Further characterization of recessive suppression in yeast

149

Biochimica et Biophysica Acta, 654 (1981) 149-155

Elsevier/North-Holland Biomedical Press BBA 99877 FURTHER CHARACTERIZATION OF RECESSIVE SUPPRESSION IN YEAST ISOLATION OF THE LOW-TEMPERATURE SENSITIVE MUTANT OF S A C C H A R O M Y C E S C E R E V I S I A E DEFECTIVE IN THE ASSEMBLY OF 60 S RIBOSOMAL SUBUNIT ANDREI P. SURGUCHOVa, ELENA S. FOMINYKCHa, VLADIMIR N. SMIRNOVa, MIKHAILD. TER-AVANESYANb, LUDMILA N. MIRONOVAb and SERGEI G. INGE-VECHTOMOVb a USSR Research Centre of Cardiology, Academy of Medical Sciences, Petroverigsky Lane 10, Moscow 101837and b Department o f Genetics, Leningrad State University, Leningrad (U.S.S.R.}

(Received September 29th, 1980) (Revised manuscript received March 17th, 1981)

Key words: Recessive suppression; Ribosomal subunit assembly; Low-temperature sensitivity; (Saccharomyces cerevisiae)

It has been shown that recessive suppressor mutations in the yeast Saccharomyees cerevisiae may cause sensitivity towards low temperatures (very slow growth or lack of growth at 10°C). One of the sup 1 low temperature sensitive (Lts-) mutants, 26-125A-P2156, was studied in detail. After a prolonged period of incubation (70 h)under restrictive conditions the protein synthesis apparatus in the mutant cells was irreversibly damaged. In addition, Lts- cells incubated under restrictive conditions synthesize unequal amounts of ribosomal subunits, the level of 60 S subunit being reduced. It has been suggested that the recessive suppression is mediated by a mutation in the gene coding for 60 S subunit component, probably a ribosomal protein. The mutation leads simultaneously to a defect in the assembly of 60 S subunit and to low-temperature sensitive growth of the mutant.

Introduction Genetic properties of the recessive suppressors suggest that they may code for proteins participating in the termination of translation [ 1 - 7 ] . Recently it has been shown that the ribosomes isolated from strains, carrying recessive suppressor mutations possess a high level of translational ambiguity in vitro. This property probably leads to suppression of all three types of nonsense mutation in vivo [8]. To study the mechanism of recessive suppression temperature-sensitive (ts-) mutants of yeast were used [9,10]. However, attempts to identify the products of sup 1 and sup 2 genes in these mutants were unsuccessful. In the present study a new class of mutants carrying recessive suppressor mutations was used, namely, low-temperature sensitive (Lts-) mutants. In these mutants low-temperature sensitivity appears to correlate with a defect of assembly of 60 S

cytoplasmic ribosomal subunit. It is suggested that the product of the recessive suppressor gene is a component of 60 S ribosomal subunit.

Materials and Methods Strains. The yeast strains used in this work were the haploid strains of S. cerevisiae described in Table I. The designation of makers was described earlier [11-12]. The classification of nonsense mutations is the following: his 7-1, leu 2-2, lys 9-A21 and pho 1-059 - ochre mutations; lys 2-A12 - amber mutation; thr 4 - probably, opal mutation [13]. sup 1 and sup 2 suppress mutations his 7-1 lys 9-A21 leu 2-2. The mutation ade 1-14 is also suppressed by recessive suppressors. The exact nature of the mutation is, however, not identified. The strains 125A-P2156 and 29V-P2156 were used for inducing suppressor mute-

0 005-2787/81[0000-0000[$02.50 © Elsevier/North-HollandBiomedical Press

150 TABLE I GENOTYPES OF PARENT AND MUTANT STRAINS OF YEAST CARRYING RECESSIVE SUPPRESSOR MUTATIONS Strain

Genotype

125A-P2156 29V-P2156 200B-P2156 99-110B-P2137 * 106-110B-P2137 * 26-125 A-P2156

~ade 1-14his 7-1 lys 2-A12 thr 4-B15 leu 2-2 pho 1-059 aade 1-14his 7-1 met Alpho 1-059 a his 7-1 lys 2-A12 met AI ura 3-B4 aphe AlOade 2-131 met Alhis 7-1 lys 9-A21 leu 2-2 sup 1 a phe A l O ade 2-131 met Al his 7-1 lys 9-A21 leu 2-2 sup 2 aade 1-14his7-1 leu 2-2sup 1 lys 2-A12thr4

* sup l andsup 2 suppress mutationshis 7-1 lys 9-A21 leu 2-2.

