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Modified ribosomes conferring resistance to cycloheximide in mutants of Saccharomyces cerevisiae
J. Mol. Biol. (1967) 26, 347-350
LETTERS TO THE EDITOR
Modified
Ribosomes conferring Resistance to Cycloheximide Mutants of Saccharomyces cevevisiae
in
Cycloheximide (trade name “Actidione”) is an antibiotic which is toxic towards many eucaryotic organisms, but not towards bacteria or blue-green algae. The inhibition of both DNA and protein synthesis is reported (Siegal & Sisler, 1964a and references therein) as being the principal biochemicaleffect ofthe drug inintact cells, while studies with cell-free systems have shown that the inhibition of the transfer of amino acids from aminoacyl-transfer RNA to polypeptide is probably the primary effect (Ennis & Lubin, 1964; Siegal & Sisler 19646). Saccharomyces fragilis, a species of yeast which is resistant to cycloheximide, was recently found to possess ribosomes which are insensitive to high levels of cycloheximide in vitro (Siegel & Sisler, 1965). Resistant strains of fungi can also be obtained from normally sensitive organisms by gene mutation, and an extensive genetic analysis of such resistance in S. cerevisiae has been reported (Wilkie & Lee, 1965). We have shown, both by chemical and biological assay of uptake of drug into the organism and by using [14C]cycloheximide (which has been prepared from Streptomyces griseus cultures, using strain 5232 of the National Collection of Industrial Bacteria with [14C]gluoose as precursor), that some of these mutants are freely permeable to the drug. We now report that a ribosomal mechanism of resistance appears to be associated with at least one of the mutant genes previously identified. Ribosomes and supernatant enzymes for protein synthesis were prepared essentially by published methods (Bretthauer, Marcus, Chaloupka, Halvorson 8z Bock, 1963) and samples of the reaction mixtures were prepared for counting as previously described (Mans & Novelli, 1961). In the presence of 250 pg/ml. of poly U, the system showed high activity, incorporating approximately 1.8 mpmoles of phenylalanine per mg of ribosomal protein into the fraction insoluble in hot trichloroacetic acid. Ribonuclease and puromycin strongly inhibited incorporation of amino acid, whereas streptomycin had no effect up to 100 pg/ml. Table 1 lists the strains studied. It was found that they could be divided into three groups, typified by curves A, B and C of Fig. 1, on the basis of the response of the initial rate of phenylalanine incorporation to increasing cycloheximide concentrations in cell-free systems. Curve A is obtained for systems derived from wild-type (sensitive) strains, or strains carrying only semi-dominant resistance genes. Cycloheximide (O-64 pM) produces a 50% inhibition of the rate of incorporation; this concentration corresponds to about 1.4 molecules of drug per ribosome in the system. Strains carrying the recessive resistance gene acg have a greatly reduced sensitivity to the drug in vitro as shown by the dose-response curve C. Here 50% inhibition requires a drug concentration of about 23 PM, or about 50 molecules per ribosome., Small variations between duplicate experiments could be attributed to the very high dependance of inhibitor-response upon the pH of the reaction medium (Cooper, unpublished work). When the ribosome fraction from strain 20 was incubated with supernatant enzymes 23
347
34%
D.
COOPER,
D.
V.
BANTHORPE TABLE
strain of S. cerevisiae
AND
Modifies
resistance
WILKIE
1 Cycloheximide resistance in viva w
Resistance genes
45 4041 (diploid) 3 20 21 4021 (diploid) $
D.
DOSWSSpOIlSe
curve irL vitro
Ccl.8 <1.8 36 3600 72
A A A c C B (Wilkie
& Lee, 1965).
20 I
!
