Saccharomyces cerevisiae

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Room 116, Bethesda, Maryland 20205; tDepartment of Biochemistry, Albert Einstein College of Medicine, Bronx, New York 10461; and tChaire de GCnetique.

Proc. Nati Acad. Sci. USA Vol. 79, pp. 4706-4708, August 1982 Genetics

Ribosomal protein L3 is involved in replication or maintenance of the killer double-stranded RNA genome of Saccharomyces cerevisiae (trichodermin resistance/mak8 gene/M double-stranded RNA)

REED B. WICKNER*, S. PORTER RIDLEY*, HOWARD M. FRIEDt, AND STEVEN G. BALL*t *Laboratory of Biochemical Pharmacology, National Institute of Arthritis, Diabetes, and Digestive and Kidney Diseases, National Institutes of Health, Building 4, Room 116, Bethesda, Maryland 20205; tDepartment of Biochemistry, Albert Einstein College of Medicine, Bronx, New York 10461; and tChaire de GCnetique Plantes, Facult6 des Sciences Agronomique de l'Etat, 5800 Gembloux, Belgium

et d'Am6lioration des

Communicated by John R. Preer, Jr., May 3, 1982

ABSTRACT Ability to secrete the K1 (or K2) toxin protein and immunity to that toxin [the K1 (or K2) killer trait] are determined by a double-stranded (ds) RNA, called M1 (or M2), whose replication and maintenance depend on at least one of the larger (L) ds RNAs and 29 chromosomal genes, called MAK genes (maintenance of killer). The location of the MAK8 gene near TCM1 (trichodermin resistance) on the yeast map suggested the possible identity of these two genes. Of six independently isolated tcml mutants, five were clearly mak-, and the sixth was weakly mak-. In each case, the mak- phenotype and the trichodermin-resistant phenotypes cosegregated in meiosis and showed the expected tight linkage to petl7. The mak- mutations in the trichodermin-resistant strains did not complement mak8-l, indicating that MAK8 and TCM1 are the same gene. The mak8-1 mutation does not make strains resistant to trichodermin, and one tcml mutation is only slightly mak-. Whereas tcml mutants lose M1 or M2 ds RNA, they do not lose L ds RNA. Because TCM1 codes for ribosomal protein L3 [Fried, H. M. & Warner, J. R. (1981) Proc NatL Acadi. Sci. USA 78, 238-242], we conclude that ribosomal protein L3 is involved in the replication and maintenance of M ds RNA. Mutations in cyh2 or cryl, producing resistance to cycloheximide and crytopleurine due to mutant ribosomal proteins, do not produce a makphenotype. In analogy with bacterial ribosome assembly mutants, yeast low-temperature-sensitive (Its) mutants may have defective ribosomes. We thus examined mutants for an effect on the killer system. An Its5 mutant, unable to grow at 5VC, also has a makphenotype (at 300C) that cosegregates in meiosis with the Its- phenotype. Mutations in seven other Its genes do not result in the mak phenotype.

Table 1. Strains of Saccharomyces cerevisiae Strain Genotype 1384 a his4[KIL-k2] 1384-4A tcml-4A, 1384-4B tcml-4B Spontaneousindependent mutants derived tcml strain 1 tcml-5A I from 1384-5A 1384 1384-5B tcml -5B J 3147-30 a as cyh2 petl7 [KIL-kj] 3147-3D a ural peRl [KIL-ki] cLP-8 a leu2 tcml-l [KIL-o] HF-T1 1874

a his4-864 tcml-2 [KIL-o] a

ural + thri

1876

a

argi leu2

1101

a his4 karl-l [KIL-kj] a adel ade2 his7 lys2 ural gall canl

SP25

+

+ tcml

-

[KIL-kl]

[-KIL-kj]

