Current Genetics 3, 27- 29 (1981)
~
~
~
© Springer-Verlag 1981
4OS Ribosomal Protein from a Saccharomyces cerevisiae Antisuppressor Mutant Exhibiting a Unique 2D Gel Pattern Susan W. Liebman and Margaret M. Cavenagh Department of Biological Sciences, University of Illinois at Chicago Circle, Box 4348, Chicago, Illinois 60680, USA
Summary. The yeast antisuppressor mutation, asu9-1 (Liebman .and Cavenagh 1980) was found to cause an alteration in the 40S ribosomal subunit. Two-dimensional polyacrylamide gel electrophoresis patterns o f the 40S ribosomal proteins from four different strains bearing the asu9-1 mutation all contained the same extra protein spot which was completely absent in five strains which did not carry the asu9 mutation. Key words: Ribosomes - Antisuppressor - Yeast
Introduction
have also appeared in Saccharomyces cerevisiae strains that lack suppressors (Smirnov et al. 1978). In an effort to isolate a new ribosomal mutation, we searched for antisuppressors that reduce the efficiencies of the omnipotent suppressors. One such antisuppressor, asu9, has recently been described (Liebman and Cavenagh 1980). Both the sup45 (Masurekar et al., in preparation) and asu9 (Liebman and Cavenagh 1980) mutations cause sensitivity to the aminoglycoside antibiotic paromomycin, a drug which binds to ribosomes. We have examined the ribosomal proteins from sup45-2 and asu9-1 yeast mutants.
Materials and Methods
Strains Used. The genealogy and complete genotypes of all In Saccharomyces cerevisiae, the omnipotent suppressors sup35 and sup45, which can suppress all types of nonsense mutants, are thought to be analogous to the ribosomal protein mutation, ramA, in E. coli. The ramA mutant (affecting ribosomal protein $4) can reverse the streptomycin dependence caused by strA mutations (affecting ribosomal protein S12) (Ozaki et al. 1969; Gorini 1974). RamA causes translational misreading leading to suppression of nonsense mutants, while strA restricts this misreading (Gorini 1969, 1974). Similarly, in S. cerevisiae, the small ribosomal subunit from sup45 strains causes misreading in a cell free poly(U)-directed protein synthesis system (Surguchov et al. 1980, Masurekar et al., in preparation). In addition, a yeast strain containing an omnipotent suppressor was reported to have an increased amount of L30 protein in its 60S ribosomal subunit relative to its suppressorless parent (Stairnov et al. 1978). However, elevated levels of L30 protein
Offprint requests to. S. W. Liebman
strains used in this paper are shown on Fig. 1. The origin of these strains has been described previously (Liebman and Cavenagh 1980).
Isolation of Ribosomal Proteins. Ribosomal subunits were isolated as described by Grant et al. (1974) except that washed cells were broken in a Braun Vibromixer and gradients were analyzed by passage through a Gilford Density Gradient analyzer. The separately collected 40S and 60S subunits were precipitated overnight at -20 °C with 0.1 volume 1 M MgCI2 and 0.7 volumes cold 95% EtOH. Subunits were then collected at 12,000 x g for 10 rain and proteins extracted by the method of Sherton and Wool (1974) and then precipitated with 80% acetone (Barritault et al. 1976).
