Functional Interaction Between the PKC1 Pathway ... - Semantic Scholar

Copyright  2000 by the Genetics Society of America

Functional Interaction Between the PKC1 Pathway and CDC31 Network of SPB Duplication Genes Waheeda Khalfan,1 Irena Ivanovska1 and Mark D. Rose Department of Molecular Biology, Princeton University, Princeton, New Jersey 08544-1014 Manuscript received February 25, 2000 Accepted for publication April 12, 2000 ABSTRACT The earliest known step in yeast spindle pole body (SPB) duplication requires Cdc31p and Kar1p, two physically interacting SPB components, and Dsk2p and Rad23p, a pair of ubiquitin-like proteins. Components of the PKC1 pathway were found to interact with these SPB duplication genes in two independent genetic screens. Initially, SLG1 and PKC1 were obtained as high-copy suppressors of dsk2⌬ rad23⌬ and a mutation in MPK1 was synthetically lethal with kar1-⌬17. Subsequently, we demonstrated extensive genetic interactions between the PKC1 pathway and the SPB duplication mutants that affect Cdc31p function. The genetic interactions are unlikely to be related to the cell-wall integrity function of the PKC1 pathway because the SPB mutants did not exhibit cell-wall defects. Overexpression of multiple PKC1 pathway components suppressed the G2/M arrest of the SPB duplication mutants and mutations in MPK1 exacerbated the cell cycle arrest of kar1-⌬17, suggesting a role for the PKC1 pathway in SPB duplication. We also found that mutations in SPC110, which encodes a major SPB component, showed genetic interactions with both CDC31 and the PKC1 pathway. In support of the model that the PKC1 pathway regulates SPB duplication, one of the phosphorylated forms of Spc110p was absent in pkc1 and mpk1⌬ mutants.

ACCHAROMYCES cerevisiae cells commit to a new round of cell division during G1. Activation of the G1-specific Cdc28p/cyclin complex promotes bud emergence, DNA replication, and duplication of the yeast microtubule organizing center, the spindle pole body (SPB; Pringle and Hartwell 1981; Reed 1992; Cross 1995). These major G1 cell cycle events occur independently of each other, but in a coordinated manner that has yet to be understood. The PKC1 cell integrity pathway has been implicated in numerous cellular processes including promoting bud emergence at the G1/S transition (Mazzoni et al. 1993; Marini et al. 1996; Zarzov et al. 1996; Gray et al. 1997; Igual et al. 1997). Pkc1p (Levin et al. 1990; Paravicini et al. 1992; Yoshida et al. 1992) activates a mitogen-activated protein (MAP) kinase cascade consisting of Bck1p (Lee and Levin 1992), Mkk1p/Mkk2p (Irie et al. 1993), and the MAP kinase, Mpk1p (Lee et al. 1993). The PKC1 pathway is most likely regulated by the small GTP-binding protein Rho1p in vivo (Nonaka et al. 1995; Kamada et al. 1996). Slg1p/Wsc1p, a plasma membrane protein, acts through Rho1p to activate the PKC1 pathway (Gray et al. 1997; Verna et al. 1997; Jacoby et al. 1998). The MAP kinase Mpk1p phosphorylates downstream targets including Rlm1p, a MADS-box transcription factor (Watanabe et al. 1995; Dodou and

S

Corresponding author: Mark D. Rose, Department of Molecular Biology, Princeton University, Princeton, NJ 08544-1014. E-mail: [email protected] 1 These authors contributed equally to this work. Genetics 155: 1543–1559 (August 2000)

Treisman 1997; Watanabe et al. 1997) that regulates transcription of a number of cell-wall components (Jung and Levin 1999). In addition, Mpk1p may also target the transcription factor complex Swi4p/Swi6p (Madden et al. 1997). Although duplication of the SPB in G1 is also Cdc28p/ Cln dependent, the specific signals that activate SPB duplication are unknown. The yeast SPB is a trilaminar disc-like structure embedded in the nuclear envelope from which nuclear and cytoplasmic microtubules are organized (Byers and Goetsch 1974, 1975). The earliest step in SPB duplication involves the formation of an electron-dense material on the cytoplasmic face of the SPB, called the satellite. The satellite is the precursor of the nascent SPB, or the spindle plaque, which forms in the cytoplasm and is then embedded into the nuclear envelope (Adams and Kilmartin 1999). The early stages of SPB duplication are independent of Cdc28p activity since mutations in CDC28 result in cell cycle arrest with a single SPB already containing a satellite (Byers and Goetsch 1974). This observation suggests that the signal to initiate SPB duplication is active before the Cdc28p/START point, but for SPB duplication to continue, a CDC28-dependent signal is required. Analysis of the early SPB duplication genes may give insight into how cell cycle control is exerted on SPB duplication because the G1 cell cycle machinery would be expected to target proteins required in the early stages of SPB duplication. Mutations in CDC31, the yeast homolog of centrin, block the earliest stage of SPB duplication, the formation of the satellite precursor