tions. For studying the allelism of the suppressor mutations arising in 125A-P2156 for sup 1 and sup 2 genes, the strains 99-110B-P2137 and 106-110BP2137 were used as testers. Allelism of the recessive suppressor mutations arising in the strain 29V-P2156 was determined in crosses with previously characterized revertants of 125A-P2156. Cultivation conditions and genetic methods. The

routine methods of yeast genetics were used. The inheritance of the recessive suppressor mutations was characterized by (1) analysis of the meiotic segregation of hybrids and (2) studying of spontaneous mitotic homozygotization of suppressor mutation [14]. Tandom samples of ascospores were obtained by the micromanipulation (for hybrid P3318) and by the use of diethyl ether (for hybrid P2739) [15]. Medium A contained (g/l): yeast extract, 10; peptone, 20; dextrose, 20; medium B contained (g/l): yeast extract, 10; agar, 20; peptone, 20; dextrose, 20. Strains were incubated at 20°C unless stated otherwise. Lowtemperature sensitivity was studied at 10°C. The difference in growth rates of cold-sensitive and low-temperature resistant strains is noticeable after 6 - 7 days of incubation on agar plates. In all biochemical experiments Lts--revertant 26-125A-P2156 was used. Chemicals and isotopes. Reagents were obtained from the same sources as described in [7,9]. a4Clabelled protein hydrolysate (specific activity, 57 mCi/mmol), [2-14C]uracil (spec. act. 60 mCi/mmol) and [6-3H]uracil (spec. act. 24 Ci/mmol) were from Amersham. MiUipore i~dters, type HA (0.45 /,tm pore size) were from MiUipore (U.S.A.). In vivo incorporation studies. (a)Shift-down experiments. The cells of parent strain and Lts--rever-

tant were transferred from agar plates into liquid medium A and grown until the middle of the exponential phase of growth with aeration at 25°C. The absorbance at 660 nm (A66o) was measured and the absorbance of the suspensions for parent strain and Lts--revertant was made equal. Suspensions were divided into two portions of 20 rnl each, which were incubated for 90 min at 10 and 25°C in the presence of 25 #Ci of ~4C-labelled amino acid mixture. From each suspension 1 ml aliquots were taken and transferred into 1 ml 10% trichloroacetic acid. The sampies were incubated at 90°C for 15 roan, precipitates were collected on Millipore filters (HA 0.45 gm), washed with 5% trichloroacetic acid and ethanol. The radioactivity incorporated into the protein was determined in a toluene scintillator. (b) Shift-up experiments. Parent and Lts--revertant cells were transferred from agar plates into liquid medium A and incubated at 10°C for 70 h for Lts-revertant or 48 h for the parent strain. The cells were harvested by centrifugation and resuspended in a fresh medium A (the ffmal A66 o was made identical). 20 ml aliquots were withdrawn from both suspensions and placed on a rotary incubator at 25°C. After 30 min preincubation 25 #Ci of ~4C-labelled amino acids mixture were added to each aliquot. Further operations are as described in section a. Kinetics o f p o l y s o m e decay at 10°C. Cells grown at 25°C were shifted to 10°C and incubation continued for 60 rain. To stop the polysome decay cycloheximide was added to each culture at the final concentration of 10 #g/ml. The cells were collected on filters, suspended in 0.05 M Tris-HC1 buffer, pH 7.6/ 10 mM MgC12/25 mM KC1/4 mM dithiothreitol and