Two-dimensional PolyaerylamMe Gel Eleetrophoresis. Gels were run as described by Waldron and Cox (1978). From 50-150 t~g of protein were loaded on the first dimension gels of 4% polyacrylamide in 8 M urea at pH 4.5 (Mets and Bogorad 1974; Subramanian 1974). Equilibration of the gel rods and second dimension electrophoresis through 12% polyacrylamide in 0.1% sodium dodeeyl sulfate were done as described by O'Farrell (1975). Gels were stained in 0.1% Coomassie Blue G, 10% acetic acid, and 25% isopropanol, and destained in 10% acetic acid and 1% glycerol. 0172-8083/81/0003/0027/$ 01.00
28
S.W. Liebman and M. M. Cavenagh: Altered 40 S Ribosomal Protein Due to a Yeast Antisuppressor
SL210-3A U.V. induced mutation L-475
X
(sup45-2)
SL421-4B
I ..... i
I meiosis; tetrad d i s s e c t i o n SL428-IC i
l L-477
(sup45-2)
|
spontaneousmutation X
(sup45-2 asug-l)
P//II/12/A
Fig. 1. Genealogy of strains used. The full genotype of SL2103A is c~ cyel-76 met8-1 leu2-1 tyr7-1 trpl-1 ade3-26 ilvl-1 lys2-1 hisS-2 canl-132 and the full genotype of SL421-4B is a eycl-76 met8-1 leu2-1 tyr7-1 trpl-1 ade3-26 ilvl-1 Iysl-1 hisS-2 can1-132. Except for mating type and lysine allele, the genotypes of all other strains in the chart vary only with respect to the suppressor and antisuppressor as indicated. Gels were run on all underlined strains I I indicates the normal wild-type 40S gel p a t t e r n . ~ indicates the presence of the dark, extra spot on the 40S gel. P77777aindicates the presence of the faint extra spot on the 40S gel
SL421-4B
I
J meiosis; tetrad d i s s e c t i o n SL434-9D
(asug~l)
Results
and
SL434-18D
(asug-l)
X
SL421-4B meiosis; tetrad d i s s e c t i o n
SL498-10A
(asu9-1)
SL498-10B
(asug-l)
SL498-IOC
(ASU9 + )
I
I
SL498-10D I
I
(ASU9 + )
Proteins extracted from purified ribosomal subunits were analyzed on a two-dimensional polyacrylamide gel electrophoresis system. In the first dimension the proteins were separated according to their charge at pH 4.5 (Mets and Bogorad 1974; Subramanian 1974) and in the second dimension according to their size (O'Farretl 19 75). In all, nine strains were examined (see Fig. 1 for genealogy): a strain lacking suppressors or antisuppressors
s. w. Liebman and M. M. Cavenagh: Altered 40 S Ribosomal Protein Due to a Yeast Antisuppressor (SL210-3A); a sup45-2 mutant induced in SL210-3A (L-475); a sup45-2 bearing segregant (SL428-1C); a sup45-2 asu9-1 double mutant induced in SL428-1C (L-477); an asu9-1 bearing segregant lacking suppressors (SL434-gD); the four segregants from a tetrad lacking suppressors but in which asu9-1 was segregating (SL 498-10A, asu9; SL498-10B, asu9; SIA98-10C, ASU9+; SL498-10D, ASU9+). The electrophoretograms of the sup45-2 strains were indistinguishable from those of the wild-type strains (Fig. 2a). However, all strains bearing the asu9-1 mutation had an extra spot on their 40S ribosomal protein gels (Fig. 2b and c). No other reproducible differences were noted. While the intensity of the new spot was approximately the same on the gels of all the asu9 strains that did not carry suppressors (SL434-9D, SL498-10A, SL498-10B), the spot was less intense on the electrophoretogram of the asu9 sup45 double mutant (L-477). Nonetheless, the spot was clearly present in the gel of the asu9 sup45 mutant, while it was completely absent in the isogenic sup45 parent (SL428-1C) (see Fig. 2a and b). Apparently, the sup 45 mutation reduces the effect o f the asu9 mutation.
Discussion These results clearly show that the asug-1 mutation causes an alteration in the 40 S ribosomal subunit, although the primary gene product of asu9 has not been determined. The protein that is present in the patterns of all asu9-containing strains, but is lacking in the gels of all other strains, may be the protein coded for b y the asu9 gene. On the other hand, the appearance of the extra spot may be due to a secondary effect o f the asu9 mutation. For instance, the protein in question may be present in wild-type cells, but only appears among the ribosomal proteins o f asu9 cells. The asu9 gene product might cause the protein to bind tightly to the ribosome when it would otherwise be washed off during the ribosome isolation. In E. coli, suppressors of streptomycin-
29
dependent mutants that do not map at the ramA locus cause an additional protein to bind to the ribosome (Dabbs 1977). Alternately, asu9 may regulate the synthesis of the extra protein. Finally, asu9 may be responsible for post-translational modification of the protein. This could alter its charge and/or size, causing it to migrate differently. Perhaps the spot is undetectable in gels o f strains lacking asu9 because it migrates along with another protein so that spots overlap, or because it migrates off the first dimension gel, as a few ribosomal proteins do. Further analysis will help us distinguish among these possibilities.
Acknowledgements. This work was supported by HEW g~ant GM24189.