Ref. 20

This work This work J. Davies (15, 16) H. Fried This work

This work

This work

[KIL-kl]

tical to TCM1 [trichodermin resistance (15-17)], whose product is ribosomal protein L3 (18). Three non-Mendelian genetic elements affecting the K1 and K2 systems.have recently been described. [HOK] (helper of killer) is defined by its ability to support the replication of an otherwise replication-defective mutant of M1 (19). [EXL] (excluder) is defined by its exclusion of M2 ds RNA when both are introduced into the same cell. [NEX] (nonexcludable) protects M2 from the action of [EXL] (20). We have found that [HOK] and [NEX] are on the same genetic element (12) and that this is a form of L ds RNA, which we call L-A-HN (refs. 12, 19, 21, and 22 and unpublished data). Distinct forms of L ds RNA, called L-B and L-C, are compatible with L-A-HN and are present in many strains (refs. 12, 19, 21, and 22 .and unpublished

The K1 killer double-stranded (ds) RNA genome (M1 ds RNA; 1.5 X 106 daltons) codes'for an 11,000-dalton protein toxin and determines immunity to that toxin (1-4). A second (K2) killerimmunity system is similarly determined by M2 ds 'RNA (1.0 X 106 daltons). M1l is dependent, for its replication and maintenance, on the products of at least 29 chromosomal genes, called MAK genes (for maintenance of killer), as well as on at least one of the larger L ds RNAs (5-12). Ofthese 29 genes, the product of only one is known. SPE2 codes for adenosylmethionine decarboxylase, an enzyme in spermine and spermidine synthesis (13), and cells bearing it lose M ds RNA when not supplied with exogenous polyamines (14). It is important to determine what are the products of the other MAK genes, what is their role in ds RNA replication or maintenance, and what is their role for the cell. This paper describes evidence that the MAK8 gene is iden-

data).

MATERIALS AND METHODS Strains of Saccharomyces cerevisiae are listed in Table 1. The media are described in ref. 8. Mutants resistant to trichodermin were isolated by plating haploid cells on rich medium containing the drug at 20 Ag/ml. The same medium was used to score trichodermin resistance. Complementation tests between different mak- mutants were done as described (7). Trichodermin was the generous gift of W. 0. Godtfredson (Leo Pharmaceutical Products, Helsingborg, Denmark).

The publication costs ofthis article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.

Abbreviation: ds RNA, double-stranded RNA. 4706

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4707

Table 2. mak property of tcml mutants Parents 3147-3C (a his4 cyh2 petl7 K1j) x 3051-lA (a tlrl leu2 tcml-1 K-) x 3072-3B (a his4 tcml-2 KW) 3147-3D (a petl7 ural K1) x 3144-15A (a his4 cyh2 tcml-4A K-) 3147-3D x 3144-3A (a ural cyh2 tcml-4A K-) 3147-3C x 3145-1C (a ural tcml-4B K-) 3147-3C x 3146-4A (a his4 cyh2 tcml-5A K-) 3147-3D x 3147-lB (a hM4 tcml-5B K-) 3147-3D x 3147-3A (a ural cyh2 tcml-5B K-) 3147-3C KW, weak killer; TcmR and Tcm', trichodermin-resistant and -sensitive. * Twenty-two parental ditype with petl 7, one tetratype with petl 7. t Nine parental ditype with petl 7, one tetratype with petl 7. t Eight parental ditype with petl 7, one tetratype with petl 7.

Cross 3176 3175 3155 3158 3157 3159 3154 3156

Spontaneous mutants resistant to cycloheximide at 3 Ag/ml or cerulenin at 3 4g/ml on rich medium were obtained by a

similar procedure.