151 disrupted in an SCP disintegrator (Sweden). The suspensions were centrifuged at 15 000 X g for 20 mist and allquots of supematants were layered on sucrose gradients for polysome analysis [9]. The ratio (polysomes/80 S ribosomes) for zero time of incubation at 10°C was taken for 100%. [14C] Uracil or [ 3H] uracil incorporation. Cells were grown in a complete synthetic medium [10] until the middle of the exponential phase under permissive conditions (25°C). Then the cells were transferred to 10°C and 30 rain later [3H]uracil was added to the final concentration of 1.67 /.tCi/ml. The cells were incubated at this temperature for 19 h. (In some experiments the incubation time was shortened to 4 or 8 h, and this did not influence the ratio of labelled uracil incorporated in the 60 S and 40 S subunits.) As a control the yeast ceils incubated under permissive conditions (25°C) in the presence of [14C]uracil were used. In this case [14C]uracil was added at the beginning of growth to the concentration of 8.3 ~Ci/ml. (When the incubation time was shortened to 4 or 8 h the ratio of labelled uracil incorporated in the 60 S and 40 S subunits did not change.) The cells were collected by filtration, washed and disrupted with glass beads in a Braun disintegrator in the buffer containing: 0.035 M Tris-HCl, pH 7.6/0.01 M MgC12[0.025 M KCI/4 mM dithiothreitol. The cell debris and mitochondria were pelleted by centrifugation (30000 Xg, 20 rnin). 1 ml aliquots were withdrawn from supernatants containing [3H]uracil and [14C]uracil, mixed and layered on sucrose gradients (6--41% sucrose (w/v)/0.05 M Tris-HCl[0.005 M MgCI2[0.5 M KCI). The subunits were separated in a SW 27 rotor for 14 h at 23 000 rev./min. After the centrifugatdon 1 ml fractions were collected and transferred into scintillation vials, containing 9 ml of dioxane scintillator and counted in a Mark III counter (Searle Analytical Inc.). For determining the ratio of subpartieles the peaks of the large and small ribosomal subunits were traced onto a piece of cardboard, cut out and weighed. The ratio of absorbances at 260 nm for the stoichiometric mixture of 60 S and 40 S subunits was found to be 2.2 : 1.

growth arrest at low temperatures. The sensitivity to low temperature is often a result of the defect in the assembly of one of the ribosomal subunits [16,17]. In our study of the phenotypic expression of the recessive suppressor mutations (the results will be published later) we have found that sometimes these mutations lead to a low temperature sensitivity of the growth. For two parent strains 125A-P2156 and 29VP2156 (for genotypes see Materials and Methods) 159 and 96 recessive suppressor mutants were obtained, respectively. The study of the ability to grow on medium B plates at 10°C has revealed that approx. 9% of revertants for both strains are low-temperature sensitive (the lack of growth or significantly decreased growth). 12 Lts--revertants were due to mutations in sup 1, and 11 Lts- due to mutations in sup 2 genes. All these revertants were sensitive only to low, and not to high temperatures. The study of inheritance of pleiotropic expressions of two suppressor mutations (sup 1-26 and sup 1-31) has demonstrated that low temperature sensitivity is a result of the recessive suppressor mutation. All 66 meiotic segregants of P3318 and P2739 hybrids (ob. tained by crosses of strains 31-125A-P2156 X200BP2156 and 26-125A-P2156X29V-P2156, respectively) bearing recessive suppressor mutation were low-temperature sensitive, and vice versa 184 segregants without recessive suppressor mutation were able to grow at 10°C (Table II). This conclusion was supported by the data on mitotic homozygotization oI

TABLEII SEGREGATION OF SUPPRESSOR ACTIVITY AND LOWTEMPERATURE SENSITIVITY IN RANDOM SAMPLES OF ASCOSPORESOF HYBRIDS P3318 AND I>2739 Hybrids

Number of segregants with phenotype * Sup+ LtV

Sup~Lt~

18 48

35 149

Results

P3318 P2739

Low-temperature sensitivity of recessive suppressor mutants The ribosomal mutations in some organisms cause

* The deviation from 1 Sup*Lts-:l Sup-Lts+ is probably due to selective advantage of segregants without suppressor mutation.

152 ressive suppressor mutation sup 1-26. Mitotic recombination was studied in diploid hybrid P2739 homozygous for ade 1-14 and his 7-1 mutations and heterozygous for recessive suppressor sup 1-26. Mitotic homozygotes in sup 1-26 were selected as spontaneously arising prototrophs for adenine and histidine as described previously [14]. It was shown that ade 1-14 and his 7-1 mutations may be suppressed simultaneously only by the recessive suppressor [18]. All 123 prototrophs obtained in this experiment had a lowtemperature sensitive phenotype. Thus, mutations in sup 1 gene may simultaneously lead to low-temperature sensitivity and to the suppressor effect. Protein synthesis in Lts--cells at a low temperature

Since low-temperature sensitivity of Lts--revertants is the result of the recessive suppressor mutation, it is reasonable to suppose that a Lts- componet participates in the process of translation. To determine the period of time necessary to effect the translation under non-permissive conditions two sets of experiments were carried out: (a) The cells of parent strain and Lts--revertant were grown till the middle of the exponential phase of growth at 25°C; then the mixture of labelled amino acids was added and the cultures were further incubated at either 25 or 10°C (Fig. la). Fig. la