I
000
+
80 60 40 20 0
I
1
I
I
I
I
1
IO
20
30
40
50
60
70
(PM) cyclohexlmlde FIG. 1. Response of cell-free systems of (1) haploid and diploid sensitive strains and a haploid strain carrying only semi-dominant resistance genes (curve A). (2) Diploid strain heterozygons for the recessive gene a$ (curve B). (3) Haploid strains carrying czci (curve C). (4) System with ribosome8 from sensitive strain, supernatant fraction from resistant strain with acs gene (curve D). (5) Reciprocal system of (4) (curve E). Reaction mixtures contained, per ml. : 20 pmoles Tris-HCl (pH 7.5) ; 60 qoles KCl; 5 /unoles magnesium acetate; 2 i+moles mercaptoethanol; 0.2 pmole spermidine; O-5 pmole ATP; 6.0 pmoles phosphoenol pyruvate; 100 pg pyruvate kinase; 0.15 qole GTP; 0.6 PC [14C]phenylalanine (specific activity 6.8 pc/pmole) purchased from the Radiochemical Centre, Amersham, England; 250 pg polyuridylic acid (Miles Chemical Co., U.S.A.); 1.2 mg ribosomal protein and 1.2 mg supernatant protein. ATP, GTP, phosphoenol pyruvate and pyruvate kinase were purchased from the Sigma Chemical Co., London, and cycloheximide from the Koch-Light Laboratories Ltd., England. Protein was estimated by a modified biuret reaction (Giles, unpublished method). Samples of 0.3 ml. were removed for radioactive assay after 0, 5, 10 and 20 min incubation at 3O’C. The initial rate of phenylalanine incorporation was then estimated from the counts obtained, and this was used in plotting the graphs above.
LETTERS
TO
THE
349
EDITOR
and transfer RNA from a sensitive strain, and vice versa, it was the ribosomes which conferred resistance to cycloheximide in the cell-free system, not the supernatant fraction (Fig. 1). To eliminate the possibility that non-specific protein sedimenting with, and contaminating, the ribosomes was a factor in resistance, ribosome preparations were washed by the method of Wettstein, Staehelin & Noll (1963). Although this procedure increased the RNA/protein ratio from about 1 : 1 to 2 : 1, resistance still followed the ribosomes, and results similar to those previously described were obtained. The third type of response to cycloheximide is represented by curve B (Fig. 1). This response to cycloheximide is obtained from extracts of the heterozygous diploid carrying aci and its wild-type allele. The strain is phenotypioally sensitive, although the ribosomes show an intermediate degree of resistance in vitro. This suggests that the diploid cells contain a mixed complement of both sensitive and resistant ribosomes, giving rise to “mixed” polysomes. Since cycloheximide does not inhibit polysome formation in mammalian systems (Williamson & Schweet, 1965) but impedes the subsequent movement of ribosomes along the messenger RNA (‘Wettstein, No11 & Venman, 1964), such mixed polysomes would be expected to be non-functional in vivo in the presence of the drug; resistant ribosomes would be blocked by sensitive ones ‘“frozen” on the messenger RNA. A similar explanation has been proposed for the recessivity of streptomycin resistance by a ribosomal mechanism in E. coli (Lederberg, Cavalli-Sforza & Lederberg, 1964). In vitro, however, the presence of excess of poly U minimizes polysome formation, allowing the resistant ribosomes to function in polypeptide synthesis. The semi-dominant genes, in this case AC,“, AC: and AC;, do not affect protein synthesis. It is possible that such mutants can convert the drug into one of the many derivatives showing reduced biological activity that have been characterized (Lee & Wilkie, 1965; Siegel, Sisler & Johnson, 1966), for these mutants are freely permeable t,o the drug. This aspect is being investigated. We thank Professor D. Lewis for the interest and encouragement he showed throughout this investigation: and also the Medical Research Council for financial assistance and a Research Assistantship for one of us (D. C.). Department of Botany Department of Chemistry Department of Botany University College London, England. Received
21 February
D. COOPER D. V. BANTHORPE D. WILEIJZ
1967 REFERENCES
Bsetthauer,
R. K.,
Marcus, L., Chaloupka, J., Halvorson, H. 0. & Rock, R. M. (1963). Biochemistry, 2, 1079. Em&, H. L. & Lubin, M. (1964). Science, 146, 1474. Lederberg, E. M., Cavalli-Sforza, L. & Lederberg, J. (1964). Proc. Nat.. Acad. SC&, Wash.