References Barritault D, Expert-Bezancon A, Guerin MF, Hayes D (1976) EurJ Biochem 63:131 135 Dabbs ER (1977) Mol Gen Genet 158:55-61 Gorini L (1969) Cold Spring Harbor Symp Quant Biol 34:101109 Gorini L (1974) In: The ribosomes. Cold Spring Harbor Lab Publ, Cold Spring Harbour, p 791 Grant P, Sanehez L, Jimgnez A (1974) J Bacteriol 123:13081314 Liebman SW, Cavenagh M (1980) Genetics 95:49-61 Mets LJ; Bogorad L (1974) Anal Biochem 57:200-210 O'Farrell PH (1975) J Biol Chem 250:4007-4027 Ozaki M, Mizushima S, Nomura M (1969) Nature 222:333339 Sherton CC, Wool IG (1974) Mol Gen Genet 135:97-112 Smirnov NV, Surguchov AP, Smirnov VV, Berestetskaya Yu V (1978) Mol Gen Genet 163:87-90 Subramanian AR (1974) Eur J Biochem 45:541-546 Surguchov AP, Berestetskaya Yu V, Fominykch ES, Pospelova EM, Smirnov VN, Ter-Avanesyan MD, Inge-Vechtomov SG (1980) FEBS Lett 111:175-178 Waldron C, Cox BS (1978) Molee Gen Genet 159:223-225 Communicated b y B.S. Cox Received September 1, 1980
~l Fig. 2. Two dimensional electrophoretograms of yeast 40S mad 60S ribosomal proteins from strains bearing and lacking the asu9 mutation. (a) 40S ribosomal proteins from the non-asu9 strain, SL428-1C (sup45-2). Patterns of the other non-asu9 strains: SL2103A, L-475, SL498-10C and SL498-10D, are identical. (b) 40S ribosomal proteins from the asu9 sup45-2 double mutant strain L-477 derived from SL428-1C shown in (a). The arrow indicates a faint spot that is reproducibly present in the sup45-2 asu9-1 double mutant, but absent in the isogenic sup45.2 parent. The lines on the right indicate the positions of the molecular weight standards, Bovine serum albumin (67,000) and carbonic anhydrase (30,000) (c) 40S ribosomal proteins from the asu9 strain, SL434-9D which lacks sup45-2. Patterns of the other asu9 strains, SL498-10A and SL498-10B are identical. The arrow indicates the spot that is present only in asu9 strains. This is the only reproducible difference between asu9 and non-asu9 strains (d) 60S ribosomal proteins the asu9 strain, SL4349D. Some 40S contaminant proteins are faint but clearly visible on this and other 60S gels. The 60S proteins, however, are virtually absent from 40S gels. The arrow indicates the characteristic asu9 spot that can be seen as a 40S contaminant on this gel of 60S proteins. This spot never appears on non-asu9 strains. Otherwise, all 60S gels are identical. Molecular weight standard positions are marked as in "(b)
~
~
~
© Springer-Verlag 1981
4OS Ribosomal Protein from a Saccharomyces cerevisiae Antisuppressor Mutant Exhibiting a Unique 2D Gel Pattern Susan W. Liebman and Margaret M. Cavenagh Department of Biological Sciences, University of Illinois at Chicago Circle, Box 4348, Chicago, Illinois 60680, USA
Summary. The yeast antisuppressor mutation, asu9-1 (Liebman .and Cavenagh 1980) was found to cause an alteration in the 40S ribosomal subunit. Two-dimensional polyacrylamide gel electrophoresis patterns o f the 40S ribosomal proteins from four different strains bearing the asu9-1 mutation all contained the same extra protein spot which was completely absent in five strains which did not carry the asu9 mutation. Key words: Ribosomes - Antisuppressor - Yeast
Introduction
have also appeared in Saccharomyces cerevisiae strains that lack suppressors (Smirnov et al. 1978). In an effort to isolate a new ribosomal mutation, we searched for antisuppressors that reduce the efficiencies of the omnipotent suppressors. One such antisuppressor, asu9, has recently been described (Liebman and Cavenagh 1980). Both the sup45 (Masurekar et al., in preparation) and asu9 (Liebman and Cavenagh 1980) mutations cause sensitivity to the aminoglycoside antibiotic paromomycin, a drug which binds to ribosomes. We have examined the ribosomal proteins from sup45-2 and asu9-1 yeast mutants.
Materials and Methods
Strains Used. The genealogy and complete genotypes of all In Saccharomyces cerevisiae, the omnipotent suppressors sup35 and sup45, which can suppress all types of nonsense mutants, are thought to be analogous to the ribosomal protein mutation, ramA, in E. coli. The ramA mutant (affecting ribosomal protein $4) can reverse the streptomycin dependence caused by strA mutations (affecting ribosomal protein S12) (Ozaki et al. 1969; Gorini 1974). RamA causes translational misreading leading to suppression of nonsense mutants, while strA restricts this misreading (Gorini 1969, 1974). Similarly, in S. cerevisiae, the small ribosomal subunit from sup45 strains causes misreading in a cell free poly(U)-directed protein synthesis system (Surguchov et al. 1980, Masurekar et al., in preparation). In addition, a yeast strain containing an omnipotent suppressor was reported to have an increased amount of L30 protein in its 60S ribosomal subunit relative to its suppressorless parent (Stairnov et al. 1978). However, elevated levels of L30 protein
Offprint requests to. S. W. Liebman
strains used in this paper are shown on Fig. 1. The origin of these strains has been described previously (Liebman and Cavenagh 1980).