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shows that the rate of amino acid incorporation into proteins in both strains at 25°C is practically identical. When the period of incubation under non-permissive conditions (10°C) is limited to about 1 h, the low-temperature sensitivity of the protein synthesis in these strains is very similar. These results agree with the kinetics of polysome decay in the parent strain and Lts--revertant after a short period of incubation of the cells at 10°C. Fig. 2 demonstrates that the decrease in the polysome content in response to a low temperature is very similar for the parent strain and Lts--revertant. (b) The cells of the parent strain and Lts--revertant were grown at 10°C (see Materials and Methods); the mixture of labelled amino acids was added and the cultures were further incubated at 25°C (Fig. lb). The prolonged incubation of the cells at 10°C was accompanied by a profound decrease in the rate of amino acid incorporation in Lts--revertant compared to that in the parent strain. The inhibition of protein synthesis in Lts--revertant is apparently irreversible, since it is preserved after a 30 min preincubation under permissive conditions (see Materials and Methods). The slow residual incorporation of amino acids in Lts--revertant cells after incubation periods of more than 60 rain (Fig. lb) could arise as a result of a leaky expression of Lts--phenotype.

153 Assembly o f ribosomal subunits in Lts--revertant at a low temperature

The low-temperature sensitivity of mutants both in proearyotes and euearyotes is often due to a defect in the assembly to one of the ribosomal subunits [19,20]. To study the subunit assembly in yeast mutants carrying recessive suppressors the rate of incorporation of labeled uracil in 60 S and 40 S subunits was determined under permissive and restrictive conditions. The results of these experiments are presented in Fig. 3. The ratio between the amount of 60 S and 40 S particles synthesized in the parent strain at 25°C is equal to 2.24; this corresponds to almost a stoichiometric ratio of the subunits (1.02 +0.05, see Materials and Methods). No significant variation of this ratio was observed when the parent strain cells were grown at 10*C (1.04 +_0.05). In contrast,

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Lts--revertant strain synthesizes disproportional amounts of the two ribosomal subunits with a slight reduction of 60 S subunits under permissive conditions (0.88-+ 0.05) (Fig. 3b) and marked reduction under restrictive conditions (0.58 +-0.06) (Fig. 3d). The reduced amount of 60 S subunits does not appear to result from its preferential decay under restrictive conditions for the following reasons: (1) in the parent strain the subunit ratio does not change at a low temperature; (2) the shortening of the incubation time at 10°C to 4 or 8 h does not affect the deficit of 60 S subunits (data not shown). At all the periods of incubation at low temperature the specific radioactivity of 60 S subunit of the revertant remains lower compared to that of parent strain. Discussion

A short exposure (up to 60 min) of Lts--revertant cells under non-permissive conditions (10°C) is not accompanied by a lesion in the translational apparatus (Fig. la); after prolonged incubation, however, low-temperature sensitivity of growth (not shown) and protein synthesis (Fig. lb) can be detected. Genetic analysis has revealed that low-temperature sensitivity is the pleiotropic effect of recessive suppressor mutation. These data suggested that Lts--component is a part of translational apparatus; if this is the case the low-temperature sensitivity of growth and protein synthesis after a long exposure under non-permissive conditions may be the result of the inactivation of sup 1 gene product. The absence of low-temperature sensitivity of protein synthesis in Lts--revertant after a short incubation period under restrictive conditions (Fig. la) and the similarity of kinetics of polysome decay in Lts--revertant and parent strain (Fig. 2) can be understood if we assume that the non-permissive temperature does not inactivate a pre-existing product of sup 1 gene but inhibits its synthesis or assembly of a multimeric complex participating in translation and containing the low-temperature sensitive component. Since the data on in viva incorporation of labelled uracil into ribosomal subunits demonstrate the defect in a 60 S subunit assembly under nonpermissive conditions, one may suppose that low-temperature sensitive component is a ribosomal protein of 60 S subunit. The temperature decrease to 10°C apparently causes a eonformational change of this