51, 678. D. (1965). Nature, 266, 90. G. D. (1961). Arch. Biochem. Biophys. 94, Siegel, M. R. & Sisler, H. D. (19640). Biochim. biophys. Acta, 87, Siegel, M. R. & Sisler, H. D. (1964b). Biochim. biophys. Actcz, 87, Siegel, M. R. & Sisler, H. D. (1965). Biochim. biophys. Acta, 103, 23* Lee,
B. K.
& Wilkie,
Mans, R. J. & Novelli,
47. 70.
83. 558.
360 Siegel, M. Wettstein, Wettstein, Wilkie, D. Williamson,
D.
COOPER,
D.
V.
BANTHORPE
AND
D.
WILKIE
R., Sisler, H. D. & Johnson, F. (1966). Biochim. Plxxrmac. 15, 1213. F. O., Noll, H. 8.z Penman, S. (1964). Bioohim. biophya. Acta, 87, 60. F. O., Staehelin, T. & Noll, H. (1963). Nature, 197, 430. & Lee, B. K. (1965). CTenet. Res. 6, 130. A. R. & Schweet, R., (1965). J. MOE. Biol. 11, 358.
PLATE I. Long
spacing
segments
from
acid-soluble
collagen.
Upper part: Actinocol. Mesogloea of the sea anemone was extracted with an acetic acid solution of pH 2.6 at 4°C. Purification of the extract by repeated precipitation (dialysis against neutral medium). Preparation by dialysis of purified actinocol solutions against ATP at pH 2.5. Positive staining with phosphotungstic acid and many1 acetate. Lower part: Calf skin collagen. Extraction of calf skin (citrate buffer, pH 3.7), purification of the collagen solution, precipitation of long spacing segments (at pH 3.5) and staining as above (electron micrograph: courtesy of Dr K. Kuhn).
LETTERS TO THE EDITOR
Modified
Ribosomes conferring Resistance to Cycloheximide Mutants of Saccharomyces cevevisiae
in
Cycloheximide (trade name “Actidione”) is an antibiotic which is toxic towards many eucaryotic organisms, but not towards bacteria or blue-green algae. The inhibition of both DNA and protein synthesis is reported (Siegal & Sisler, 1964a and references therein) as being the principal biochemicaleffect ofthe drug inintact cells, while studies with cell-free systems have shown that the inhibition of the transfer of amino acids from aminoacyl-transfer RNA to polypeptide is probably the primary effect (Ennis & Lubin, 1964; Siegal & Sisler 19646). Saccharomyces fragilis, a species of yeast which is resistant to cycloheximide, was recently found to possess ribosomes which are insensitive to high levels of cycloheximide in vitro (Siegel & Sisler, 1965). Resistant strains of fungi can also be obtained from normally sensitive organisms by gene mutation, and an extensive genetic analysis of such resistance in S. cerevisiae has been reported (Wilkie & Lee, 1965). We have shown, both by chemical and biological assay of uptake of drug into the organism and by using [14C]cycloheximide (which has been prepared from Streptomyces griseus cultures, using strain 5232 of the National Collection of Industrial Bacteria with [14C]gluoose as precursor), that some of these mutants are freely permeable to the drug. We now report that a ribosomal mechanism of resistance appears to be associated with at least one of the mutant genes previously identified. Ribosomes and supernatant enzymes for protein synthesis were prepared essentially by published methods (Bretthauer, Marcus, Chaloupka, Halvorson 8z Bock, 1963) and samples of the reaction mixtures were prepared for counting as previously described (Mans & Novelli, 1961). In the presence of 250 pg/ml. of poly U, the system showed high activity, incorporating approximately 1.8 mpmoles of phenylalanine per mg of ribosomal protein into the fraction insoluble in hot trichloroacetic acid. Ribonuclease and puromycin strongly inhibited incorporation of amino acid, whereas streptomycin had no effect up to 100 pg/ml. Table 1 lists the strains studied. It was found that they could be divided into three groups, typified by curves A, B and C of Fig. 1, on the basis of the response of the initial rate of phenylalanine incorporation to increasing cycloheximide concentrations in cell-free systems. Curve A is obtained for systems derived from wild-type (sensitive) strains, or strains carrying only semi-dominant resistance genes. Cycloheximide (O-64 pM) produces a 50% inhibition of the rate of incorporation; this concentration corresponds to about 1.4 molecules of drug per ribosome in the system. Strains carrying the recessive resistance gene acg have a greatly reduced sensitivity to the drug in vitro as shown by the dose-response curve C. Here 50% inhibition requires a drug concentration of about 23 PM, or about 50 molecules per ribosome., Small variations between duplicate experiments could be attributed to the very high dependance of inhibitor-response upon the pH of the reaction medium (Cooper, unpublished work). When the ribosome fraction from strain 20 was incubated with supernatant enzymes 23
347
34%
D.
