Proc. Natl. Acad. Sci. USA Vol. 88, pp. 9392-9396, November 1991 Genetics
FUS3 represses CLN1 and CLN2 and in concert with KSS1 promotes signal transduction (cell cycle control/cyclin/protein kinase/mating/yeast)
ELAINE A. ELION*, JULIE A. BRILL, AND GERALD R. FINK Whitehead Institute for Biomedical Research and Department of Biology, Massachusetts Institute of Technology, 9 Cambridge Center, Cambridge, MA 02142
Contributed by Gerald R. Fink, July 23, 1991
al. (17)] in pRS313 (18), a CEN3 HIS3 plasmid; (ii) pYEE117, a 3.5-kb EcoRV-Sal I fragment of the GALJ-STE12 gene (provided by B. Errede, University of North Carolina) in pRS313; (iii) pYEE118, a 3.8-kb EcoRI-Sal I fragment of the KSS1 gene (6) in pRS313; (iv) pJB207, a 4.5-kb Nco I-Nco I fragment with Fusl-lacZ (ref. 19; fusion of 5' region of FUSJ to Escherichia coli lacZ) in YEp423 (20), a LEU2 2-Acm plasmid; (v) pJB220, which replaces a 1.5-kb Bgl II-Bgl II fragment internal to STE5 with a 0.8-kb Bgl II-BamHI TRPJ fragment; and (vi) pJB225, a 1.8-kb HIS3 BamHI-BamHI fragment inserted in the Bgl II site offus3AXmnI in pYEE97 (1). All yeast strains are isogenic derivatives of W303 strain EY699 MATa FUS3 KSSJ ura3-1 his3-11,15 leu2-3,112 trpl-1 ade2 canl-100 Gal' (from R. Rothstein, Columbia University) or S288C strain EY441 FUS3 kssl ura3-52 leu23,112 his3A200 ade2-1 lys2A201 (1) unless noted. Two FUS3 null alleles (fus3A) were used interchangeably,fus3-6::LEU2 (1) andfus3-7::HIS3 (pJB225). Sterilefus3A strains were first transformed with the FUS3 gene on a plasmid for crosses. Diploids that had lost the FUS3 plasmid were then sporulated. ksslA:: URA3 was made with pBC65 (6), cln3A:: URA3 and ste4::URA3 were made as described (1), ste2A::LEU2 was made with pAB506 (from J. Konopka, University of Washington), stel2Al:: URA3 was made with pNC163 (from B. Errede), steSA101::TRPJ was made with pJB220, and clnl:: TRPI and cln2: :LEU2 were made with 419 and 810 (21). All deletion alleles were confirmed by Southern analysis. Viable combinations of fus3A, clnl, cln2, and cln3A were progeny from crosses between isogenic MATa and MATa EY699-derived parents: EEX227:MATa clni::TRPI X MATa fus3-7::HIS3 cln2::LEU2; EEX228: MATa clnl::TRPI x MATa fus3-6::LEU2 cln3::URA3; EEX229 EY739 MATa fus3-7::HIS3 cln2::LEU2 x EY709 MATa cln3::URA3. The MATa KSSJ fus3A clni cln2 strain was made by a cross of nonisogenic strains EY738 MATa cln2 fus3A KSSJ and JBY511 MATa clmi cln2 kssi and was compared to a MATa KSSJ FUS3 clmi cln2 sister ascospore in Fig. 1. The barn- strains in Fig. 2 are isogenic derivatives of EEX233-5C MATa bar)-1 fus3-7::HIS3 ura3-52 meti. Quantitation of Pheromone Response. Cells harboring either pYEE116 or pYEE117 and Fusl-lacZ (pSB234 or pJB207) were grown at 30'C in SC medium containing 2% raffinose and lacking histidine and either uracil or leucine to an OD6w of 0.4-0.6, pelleted, resuspended at an OD600 of 0.25 in SC medium containing 2% galactose, lacking histidine, uracil or leucine, and agitated at 300C. At the indicated time intervals, one aliquot was fixed and assayed for the
FUS3 is functionally redundant with KSS1, a ABSTRACT homologous yeast protein kinase, for a step(s) in signal transduction between the 13 subunit of the guanine nucleotide binding protein (G protein), STE4, and the mating typespecific transcriptional activator, STE12. Either FUS3 or KSS1 can execute this function; when neither gene encoding these protein kinases is present, signal transduction is blocked, causing sterility. This functional redundancy is strain dependent; some standard laboratory strains (S288C) are kssl-. FUS3 has additional functions required for cell cycle arrest and vegetative growth that do not overlap with KSS1 functions. FUS3 mediates cell cycle arrest during mating through transcriptional repression of two G, cyclins (CLN1 and CLN2) and through posttranscriptional inhibition of a third G, cyclin (CLN3). FUS3 is also required for vegetative growth in haploid strains dependent upon CLN3 for cell cycle progression but is not required in strains dependent upon either CLN1 or CLN2, suggesting a functional divergence among the three G, cyclins. The diverse roles for FUS3 suggest that the FUS3 protein kinase has multiple substrates, some of which may be shared with KSS1.