RESULTS Both mak8-1 (6) and tcml (16) are closely linked to PET17 (a gene required for respiration) on chromosome XV, suggesting the possibility that these two genes are identical. Six independently isolated tcml mutants were tested for the mak- phenotype by crossing them with killer strains (marked with peti 7 to identify the tcml locus by linkage). The results (Table 2) show that five of the tcml mutants have a mak- phenotype that cosegregates in all tetrads examined (122) with the trichoderminresistant phenotype. In each case, tight linkage with the peti 7 gene was observed, as expected. A sixth tcml mutant showed only a weakly mak- phenotype in many segregants. Strains carrying the mak8-1 mutation did not show resistance to trichodermin. To determine whether the mak- mutation in the tcml trichodermin-resistant mutants was in the same gene as that in the mak8-1 strains, complementation tests were carried out. Complementation tests cannot be carried out by simply mating two mak- mutants with each other, because both parents lack the killer plasmid. The method used (7) takes advantage of the fact that although vegetative maka- cells do not have M ds RNA, maka& spores from a makaj + [KIL-k1] diploid do have the killer ds RNA genome. When the spores from this diploid are mated with a haploid makb- strain, the diploids formed will be about 50% K+ if a = b and 100% Ki if a # b (7). In practice, the proportion of K- diploids formed in control matings of maka/+ spores with maka haploid strains varies between 14% and 57% (7). The results (Table 3) showed that mak8-1 and tcml-l are recessive and do not complement each other, and neither complemented the nmk- mutations in tcml4A, tcml4B, tcml-5A, or tcml-5B strains. These results indicate that mak8-1 strains and the tcml mutants are all defective in the same gene. In addition to M1 and M2 ds RNAs, killer and nonkiller yeast can contain at least two kinds of L ds RNA. One, called L-A-HN, carries the [HOK] and [NEX] cytoplasmic genes and depends on the products of MAK3, MAK10, and PET18. The other, L-B or L-C, carries neither [HOK] nor [NEX] and requires none of the MAK genes tested, including MAK3, MAKJO, and PET18 (refs. 12 and 19-22 and unpublished data). Some strains carry a cytoplasmic gene, [EXL], that excludes M2 ds RNA. [EXL] is also located on a form of L ds RNA called LA-E. Trichodermin-resistant mutants were isolated from a series

Meiotic segregation 2 TcmR K1- PET4:2 Tcms K1+ petl7 2 TcmR K1- or Kjw PET+:2 Tcms K1+ petl7 2 TcmR K1-:2 Tcms K1j 2 TcmR Kj-:2 Tcms K1+ 2 TcmR Kj:2 TcmsKj+ 2 TcmR K1- PET4:2 Tcms K1+ petl7 2 TcmR K1- PET+:2 Tcms K1+ petl7 2 TcmR K1- PET+:2 Tcms K1+ petl7

No. of tetrads examined 11 24 23* lot 9t

24 24 21

of different strains to determine which of the ds RNAs depend on the MAK8 (TCM1) gene. M1 and M2 were lost from all trichodermin-resistant mutants, but [HOK], [NEX], and [EXL] remained. In strains carrying only L-B or L-C, this also remained in the trichodermin-resistant derivatives. Thus, while the MAK8 (TCM1) gene is needed for M1 and M2 replication, there is no evidence that it is involved in maintenance of L-AHN, L-A-E, or other L ds RNAs. The involvement of ribosomal protein L3 in killer ds RNA maintenance or replication suggested that other MAK genes might code for ribosomal proteins. Cycloheximide (23), cryptopleurine, and trichodermin (24) all cure the killer plasmid and all are inhibitors of protein synthesis acting on ribosomal proteins (15, 16, 18, 25, 26). We tested three independent cryl Table 3. Complementation tests among tcml mutants and the mak8-1 mutant* Source of spores Strain 1876 Strain 1874 tcml -1 mak8-1 +

Haploid strain 201 MAK+ [KIL-o] AN33 MAKE [KIL-o] makx [KIL-o]t 3C 2988-7B mak8-1 2988-3A mak8-1 2988-7A mak8-1 3015-10A tcml-l 2994-6A tcml-l 3144-15B tcml-4A 3144-13A tcml-4A 3145-6B tcml-4B

3145-IA tcml-4B

[KIL-k1l], K- diploids/ K+ diploids 0/222 0/163 0/41 40/68 25/30 10/33 2/19 46/39 13/26 16/43 11/27 32/105

[KIL-k1], K- diploids/ K' diploids 0/109

0/45 14/43 19/45 16/51 4/34 15/66 32/74 23/71

11/26

27/58 8/19 3146-18B tcml-5A 9/35 3146-4B tcml-5A 11/42 3147-1B tcml-5B 6/28 54/103 16/22 3147-9A tcml-5B. * To carry out complementation tests, meiotic spores from the diploid strains 1874 and 1876 were mated for 8 hr at 30°C on YPAD medium (8) with an excess of cells of the indicated haploid strains and plated for single colonies on minimal media to select against unmated spores or haploids or unsporulated 1874 or 1876 cells. The clones appearing were tested for the killer phenotype and the numbers of K- diploids and K+ diploids are tabulated. t The makx gene is an unmapped mak- mutant that is unlinked to petl7 and is thus distinct from mak8 or tcml.