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(Byers 1981; Schild et al. 1981; Baum et al. 1986). In addition to SPB duplication, Cdc31p also plays a role in morphogenesis and cell integrity via interaction with Kic1p (Sullivan et al. 1998). Mutations in the kinase domain of Kic1p and certain cdc31 alleles result in abnormal bud morphology and lysis. However, kic1 and cdc31 mutants exhibit cell-wall defects that appear to be different than those observed in pkc1 pathway mutants (Sullivan et al. 1998). Another SPB component, Kar1p, is required at the same step of SPB assembly as Cdc31p (Rose and Fink 1987). Kar1p genetically and physically interacts with Cdc31p and is required to localize Cdc31p to the SPB (Biggins and Rose 1994; Vallen et al. 1994; Spang et al. 1995). The temperature-sensitive mutation kar1-⌬17 mislocalizes Cdc31p at high temperature and can be rescued by high dosage of CDC31 and the dominant allele CDC31-16. Formally, therefore, Kar1p functions upstream of Cdc31p. DSK2 is a nonessential ubiquitinlike protein identified as a dominant suppressor that also relocalizes Cdc31p in kar1-⌬17 mutants (Biggins and Rose 1994; Vallen et al. 1994; Biggins et al. 1996). Deletion of DSK2 in combination with deletion of another ubiquitin-like gene, RAD23, results in a temperature-sensitive block in SPB duplication prior to satellite formation (Biggins et al. 1996). Unlike mutations in KAR1, the dsk2⌬ rad23⌬ strain is not defective for Cdc31p localization at the SPB. However, like kar1-⌬17 mutants, the SPB duplication defect of dsk2⌬ rad23⌬ can be suppressed by high-copy CDC31 and by CDC3116. These results suggest that Dsk2p/Rad23p also function upstream of Cdc31p to mediate an important function of Cdc31p at the SPB (Biggins et al. 1996). By employing the SPB duplication mutants kar1-⌬17 and dsk2⌬ rad23⌬ in different genetic screens, we identified multiple genetic interactions between the PKC1 and the SPB duplication pathways. Members of the PKC1 pathway, in high copy, could suppress the temperature sensitivity of kar1-⌬17, dsk2⌬ rad23⌬, and cdc31-2. Overexpression of different members of the PKC1 pathway specifically suppressed the SPB duplication mutants but not other G2/M-arrested cells. In addition, we analyzed many combinations of double mutants and found extensive synthetic lethality and synthetic growth defects. To understand the functional basis of the synthetic interactions, we analyzed the phenotypes of the double mutants with respect to cell integrity and G2/M arrest. In a mpk1⌬ kar1-⌬17 double mutant, the G2/M arrest defect was clearly exacerbated. We also found that phosphorylation of Spc110p, an essential SPB component, was defective in pkc1ts and mpk1⌬ mutants, suggesting that the PKC1 pathway may directly or indirectly regulate Spc110p phosphorylation. Our results demonstrate an important functional link between PKC1 pathway activity and SPB duplication. We propose that PKC1 MAP kinase signaling positively regulates an SPB component,

