J2006 and J2022 contained recessive antisuppressors. An antisuppressor that was carried in the strain, J2006, was denoted asull, and the mutant strain was studied further (see Table 2). In order to examine the segregation of asu11, a diploid strain homozygous for leu2-1 and SUP46 and heterozygous for asull was constructed (J2006 x IS14-9D) and sporulated. How-
ever, most of the asci gave rise to two or three spores. This observation suggests that in most cells meiosis was abnormally performed because of the presence of SUP46 homo-allele in the diploid. The SUP46 and other omnipotent suppressors generally cause retardation of mating, sporulation, spore germination and cell growth (Ono et al. in preparation). Only two sets of complete tetrads were obtained and they segregated 2+ and 2 - for the suppression of leu2-1. The segregation of asul 1 was also examined by using a diploid strain which was homozygous for leu2-1 and heterozygous for SUP46 and asull, respectively. The meiotic progenies obtained from the diploid strain were scored by the suppression of leu2-1, and the types of the segregation were determined in each of the tetrads as follows. Parental ditype (P): SUP46/asull X2, +/+ X2; Nonparental ditype (NP):
J. Ishiguro: Characterization of Yeast Antisuppressor
a
201
C
b
2,r"
E o ¢.D 1.5
e-. ~;~ 1 . 0
//
o5 / 8 /
I
B
I 1.,1 2 4
10
'
5
' I IR-
10
I
5
*
I n
"'
I#E4
Hours Fig. 2a-c. The effects of high temperature (38 °C) on cell growth. Cells were incubated at 27 °C ( o - o ) or 38 °C (*-*). BO133-3B strain (a), 0-9003 (SUP46) strain (b) and J2006 (asu11) strain (c)
SUP46~+ X2, +/asul 1 X2; Tetratype (T): SUP46/asul 1, SUP46~+, +/asull, +/+. Since only the meiotic progeny with the genotype of SUP46/+ can grow up on Sc-leu plate, P, NP and T types of tetrads might reveal 0 + : 4 - , 2+ : 2 and 1+ : 3 - segregations for supprssion of leu2-1, respectively. As seen in Table 3, the antisuppressor, asull, segregated independently from SUP46 suppressor. The ratio of P : NP : T was nearly 1 : 1:4. These results demonstrate that a single recessive gene might be responsible for the asull mutation.
Effects of Aminoglycoside Antibiotics and High Temperature on Cell Growth Effects of aminoglycoside antibiotics on cell growth are shown in Fig. 1. Cell growth of SUP46 strain (0-9003) was completely inhibited by paromomycin (500 #g/ml) or hygromycin B (200/lg/ml), while that of the parental wild strain (BO133-3B) was not significantly affected at the drag concentrations. The asull strain (J2006) which was isolated as hygromycin B-resistant revertant appeared to have cross-resistance to paromomycin. It also can be seen that in the J2006 strain, retardation of cell growth caused by SUP46 mutation was restored to almost the normal level. Effects of high temperature on cell growth are shown in Fig. 2. The growth of the 0-9003 strain was significantly inhibited at 38 °C, while that of the BO133-3B strain was not affected. The J2006 strain also appeared to gain the resistance to the high temperature.
Ribosomal Proteins from A n tisuppressor Mutants Ribosomal proteins obtained from five antisuppressor mutants were analyzed by two-dimensional gel electrophoresis. The results are summarized in Table 2. The ribosomes of all mutants had an altered S11 protein, which had been shown to be responsible for SUP46 mutation (Ishiguro et al. 1981, and see Fig. 3a, b in this paper). This result consists with the genetic evidences that SUP46 suppressor is still present in the five revertants. Added to this, another altered ribosomal protein, $27, was found in the strain, J2006, as shown in Fig. 3c. This finding was also confirmed in the 40S ribosomal subunits (Fig. 4). The mutated protein moved less towards the cathode in the pH gradient gel as compared with the corresponding wild protein, indicating that the mutant protein has a decreased pH of its isoelectric point. A diploid strain heterozygous for asull and homozygous for SUP46 was constructed (J2006 x IS14-9D) and the ribosomal proteins were examined. As seen in Fig. 3d, both altered and normal $27 proteins and only altered S11 protein were clearly observed in the gel. This result suggests that the structural gene coding for the $27 protein might be altered but not for the modifying enzyme acting on the protein.