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(cryptopleurine resistance) mutants (27) for a possible makphenotype by crossing them with the K1 killer strain 1101. The diploids and all meiotic segregants (12 tetrads for each cross) were all killers, indicating these cryl mutants were MAK+. Spontaneous mutants resistant to cycloheximide at 3 tkg/ml were isolated from three single colonies ofstrain SP25. Twentythree mutations (5, 8, and 10 from the three single colonies) were recessive and did not complement a known cyh2 (cycloheximide resistance) mutation. These were each crossed with killer strain 1101 and meiotic segregants were examined by tetrad dissection or random spore. analysis. About half of the segregants in each case were cycloheximide resistant but none were nonkillers, indicating that all were MAK+. The cyh2 mutant obtained from the Yeast Genetic Stock Center (Berkeley, CA) and whose identity was confirmed by mapping data was also MAK+. We also isolated, from a killer strain, 40 independently arising mutants resistant to cerulenin at 3 ug/ml, another protein synthesis inhibitor (28). .All mutants remained killers. Singh and Manney (29) isolated a series of cold-sensitive mutants, suggesting that these might include ribosome assembly mutants in analogy with such bacterial mutants (30, 31). Among these mutations (Its = low-temperature-sensitive), we found that only one, lts5, conferred a weak make phenotype at 30TC. lts5 was linked to aro7 (tetrad analysis: parental ditype = 11, nonparental ditype = 0, tetratype = 27). Mutations of the other seven Its genes tested did not produce the makphenotype. DISCUSSION At least 29 chromosomal genes are required for replication of M1 ds RNA under conditions permissive for cell growth. Of these genes, one (SPE2) is required for spermidine and spermine synthesis. This report shows that. another of these genes, MAK8, is identical to the TCM1 gene, which codes for the ribosomal protein L3. The cryl (cryptopleurine resistance) and cyh2 (cycloheximide resistance) genes also code for ribosomal proteins, but mutations in these genes do not affect killer ds RNA maintenance or replication. The its5-1 mutant (29) has the make phenotype, although other Its mutants do not. These mutants-were isolated with the idea that they might have altered ribosomal assembly, but there is no direct evidence on this point. The loss of Ml or M2 from tcml mutants is to be distinguished from the curing of M1 or M2 by various inhibitors of protein synthesis, including cycloheximide, cryptopleurine, and trichodermin. The curing occurs only when cells are dramatically slowed in their growth rate. In contrast, the drug-resistant mutants grow normally, and among these only those resistant to trichodermin lose the M1 (and M2) ds RNA. Thus, -we are inclined to believe that the curing effect is an indirect one, whereas the role of ribosomal protein L3 could be direct. The. mechanism of involvement of ribosomal protein L3 in killer plasmid replication could resemble the situation in the RNA phage QP3, in which three ofthe four subunits of the replicase are borrowed from the host's protein synthetic apparatus-namely, ribosomal protein S1 and elongation factors Ts and Tu (32). Another precedent is the finding that spectinomycin resistance mutations ofEscherichia coli result in the instability of the F plasmid (33, 34). It is also possible that the tcml mutants