Isolation of Ribosomal Proteins. Ribosomal subunits were isolated as described by Grant et al. (1974) except that washed cells were broken in a Braun Vibromixer and gradients were analyzed by passage through a Gilford Density Gradient analyzer. The separately collected 40S and 60S subunits were precipitated overnight at -20 °C with 0.1 volume 1 M MgCI2 and 0.7 volumes cold 95% EtOH. Subunits were then collected at 12,000 x g for 10 rain and proteins extracted by the method of Sherton and Wool (1974) and then precipitated with 80% acetone (Barritault et al. 1976).
Two-dimensional PolyaerylamMe Gel Eleetrophoresis. Gels were run as described by Waldron and Cox (1978). From 50-150 t~g of protein were loaded on the first dimension gels of 4% polyacrylamide in 8 M urea at pH 4.5 (Mets and Bogorad 1974; Subramanian 1974). Equilibration of the gel rods and second dimension electrophoresis through 12% polyacrylamide in 0.1% sodium dodeeyl sulfate were done as described by O'Farrell (1975). Gels were stained in 0.1% Coomassie Blue G, 10% acetic acid, and 25% isopropanol, and destained in 10% acetic acid and 1% glycerol. 0172-8083/81/0003/0027/$ 01.00
28
S.W. Liebman and M. M. Cavenagh: Altered 40 S Ribosomal Protein Due to a Yeast Antisuppressor
SL210-3A U.V. induced mutation L-475
X
(sup45-2)
SL421-4B
I ..... i
I meiosis; tetrad d i s s e c t i o n SL428-IC i
l L-477
(sup45-2)
|
spontaneousmutation X
(sup45-2 asug-l)
P//II/12/A
Fig. 1. Genealogy of strains used. The full genotype of SL2103A is c~ cyel-76 met8-1 leu2-1 tyr7-1 trpl-1 ade3-26 ilvl-1 lys2-1 hisS-2 canl-132 and the full genotype of SL421-4B is a eycl-76 met8-1 leu2-1 tyr7-1 trpl-1 ade3-26 ilvl-1 Iysl-1 hisS-2 can1-132. Except for mating type and lysine allele, the genotypes of all other strains in the chart vary only with respect to the suppressor and antisuppressor as indicated. Gels were run on all underlined strains I I indicates the normal wild-type 40S gel p a t t e r n . ~ indicates the presence of the dark, extra spot on the 40S gel. P77777aindicates the presence of the faint extra spot on the 40S gel
SL421-4B
I
J meiosis; tetrad d i s s e c t i o n SL434-9D
(asug~l)
Results
and
SL434-18D
(asug-l)
X
SL421-4B meiosis; tetrad d i s s e c t i o n
SL498-10A
(asu9-1)
SL498-10B
(asug-l)
SL498-IOC
(ASU9 + )
I
I
SL498-10D I
I
(ASU9 + )
Proteins extracted from purified ribosomal subunits were analyzed on a two-dimensional polyacrylamide gel electrophoresis system. In the first dimension the proteins were separated according to their charge at pH 4.5 (Mets and Bogorad 1974; Subramanian 1974) and in the second dimension according to their size (O'Farretl 19 75). In all, nine strains were examined (see Fig. 1 for genealogy): a strain lacking suppressors or antisuppressors
s. w. Liebman and M. M. Cavenagh: Altered 40 S Ribosomal Protein Due to a Yeast Antisuppressor (SL210-3A); a sup45-2 mutant induced in SL210-3A (L-475); a sup45-2 bearing segregant (SL428-1C); a sup45-2 asu9-1 double mutant induced in SL428-1C (L-477); an asu9-1 bearing segregant lacking suppressors (SL434-gD); the four segregants from a tetrad lacking suppressors but in which asu9-1 was segregating (SL 498-10A, asu9; SL498-10B, asu9; SIA98-10C, ASU9+; SL498-10D, ASU9+). The electrophoretograms of the sup45-2 strains were indistinguishable from those of the wild-type strains (Fig. 2a). However, all strains bearing the asu9-1 mutation had an extra spot on their 40S ribosomal protein gels (Fig. 2b and c). No other reproducible differences were noted. While the intensity of the new spot was approximately the same on the gels of all the asu9 strains that did not carry suppressors (SL434-9D, SL498-10A, SL498-10B), the spot was less intense on the electrophoretogram of the asu9 sup45 double mutant (L-477). Nonetheless, the spot was clearly present in the gel of the asu9 sup45 mutant, while it was completely absent in the isogenic sup45 parent (SL428-1C) (see Fig. 2a and b). Apparently, the sup 45 mutation reduces the effect o f the asu9 mutation.