COOPER,
D.
V.
BANTHORPE TABLE
strain of S. cerevisiae
AND
Modifies
resistance
WILKIE
1 Cycloheximide resistance in viva w
Resistance genes
45 4041 (diploid) 3 20 21 4021 (diploid) $
D.
DOSWSSpOIlSe
curve irL vitro
Ccl.8 <1.8 36 3600 72
A A A c C B (Wilkie
& Lee, 1965).
20 I
!
I
000
+
80 60 40 20 0
I
1
I
I
I
I
1
IO
20
30
40
50
60
70
(PM) cyclohexlmlde FIG. 1. Response of cell-free systems of (1) haploid and diploid sensitive strains and a haploid strain carrying only semi-dominant resistance genes (curve A). (2) Diploid strain heterozygons for the recessive gene a$ (curve B). (3) Haploid strains carrying czci (curve C). (4) System with ribosome8 from sensitive strain, supernatant fraction from resistant strain with acs gene (curve D). (5) Reciprocal system of (4) (curve E). Reaction mixtures contained, per ml. : 20 pmoles Tris-HCl (pH 7.5) ; 60 qoles KCl; 5 /unoles magnesium acetate; 2 i+moles mercaptoethanol; 0.2 pmole spermidine; O-5 pmole ATP; 6.0 pmoles phosphoenol pyruvate; 100 pg pyruvate kinase; 0.15 qole GTP; 0.6 PC [14C]phenylalanine (specific activity 6.8 pc/pmole) purchased from the Radiochemical Centre, Amersham, England; 250 pg polyuridylic acid (Miles Chemical Co., U.S.A.); 1.2 mg ribosomal protein and 1.2 mg supernatant protein. ATP, GTP, phosphoenol pyruvate and pyruvate kinase were purchased from the Sigma Chemical Co., London, and cycloheximide from the Koch-Light Laboratories Ltd., England. Protein was estimated by a modified biuret reaction (Giles, unpublished method). Samples of 0.3 ml. were removed for radioactive assay after 0, 5, 10 and 20 min incubation at 3O’C. The initial rate of phenylalanine incorporation was then estimated from the counts obtained, and this was used in plotting the graphs above.