Proc. Natl. Acad. Sci. USA 79 (1982)

lose the M1 or M2 ds RNA because of a specific effect on translation, impeding the synthesis of some proteins not essential to the host but needed by M1 and M2, or because of a direct role of ribosomes in the maintenance of M1 and M2. We thank John McCusker for cryl mutants and Thomas Manney for cryl and Its mutants. S. P. R. was supported by American Cancer Society Postdoctoral Fellowship PF1667. 1. Bevan, E. A., Herring, A. J. & Mitchell, D. S. (1973) Nature (London) 245, 81-86. 2. Vodkin, M., Katterman, F. & Fink, G. R. (1974)J. Bacteriol. 117, 681-686. 3. Palfiee, R. G. E. & Bussey, H. (1979) Eur. J. Biochem. 93, 487-493. 4. Bostian, K. A., Hopper, J. E., Rogers, D. T. & Tipper, D. J. (1980) Cell 19, 403-414. 5. Sommers, J. M. & Bevan, E. A. (1969) Genet. Res. 13, 71-83. 6. Wickner, R. B. & 'Leibowitz, M. J. (1976) J. Mol. Biol 105, 427-443. 7. Wickner, R. B. (1978) Genetics 88, 419-425. 8. Wickner, R. B. (1979) Genetics 92, 803-821. 9. Wickner, R. B. & Leibowitz, M. J. (1979) J. Bacteriol 140, 154-160. 10. Guerry-Kopecko, P. & Wickner, R. B. (1980) J. Bacteriol 144, 1113-1118. 11. Hopper, J. E., Bostian, K. A., Rowe, L. B. & Tipper, D. J. (1977)J. Biol Chem. 252, 9010-9017. 12. Sommer, S. S. & Wickner, R. B. (1982)J. Bacteriol 150, 545551. 13. Cohn, M. S., Tabor, C. W. & Tabor, H. (1978)J. Bacteriol 134, 208-213. 14. Cohn, M. S, Tabor, C. W., Tabor, H. & Wickner, R. B. (1978) J. Biol Chem. 253, 5225-5227. 15. Schindler, D., Grant, P. & Davies, J. (1974) Nature (London) 248, 535-536. 16. Grant, P. G., Schindler, D. & Davies, J. (1976) Genetics 83, 667-673. 17. Corrasco, L., Barbacid, M. &,Vasquez, D. (1973) Biochim. Biophys. Acta 312, 368-376. 18. Fried, H. M. & Warner, J. R. (1981) Proc. Nati Acad. Sci. USA 78, 238-242. 19. Wickner, R. B. & Toh-e, A. (1982Y Genetics 100, 159-174. 20. Wickner, R. B. (1980) Cell 21, 217-226. 21. Sommer, S. S. & Wickner, R. B. (1981) in Cold Spring Harbor Meeting on the Molecular Biology of Yeast (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY), p. 200 (abstr.). 22. Wickner, R. B. (1981),in Cold Spring Harbor Meeting on the Molecular Biology of Yeast (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY), p. 251 (abstr.). 23. Fink, G. R. & Styles, C. A. (1972) Proc. Nati Acad. Sci. USA 69, 2846-2849. 24. Leibowitz,' M. J. (1982) Curr. Genet. 5, in press. 25. Skogerson, 'L., McLaughlin, C. &'Wakatama, E. (1973)J. Bacteriol 116, 818-822. 26. Stocklein, W. & Pieperberg, W. (1979) Curr. Genet. 1, 177-183. 27. Meade, J. 'H., Riley, M. I. & Manney, T. R.'(1977)J. Bacteriol 129, 1428-1434. 28. Rouslin, W. (1979) J. Bacteriol 139, 502-506. 29. Singh, A. & Manney, T. R. (1974) Genetics 77, 651-659. 30. Guthrie, C., Nashimoto, H. & Nomura, M. (1969) Proc. Natt Acad. Sci. USA 63, 384-391. 31. Tai, P.-C., Kessler, D. P. & Ingraham, J. (1969)J. Bacteriol. 97, 1298-1304. 32. Blumenthal, T. & Carmichael, G. G. (1979) Annu. Rev. Biochem. 48, 525-548. 33. Yamagata, H. & Uchida, H. '(1972) J. Mol. Biol 67, 533-535. 34. Yamagata, H., Dombou, M., Sato, T., Mizushima, S. & Uchida, H. (1980) J. Bacteriol 143, 661-667.