Saccharomyces cerevisiae cells with a functional FUS3 gene arrest at the G1 portion of the cell cycle termed START in response to mating pheromone (e.g., a factor and a factor). In contrast,fas3 mutants fail to undergo cell cycle arrest and continue to divide mitotically although they induce the expression of mating-specific genes and form mating-specific structures (1, 2). Deletion of the CLN3 gene restores G1 arrest to fus3 mutants, suggesting a connection between FUS3mediated cell cycle arrest and inhibition of CLN3 (1), a G1 cyclin (3-5). FUS3 is a member of a family of highly related protein kinases (1) conserved from yeast to mammals that includes KSS1 (6), Spk1' (7), ERK1 (extracellular signalregulated kinase 1) (8), and ERK2 (9). KSS1 is thought to be involved in recovery from a factor-induced G1 arrest in S. cerevisiae (6); Spk1' is required for mating in Schizosaccharomyces pombe (7). ERK1 [ref. 8; also termed MAP2 kinase for microtubule-associated protein 2 kinase (or mitogenactivated protein kinase) and myelin basic protein kinase] acts in diverse signal-transduction pathways in mammals and Sea Star oocytes (10-15). In this report we show that FUS3 has multiple functions in haploid cells, some of which are redundant with KSS1.
MATERIALS AND METHODS Media, Plasmids, and Strains. SC media with 2% dextrose, 2% galactose, or 2% raffinose were prepared as described (16). Plasmids include (i) pYEE116, a 2.0-kilobase (kb) EcoRI-Sal I fragment of the GALl-STE4 gene [Whiteway et
Abbreviations: ERK1, extracellular signal-regulated kinase 1; MAP2 kinase, microtuble-associated protein 2 kinase (also mitogenactivated protein kinase); G protein, guanine nucleotide binding protein. *To whom reprint requests should be sent at the present address: Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 25 Shattuck Street, Boston, MA 02115.
The publication costs of this 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.
9392
Genetics: Elion
et
Proc. Natl. Acad. Sci. USA 88 (1991)
al.
number of budded and unbudded cells (1), and another aliquot was assayed for P-galactosidase activity (19). In the STE12 overexpression study, the 6-hr time point was used for comparison because long-term overproduction of STE12 is toxic (22). RESULTS FUS3 and KSS1 Are Functionally Redundant for Signal Transduction. Haploid MATa strains lacking both FUS3 and KSS1 (fus3A ksslA) are sterile, produce reduced basal levels of a factor, and fail to express FUSI upon exposure to mating pheromone (Table 1). Isogenic strains containing either a functional FUS3 or KSSI gene (FUS3 kssl A andfus3A KSSI) mate, produce normal basal levels of a factor, and induce FUSI expression in the presence of a factor. The basal level of a factor secreted from MATa cells is a measure of the integrity of the signal-transduction pathway in the absence of pheromone (22, 24), whereas the induction of FUSI (19, 25) is a measure of the ability of this pathway to be activated by mating pheromone. The block in mating-specific transcription in thefus3A ksslA double mutant and the ability of either the FUS3 gene or the KSSI gene to restore both basal and induced mating-specific transcription suggest that either FUS3 or KSS1 is sufficient to perform a shared function required for signal transduction. FUS3 and KSS1 Act After STE4 and Before STE12. The position of FUS3 and KSS1 in the signal-transduction pathway was determined by examining the phenotype offus3A and ksslA strains overproducing either STE4 (G protein f8 subunit; ref. 26) or STE12 (mating-specific transcription factor; refs. 27 and 28). STE4 precedes STE12 in the pathway (29, 30). Overexpression of either STE4 or STE12 in FUS3 KSSI strains induces transcription of mating-specific genes (e.g., FUSI), morphological changes, and G1 arrest (refs. 17, 22, 31, 32; Table 2). Overexpression of either STE4 or STE12 infus3A KSSI or FUS3 ksslA strains induces FUSI expression and morphological changes, confirming that either FUS3 or KSS1 is sufficient for signal transduction. Thefus3A ksslA double mutant blocks the STE4 overexpression phenotypes but does not block the STE12 overexpression phenotype, placing FUS3 and KSS1 after STE4 but prior to STE12 in the pathway (Table 2). The effects of FUS3 and KSSJ on signal transduction can be distinguished and suggest a negative