LETTERS
TO
THE
349
EDITOR
and transfer RNA from a sensitive strain, and vice versa, it was the ribosomes which conferred resistance to cycloheximide in the cell-free system, not the supernatant fraction (Fig. 1). To eliminate the possibility that non-specific protein sedimenting with, and contaminating, the ribosomes was a factor in resistance, ribosome preparations were washed by the method of Wettstein, Staehelin & Noll (1963). Although this procedure increased the RNA/protein ratio from about 1 : 1 to 2 : 1, resistance still followed the ribosomes, and results similar to those previously described were obtained. The third type of response to cycloheximide is represented by curve B (Fig. 1). This response to cycloheximide is obtained from extracts of the heterozygous diploid carrying aci and its wild-type allele. The strain is phenotypioally sensitive, although the ribosomes show an intermediate degree of resistance in vitro. This suggests that the diploid cells contain a mixed complement of both sensitive and resistant ribosomes, giving rise to “mixed” polysomes. Since cycloheximide does not inhibit polysome formation in mammalian systems (Williamson & Schweet, 1965) but impedes the subsequent movement of ribosomes along the messenger RNA (‘Wettstein, No11 & Venman, 1964), such mixed polysomes would be expected to be non-functional in vivo in the presence of the drug; resistant ribosomes would be blocked by sensitive ones ‘“frozen” on the messenger RNA. A similar explanation has been proposed for the recessivity of streptomycin resistance by a ribosomal mechanism in E. coli (Lederberg, Cavalli-Sforza & Lederberg, 1964). In vitro, however, the presence of excess of poly U minimizes polysome formation, allowing the resistant ribosomes to function in polypeptide synthesis. The semi-dominant genes, in this case AC,“, AC: and AC;, do not affect protein synthesis. It is possible that such mutants can convert the drug into one of the many derivatives showing reduced biological activity that have been characterized (Lee & Wilkie, 1965; Siegel, Sisler & Johnson, 1966), for these mutants are freely permeable t,o the drug. This aspect is being investigated. We thank Professor D. Lewis for the interest and encouragement he showed throughout this investigation: and also the Medical Research Council for financial assistance and a Research Assistantship for one of us (D. C.). Department of Botany Department of Chemistry Department of Botany University College London, England. Received
21 February
D. COOPER D. V. BANTHORPE D. WILEIJZ
1967 REFERENCES
Bsetthauer,
R. K.,
Marcus, L., Chaloupka, J., Halvorson, H. 0. & Rock, R. M. (1963). Biochemistry, 2, 1079. Em&, H. L. & Lubin, M. (1964). Science, 146, 1474. Lederberg, E. M., Cavalli-Sforza, L. & Lederberg, J. (1964). Proc. Nat.. Acad. SC&, Wash.
51, 678. D. (1965). Nature, 266, 90. G. D. (1961). Arch. Biochem. Biophys. 94, Siegel, M. R. & Sisler, H. D. (19640). Biochim. biophys. Acta, 87, Siegel, M. R. & Sisler, H. D. (1964b). Biochim. biophys. Actcz, 87, Siegel, M. R. & Sisler, H. D. (1965). Biochim. biophys. Acta, 103, 23* Lee,
B. K.
& Wilkie,
Mans, R. J. & Novelli,
47. 70.
83. 558.
360 Siegel, M. Wettstein, Wettstein, Wilkie, D. Williamson,
D.
COOPER,
D.
V.
BANTHORPE
AND
D.
WILKIE
R., Sisler, H. D. & Johnson, F. (1966). Biochim. Plxxrmac. 15, 1213. F. O., Noll, H. 8.z Penman, S. (1964). Bioohim. biophya. Acta, 87, 60. F. O., Staehelin, T. & Noll, H. (1963). Nature, 197, 430. & Lee, B. K. (1965). CTenet. Res. 6, 130. A. R. & Schweet, R., (1965). J. MOE. Biol. 11, 358.
PLATE I. Long
spacing
segments
from
acid-soluble
collagen.
Upper part: Actinocol. Mesogloea of the sea anemone was extracted with an acetic acid solution of pH 2.6 at 4°C. Purification of the extract by repeated precipitation (dialysis against neutral medium). Preparation by dialysis of purified actinocol solutions against ATP at pH 2.5. Positive staining with phosphotungstic acid and many1 acetate. Lower part: Calf skin collagen. Extraction of calf skin (citrate buffer, pH 3.7), purification of the collagen solution, precipitation of long spacing segments (at pH 3.5) and staining as above (electron micrograph: courtesy of Dr K. Kuhn).