Correlation between A ntisuppressor (asul 1) and Alteration of Ribosomal Protein ($27) A diploid strain was constructed to be homozygous for the suppressible marker, leu2-1, and heterozygohs for
202
J. Ishiguro: Characterization of Yeast Antisuppressor
Fig. 3a-d. Sections of two-dimensional gel electrophoretograms of 80S ribosomal proteins. Nonequilibrium pH gradient gel electrophoresis (pH 3.5-10) in 1-D and acidic gel electrophoresis (pH 4.6) in 2-D were carried out as described in the Materials and Methods. Typical examples obtained from BO133-3B strain (a) 0-9003 (SUP46), J2001, J2002, J2004 and J2022 strains (b) J 2006 (asu11) strain (c) and Heterodiploid (asu11/+) strain (d). The S l l and $27 proteins are indicated by the arrows
the SUP46, asull and other markers, respectively, in order to ascertain correlation between asull mutation and alteration of $27 protein. The meiotic segregants from the diploid were scored by the appropriate markers, and their ribosomal proteins were analyzed above. The result is shown in Table 4. None of the segregants grew up on the SC-leu plate, indicating that the tetrad has a presumable genotype of P type (SUP46/asu11 X2, +/+ X2). Segregation of other markers indicated that meiosis was normal. In this case, it was shown that two out of four segregants contained altered S l l and altered $27 proteins, and the remaining two segregants contained normal S l l and normal $27 proteins. In the case o f a T type-tetrad, it was also observed that only one segregant was able to grow up on the SC-leu plate of which ribosomes contained altered S11 and normal $27 proteins (data not shown). In certain progenies with a presumable genotype of SUP46/asull, normal $27 protein was found to be somewhat leaky (Table 4).
, F i g . 4a and b. Two-dimensional gel electrophoretograms of proteins of 40S ribosomal subunits obtained from 0-9003 (SUP46) strain (a) and J2006 (asu11) strain (b). The $27 proteins are indicated by the arrows
J. Ishiguro: Characterization of Yeast Antisuppressor
203
Table 4. Correlation between Antisuppressor (asu11) and Alteration of Ribosomal Protein ($27) Strain
Growth on SC-
presumable genotype
leu
lys
ura
his
+
-
+ -
+ +
Ribosomal Protein S 11
$27
Altered Altered Normal Normal
Altered Altered & Normal Normal Normai
D6 (J2006 x OG7-2C)a Segregant
a
11-A -B -C -D
.
-
.
. + +
.