Table 1. Quantitation of pheromone response
a-Ft Strains* S288C FUS3 kssJ fus3A kssl W303 FUS3 KSSI fus3A KSSI FUS3 kssJA fus3A ksslA
% 2Nt Halo 38
+++
FUS3 represses CLN1 and CLN2 and in concert with KSS1 promotes signal transduction (cell cycle control/cyclin/protein kinase/mating/yeast)
ELAINE A. ELION*, JULIE A. BRILL, AND GERALD R. FINK Whitehead Institute for Biomedical Research and Department of Biology, Massachusetts Institute of Technology, 9 Cambridge Center, Cambridge, MA 02142
Contributed by Gerald R. Fink, July 23, 1991
al. (17)] in pRS313 (18), a CEN3 HIS3 plasmid; (ii) pYEE117, a 3.5-kb EcoRV-Sal I fragment of the GALJ-STE12 gene (provided by B. Errede, University of North Carolina) in pRS313; (iii) pYEE118, a 3.8-kb EcoRI-Sal I fragment of the KSS1 gene (6) in pRS313; (iv) pJB207, a 4.5-kb Nco I-Nco I fragment with Fusl-lacZ (ref. 19; fusion of 5' region of FUSJ to Escherichia coli lacZ) in YEp423 (20), a LEU2 2-Acm plasmid; (v) pJB220, which replaces a 1.5-kb Bgl II-Bgl II fragment internal to STE5 with a 0.8-kb Bgl II-BamHI TRPJ fragment; and (vi) pJB225, a 1.8-kb HIS3 BamHI-BamHI fragment inserted in the Bgl II site offus3AXmnI in pYEE97 (1). All yeast strains are isogenic derivatives of W303 strain EY699 MATa FUS3 KSSJ ura3-1 his3-11,15 leu2-3,112 trpl-1 ade2 canl-100 Gal' (from R. Rothstein, Columbia University) or S288C strain EY441 FUS3 kssl ura3-52 leu23,112 his3A200 ade2-1 lys2A201 (1) unless noted. Two FUS3 null alleles (fus3A) were used interchangeably,fus3-6::LEU2 (1) andfus3-7::HIS3 (pJB225). Sterilefus3A strains were first transformed with the FUS3 gene on a plasmid for crosses. Diploids that had lost the FUS3 plasmid were then sporulated. ksslA:: URA3 was made with pBC65 (6), cln3A:: URA3 and ste4::URA3 were made as described (1), ste2A::LEU2 was made with pAB506 (from J. Konopka, University of Washington), stel2Al:: URA3 was made with pNC163 (from B. Errede), steSA101::TRPJ was made with pJB220, and clnl:: TRPI and cln2: :LEU2 were made with 419 and 810 (21). All deletion alleles were confirmed by Southern analysis. Viable combinations of fus3A, clnl, cln2, and cln3A were progeny from crosses between isogenic MATa and MATa EY699-derived parents: EEX227:MATa clni::TRPI X MATa fus3-7::HIS3 cln2::LEU2; EEX228: MATa clnl::TRPI x MATa fus3-6::LEU2 cln3::URA3; EEX229 EY739 MATa fus3-7::HIS3 cln2::LEU2 x EY709 MATa cln3::URA3. The MATa KSSJ fus3A clni cln2 strain was made by a cross of nonisogenic strains EY738 MATa cln2 fus3A KSSJ and JBY511 MATa clmi cln2 kssi and was compared to a MATa KSSJ FUS3 clmi cln2 sister ascospore in Fig. 1. The barn- strains in Fig. 2 are isogenic derivatives of EEX233-5C MATa bar)-1 fus3-7::HIS3 ura3-52 meti. Quantitation of Pheromone Response. Cells harboring either pYEE116 or pYEE117 and Fusl-lacZ (pSB234 or pJB207) were grown at 30'C in SC medium containing 2% raffinose and lacking histidine and either uracil or leucine to an OD6w of 0.4-0.6, pelleted, resuspended at an OD600 of 0.25 in SC medium containing 2% galactose, lacking histidine, uracil or leucine, and agitated at 300C. At the indicated time intervals, one aliquot was fixed and assayed for the
FUS3 is functionally redundant with KSS1, a ABSTRACT homologous yeast protein kinase, for a step(s) in signal transduction between the 13 subunit of the guanine nucleotide binding protein (G protein), STE4, and the mating typespecific transcriptional activator, STE12. Either FUS3 or KSS1 can execute this function; when neither gene encoding these protein kinases is present, signal transduction is blocked, causing sterility. This functional redundancy is strain dependent; some standard laboratory strains (S288C) are kssl-. FUS3 has additional functions required for cell cycle arrest and vegetative growth that do not overlap with KSS1 functions. FUS3 mediates cell cycle arrest during mating through transcriptional repression of two G, cyclins (CLN1 and CLN2) and through posttranscriptional inhibition of a third G, cyclin (CLN3). FUS3 is also required for vegetative growth in haploid strains dependent upon CLN3 for cell cycle progression but is not required in strains dependent upon either CLN1 or CLN2, suggesting a functional divergence among the three G, cyclins. The diverse roles for FUS3 suggest that the FUS3 protein kinase has multiple substrates, some of which may be shared with KSS1.