154 protein and the concomitant defect of the 60 S subunit assembly. As a result, a deficiency of the large ribosomal subunit is observed (Fig. 3). Once assembled, 60 S subunits from Lts--revertant are not lowtemperature sensitive, since the short period of incubation of the cells under restrictive conditions does not inhibit protein synthesis (Fig. la). The slight decrease in the ratio of 60 S : 40 S subunits in Lts-revertant is observed under permissive conditions; a non-permissive temperature only accentuates the phenotypic effect of this mutation. Since Lts--revertant is able to grow at 10°C (results not shown) and to synthesize 60 S ribosomal subunit (however, at a reduced rate) we can conclude that the formation of 60 S subunit is not completely blocked but proceeds at a reduced rate; therefore the expression of low temperature sensitivity is leaky. It should be mentioned that the conclusion about the 60 S subunit assembly defect as a result of a conformational change of a ribosomal protein needs a more direct experimental evidence. Bayliss and Vinopal [19] and Bayliss and Ingraham [20] isolated Lts- strains of the yeast S. cerevisiae with altered sensitivity to streptomycine. At non-permissive temperature 28 S ribonucleoproteinparticles were found in this strain, whereas 60 S and 40 S subunits were not synthesized under these conditions. It is obvious that the defect of ribosome assembly in Lts- strains described in our paper is not similar to that found by Bayliss et al. The exact mechanism of the defect in 60 S ribosomal subunit assembly remains to be established. Since we cannot detect any precursors of subunits in mutant cells it is possible that one of the early stages of the subunit assembly is impaired. A similar mutation blocking the assembly of a ribosomal subunit in Escherichia coli was described by Herzog et al. [21]. The data concerning the defect of assembly of a large ribosomal subunit in Lts--revertant are important for localizing the gene sup 1 product. Since lowtemperature sensitivity and the ability to suppress nonsense codons are strictly coupled we conclude that both effects are the result of the same mutation. The low-temperature sensitivity of 60 S subunit assembly demonstrates that a mutationally altered component may be localized in this ribosomal subunit. We demonstrated earlier that the products of sup 1 and sup 2 genes are proteins but not RNAs

[6,7]; the same conclusion was drawn by Gerlach, who characterized similar recessive suppressor strain of yeast [4,5]. Since here we found that the sup 1 gene product is very likely localized in the 60 S ribosomal subunit we can suggest that this product is a ribosomal protein of the large subunit. This conclusion is supported by indirect data obtained earlier in this lab oratory: 1. The alteration of some functional properties of 60 S subunit after incubation under non-permissive conditions [9]. 2. The alteration of the ribosomal proteins pattern of 60 S subunit in recessive suppressor strain compared to that of parent strain [22]. 3. Isolation of cycloheximide dependent strain of yeast carrying a recessive suppressor mutation (TerAvanesyan, M.D., unpublished data); cycloheximide is known to bind preferentially to the large ribosomal subunit [23,24]. Taken together these data enable us to suggest that the product of sup 1 gene is a ribosomal protein of the 60 S subunit. In this connection the following point may be discussed. According to a widespread opinion the fidelity of translation is determined by the small ribosomal subunit [25]. In our previous paper we have shown that the ribosomes from suppressor strain possess a high level of misreading [8]. In this paper we have demonstrated that the mutation alters the large ribosomal subunit. Taking into account these two observations we can propose that 60 S subunit of yeast ribosomes can take part in the control of the translation fidelity. However, bearing in mind the complex interaction between the ribosomal subunits, one cannot exclude the possiblity that the effect of the large ribosomal subunit on the translation fidelity is mediated by the small subunit. Acknowledgement The authors would like to express their gratitude to Mrs. E.M. Pospelova for the assistance in experiments. References

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15 Daves, I.W. and Hardie, I.D. (1974) Mol. Gen. Genet. 131,281-289 16 Nomura, M. and Morgan, E.A. (1977) Annu. Rev. Genet. 11,297-347 17 Smith, I. (1977) in Molecular Mechanismsof Protein Biosynthesis (Weissbach, H. and Pestka, S., eds.), pp. 627701, Academic Press, New York 18 Inge-Vectomov, S.G. and Andrianova, V.M. (1972) in Molecular Mechanisms of Genetic Processes, pp. 189195, Nauka, Moscow 19 Bayliss, F.T. and Vinopal, R.T. (1971) Science 174, 1339-1341 20 Bayliss, F.T. and Ingraham, J.L. (1974) J. Bacteriol. 118, 319-325 21 Herzog, A., Yaguchi, M., Cabezon, T., Corchuelo, M.-C., Petre, J. and Bollen, A. (1979) Mol. Gen. Genet. 171, 15-22 22 Smirnov, V.N., Surguchov, A.P., Smirnov, V.V., Berestetskaya, Yu.V. and Inge-Vechtomov, S.G. (1978) Mol. Gen. Genet. 163, 87-90 23 Warner, J.R. (1974) in Ribosomes (Nomura, M., Tissieres, A. and Lengyel, P., eds.), pp. 461-468, Cold Spring Harbor Publishing, New York 24 PiSch, H., Zakrewski, S. and Nierhaus, K.H. (1979) Mol. Gen. Genet. 175,181-186 25 Gorini, L. (1974) in Ribosomes (Nomura, M., Tissieres, A. and Lengyel, P., eds.), pp. 793-803, Cold Spring Harbor Publishing, New York