SUP46/asul i SUP46/asul 1 +/+ +/+
diploid strain is homozygous for leu2-1 and heterozygous for other markers. + indicates good growth no sign of growth by 7 days D6
Discussion
The SUP46 suppressor mutant was previously shown to have ribosomes which cause abnormally high rates of mistranslation, and therefore, to be unusually sensitive to paromomycin which hase been known to stimulate translational errors (Masurekar et al. 1981). The SUP46 xibosomes were also found to contain altered S l l protein of 40S subunits, which was responsible for the suppression by misreading (Ishiguro et al. 1981). In the present study, the mutant carrying an antisuppressor, designated asull, which reduces the efficiency of SUP46 suppressor has been shown to have additional alteration in $27 protein of 40S ribosomal subunits. The antisuppressor mutant which was isolated from hygromycin B-resistant revertants displayed cross-resistance to paromomycin, suggesting that the mutation renders restrictive effect to translational errors caused by alteration of the S11 protein. The correspondence between the asull mutation and the alteration-of $27 protein was ascertained by examining meiotic segregants from a heterozygous diploid. In this experiment, however, it was observed that both normal and abnormal $27 protein were observed in certain meiotic segregants carrying SUP46/asull mutations. The cause of this "leakiness" of the asull mutation in certain meiotic progenies is unknown. One possibility is that asull is not the structural gene for $27, but codes for a modifying enzyme acting on $27. However this is inconsistent with the production of both normal and abnormal $27 proteins in a diploid heterozygous asul 1/+ (Fig. 3d). An alternative explanation is that the "leakiness" is caused by misreading of asull mRNA enhanced by the SUP46 mutation. The SUP46 suppressor mutant was found to be temperature sensitive at 38 °C. The asu11 mutation was accompanied with restoration of the temperature sensitivity, suggesting that the alteration of ribosomal components, S l l and $27 proteins, would participate in the temperature sensitivity of cell growth. It has been demonstrated that ts-revertant which carries recessive sup-
by
5 days and - indicates
pressor, sup2, has a defect in termination of protein synthesis and accumulates 80S ribosomes bearing peptidyl tRNA at 36 °C (Smimov et al. 1974, 1976 ; Surguchov et al. 1980). In the SUP46 mutant, however, polysome decay was not observed under the restrictive temperature (results not shown). Of five antisuppressor mutants isolated independently, one was found to have alteration in ribosomal protein, $27, and the remaining four mutants were not. This result suggests the possibility that at least two kinds of antisuppressors which are effective on SUP46 suppressor would be present, although allelism tests were not performed. Recently two antisuppressors have been found and designated asu9 and ASU10 (Liebman and Cavenagh 1980; Liebman et al. 1980). The former acts on recessive omnipotent suppressors, sup35 and sup45, and the latter is specific for only sup35 suppressor. In the present study, it is not known if asull antisuppressor acts on sup35 or sup45. All five antisuppressors including asull were recessive as well as asu9 when scored on the basis of suppression of leu2-1. Thus, the possibility remains that some of them would be the same as asu9 mutation. Liebman and Cavenagh (1981) have showed by using two-dimensional gel electrophoresis that 40S ribosomal subunits from the strains carrying asu9 mutation contain a small amount of extra protein which is absent in the asu9 non-carrying-strains. However, no other significant differences were observed between them, and the extra protein may not be an intrinsic ribosomal protein. Therefore, the asu11 mutation is considered to be different from the asu9 mutation. The SUP46 mutation resembles ram mutations in Escheriehia coli in the sense that both mutations cause alterations of ribosomal proteins (S11 in yeast and $4 or $5 in E. coli) and lead to translational ambiguity (Masurekar et al. 1981; Ishiguro et al. 1981). Amino acids composition of S11 protein (YS11 of Hiroshima Nomenclature, Otaka and Osawa 1981) seems to be highly analogous to that of $4 p r o t d n of E. coli (Kaltschmidt et al. 1970; Higo and Otaka 1979). Indeed, structural ho-
204 mology between them can be assumed by using Difference Index Method (Difference Index = 7.6, Higo and Otaka at Hiroshima Univ., personal communication). The S11 protein hase been known to be a rRNA bindingprotein as well as in the case of $4 protein of E. coli (Zimmermann 1974; Reyes et al. 1978). In contrast, strA mutations in E. coli restrict ribosomal ambiguity caused by the ram mutations, and alter a ribosomal protein, S12 (Gorini 1974 see review). The antisuppressor mutation described in this paper is thought to be analogous to the strA mutations. Interestingly, the $27 protein of yeast has a similar electrophoretic mobility to the S12 protein ofE. coli in two-dimensional gel. Thus, our findings would be useful for expanding not only biochemical but also evolutional knowledges about eukaryotic ribosomal proteins.
Acknowledgement. The author wishes to thank Dr. B.-I. Ono (Okayama Univ., Okayama, Japan) for supplying the strains and for helpful discussions and comments, and the author is also grateful to Dr. Y. Arakatsu (Konan Univ., Kobe, Japan) for supporting this work and for critical reading of the manuscript.
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Communicated b y B. Cox Received July 3, 1981