Saccharomyces cerevisiae cells with a functional FUS3 gene arrest at the G1 portion of the cell cycle termed START in response to mating pheromone (e.g., a factor and a factor). In contrast,fas3 mutants fail to undergo cell cycle arrest and continue to divide mitotically although they induce the expression of mating-specific genes and form mating-specific structures (1, 2). Deletion of the CLN3 gene restores G1 arrest to fus3 mutants, suggesting a connection between FUS3mediated cell cycle arrest and inhibition of CLN3 (1), a G1 cyclin (3-5). FUS3 is a member of a family of highly related protein kinases (1) conserved from yeast to mammals that includes KSS1 (6), Spk1' (7), ERK1 (extracellular signalregulated kinase 1) (8), and ERK2 (9). KSS1 is thought to be involved in recovery from a factor-induced G1 arrest in S. cerevisiae (6); Spk1' is required for mating in Schizosaccharomyces pombe (7). ERK1 [ref. 8; also termed MAP2 kinase for microtubule-associated protein 2 kinase (or mitogenactivated protein kinase) and myelin basic protein kinase] acts in diverse signal-transduction pathways in mammals and Sea Star oocytes (10-15). In this report we show that FUS3 has multiple functions in haploid cells, some of which are redundant with KSS1.
MATERIALS AND METHODS Media, Plasmids, and Strains. SC media with 2% dextrose, 2% galactose, or 2% raffinose were prepared as described (16). Plasmids include (i) pYEE116, a 2.0-kilobase (kb) EcoRI-Sal I fragment of the GALl-STE4 gene [Whiteway et
Abbreviations: ERK1, extracellular signal-regulated kinase 1; MAP2 kinase, microtuble-associated protein 2 kinase (also mitogenactivated protein kinase); G protein, guanine nucleotide binding protein. *To whom reprint requests should be sent at the present address: Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 25 Shattuck Street, Boston, MA 02115.
The publication costs of this 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.
9392
Genetics: Elion
et
Proc. Natl. Acad. Sci. USA 88 (1991)
al.
number of budded and unbudded cells (1), and another aliquot was assayed for P-galactosidase activity (19). In the STE12 overexpression study, the 6-hr time point was used for comparison because long-term overproduction of STE12 is toxic (22). RESULTS FUS3 and KSS1 Are Functionally Redundant for Signal Transduction. Haploid MATa strains lacking both FUS3 and KSS1 (fus3A ksslA) are sterile, produce reduced basal levels of a factor, and fail to express FUSI upon exposure to mating pheromone (Table 1). Isogenic strains containing either a functional FUS3 or KSSI gene (FUS3 kssl A andfus3A KSSI) mate, produce normal basal levels of a factor, and induce FUSI expression in the presence of a factor. The basal level of a factor secreted from MATa cells is a measure of the integrity of the signal-transduction pathway in the absence of pheromone (22, 24), whereas the induction of FUSI (19, 25) is a measure of the ability of this pathway to be activated by mating pheromone. The block in mating-specific transcription in thefus3A ksslA double mutant and the ability of either the FUS3 gene or the KSSI gene to restore both basal and induced mating-specific transcription suggest that either FUS3 or KSS1 is sufficient to perform a shared function required for signal transduction. FUS3 and KSS1 Act After STE4 and Before STE12. The position of FUS3 and KSS1 in the signal-transduction pathway was determined by examining the phenotype offus3A and ksslA strains overproducing either STE4 (G protein f8 subunit; ref. 26) or STE12 (mating-specific transcription factor; refs. 27 and 28). STE4 precedes STE12 in the pathway (29, 30). Overexpression of either STE4 or STE12 in FUS3 KSSI strains induces transcription of mating-specific genes (e.g., FUSI), morphological changes, and G1 arrest (refs. 17, 22, 31, 32; Table 2). Overexpression of either STE4 or STE12 infus3A KSSI or FUS3 ksslA strains induces FUSI expression and morphological changes, confirming that either FUS3 or KSS1 is sufficient for signal transduction. Thefus3A ksslA double mutant blocks the STE4 overexpression phenotypes but does not block the STE12 overexpression phenotype, placing FUS3 and KSS1 after STE4 but prior to STE12 in the pathway (Table 2). The effects of FUS3 and KSSJ on signal transduction can be distinguished and suggest a negative
Table 1. Quantitation of pheromone response
a-Ft Strains* S288C FUS3 kssJ fus3A kssl W303 FUS3 KSSI fus3A KSSI FUS3 kssJA fus3A ksslA
% 2Nt Halo 38
+++