Discussion These results clearly show that the asug-1 mutation causes an alteration in the 40 S ribosomal subunit, although the primary gene product of asu9 has not been determined. The protein that is present in the patterns of all asu9-containing strains, but is lacking in the gels of all other strains, may be the protein coded for b y the asu9 gene. On the other hand, the appearance of the extra spot may be due to a secondary effect o f the asu9 mutation. For instance, the protein in question may be present in wild-type cells, but only appears among the ribosomal proteins o f asu9 cells. The asu9 gene product might cause the protein to bind tightly to the ribosome when it would otherwise be washed off during the ribosome isolation. In E. coli, suppressors of streptomycin-
29
dependent mutants that do not map at the ramA locus cause an additional protein to bind to the ribosome (Dabbs 1977). Alternately, asu9 may regulate the synthesis of the extra protein. Finally, asu9 may be responsible for post-translational modification of the protein. This could alter its charge and/or size, causing it to migrate differently. Perhaps the spot is undetectable in gels o f strains lacking asu9 because it migrates along with another protein so that spots overlap, or because it migrates off the first dimension gel, as a few ribosomal proteins do. Further analysis will help us distinguish among these possibilities.
Acknowledgements. This work was supported by HEW g~ant GM24189.
References Barritault D, Expert-Bezancon A, Guerin MF, Hayes D (1976) EurJ Biochem 63:131 135 Dabbs ER (1977) Mol Gen Genet 158:55-61 Gorini L (1969) Cold Spring Harbor Symp Quant Biol 34:101109 Gorini L (1974) In: The ribosomes. Cold Spring Harbor Lab Publ, Cold Spring Harbour, p 791 Grant P, Sanehez L, Jimgnez A (1974) J Bacteriol 123:13081314 Liebman SW, Cavenagh M (1980) Genetics 95:49-61 Mets LJ; Bogorad L (1974) Anal Biochem 57:200-210 O'Farrell PH (1975) J Biol Chem 250:4007-4027 Ozaki M, Mizushima S, Nomura M (1969) Nature 222:333339 Sherton CC, Wool IG (1974) Mol Gen Genet 135:97-112 Smirnov NV, Surguchov AP, Smirnov VV, Berestetskaya Yu V (1978) Mol Gen Genet 163:87-90 Subramanian AR (1974) Eur J Biochem 45:541-546 Surguchov AP, Berestetskaya Yu V, Fominykch ES, Pospelova EM, Smirnov VN, Ter-Avanesyan MD, Inge-Vechtomov SG (1980) FEBS Lett 111:175-178 Waldron C, Cox BS (1978) Molee Gen Genet 159:223-225 Communicated b y B.S. Cox Received September 1, 1980
~l Fig. 2. Two dimensional electrophoretograms of yeast 40S mad 60S ribosomal proteins from strains bearing and lacking the asu9 mutation. (a) 40S ribosomal proteins from the non-asu9 strain, SL428-1C (sup45-2). Patterns of the other non-asu9 strains: SL2103A, L-475, SL498-10C and SL498-10D, are identical. (b) 40S ribosomal proteins from the asu9 sup45-2 double mutant strain L-477 derived from SL428-1C shown in (a). The arrow indicates a faint spot that is reproducibly present in the sup45-2 asu9-1 double mutant, but absent in the isogenic sup45.2 parent. The lines on the right indicate the positions of the molecular weight standards, Bovine serum albumin (67,000) and carbonic anhydrase (30,000) (c) 40S ribosomal proteins from the asu9 strain, SL434-9D which lacks sup45-2. Patterns of the other asu9 strains, SL498-10A and SL498-10B are identical. The arrow indicates the spot that is present only in asu9 strains. This is the only reproducible difference between asu9 and non-asu9 strains (d) 60S ribosomal proteins the asu9 strain, SL4349D. Some 40S contaminant proteins are faint but clearly visible on this and other 60S gels. The 60S proteins, however, are virtually absent from 40S gels. The arrow indicates the characteristic asu9 spot that can be seen as a 40S contaminant on this gel of 60S proteins. This spot never appears on non-asu9 strains. Otherwise, all 60S gels are identical. Molecular weight standard positions are marked as in "(b)