possibly to coordinate SPB duplication and bud emergence during G1. MATERIALS AND METHODS Microbial techniques and yeast strain construction: Yeast media and microbial techniques were essentially as previously described (Rose et al. 1990). Bacterial media were as described (Sambrook et al. 1989), and bacterial strain XL1-Blue was used for all bacterial manipulations. All bacterial strains are listed in Table 1. All yeast strains used are listed in Table 2. Yeast strains were constructed using standard genetic techniques. All yeast strains were isogenic to S288c. To generate double mutants for synthetic lethality studies, strains with mutations in cdc31-1, kar1-⌬17, spc10-220, mps2-1, and dsk2⌬ rad23⌬ were kept covered with a URA3 plasmid bearing the appropriate wild-type gene to prevent increased ploidy. PKC1 pathway components were then disrupted in the covered mutant strains. The resulting double mutant strains containing wild-type (WT) URA3 plasmids were streaked on 5-fluoroorotic acid (5-FOA) at 23␱ to select for plasmid loss (Boeke et al. 1984). When double or triple mutant combinations were viable on 5-FOA at 23⬚, growth at higher temperatures was assayed by spotting 10-fold serial dilutions. In most cases, double mutants between PKC1 pathway components and SPB duplication genes were generated either by PCR-mediated gene disruption (Baudin et al. 1993) or with disruption plasmids listed in Table 1. In a few cases, the double/triple mutants were generated as segregants of genetic crosses. Specifically, the slg1⌬ kar1-⌬17 strain (MY4646) was constructed by mating a kar1-⌬17 [KAR1 URA3] (MS2373) strain to a slg1⌬ (MY4455), a cdc31-1 slg1⌬ double mutant (MY6862) by crossing MY4455 to MY3885, the dsk2⌬ rad23⌬ slg1⌬ (MY4916) strain by mating MY4449 to MY4455, the pkc1ts kar1-⌬17 strain (MY6805) by crossing DL523 (Levin and Bartlett-Heubusch 1992) to MS2374, and a dsk2⌬ rad23⌬ pkc1ts strain (MY6296) by mating DL523 (Levin and Bartlett-Heubusch 1992) to MY4197. kar1-⌬17 synthetic lethality screen and identification of mpk1: To identify mutations that result in synthetic lethality with kar1-⌬17, a “shuffle-mutagenesis” screening strategy was employed. MS2373 and MS2376, ura3 kar1-⌬17 strains of both mating types, were constructed, each carrying a KAR1 URA3based centromeric plasmid to suppress the mutant defect. The strains were mutagenized with ethyl methanesulfonate (Rose et al. 1990) to 40% viability and plated on synthetic complete medium lacking uracil (SC-ura) to select for the covering plasmid. Mutations that cause slow growth or lethality after loss of the covering plasmid were assayed by their sensitivity to 5-FOA media, which selects against the URA3 plasmid. Out of 60,000 colonies screened, we identified four 5-FOA-sensitive mutants whose 5-FOA sensitivity was recessive and segregated as single genes. The 5-FOA sensitivity of the four mutants was rescued by a second plasmid bearing KAR1 on a LEU2-based vector, as expected for mutations that affect KAR1 function. Complementation analysis revealed that two of the mutations defined unique linkage groups and were subsequently identified as mutations in REG1 and NEM1 (W. Khalfan and M. D. Rose, unpublished observations). The remaining two mutations, SL6 and SL18, belonged to the same linkage group and caused temperature sensitivity at 37⬚, and the temperature sensitivity was linked to 5-FOA sensitivity because they cosegregated in crosses (⬍3.1 cM). We cloned the wild-type gene corresponding to the SL6 mutation by complementation of the temperature sensitivity of kar1-⌬17 SL6 strain bearing the KAR1 LEU2 plasmid

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TABLE 1 Bacterial Plasmids Plasmid p10 p636 p666 p759 p784 pBG168 pHS40 pJG109 pMR2012 pMR2223 pMR2745 pMR2757 pMR4090 pMR4290 pMR4481 pMR76 pMR77 pRH102 pRS416 pRS426 pSJ0

Yeast marker

Source

PKC1 URA3 2␮ BCK1-20 URA3 CEN MPK1 LEU2 CEN mpk1⌬::TRP bck1⌬::HIS3 CDC13 URA3 CEN spc110-220 URA3 YIP SPC110 URA3 CEN CDC31 URA3 2␮ CDC31-16 URA3 CEN ARS DSK2-1 URA3 CEN ARS DSK2 URA3 CEN ARS SLG1 URA3 2␮ MPK1 URA3 2␮ MPS2 URA3 CEN KAR1 URA3 CEN ARS KAR1 URA3 CEN ARS CDC7 URA3 CEN URA3 CEN URA3 2␮ CDC15 URA3 CEN

D. Levin (Johns Hopkins University) D. Levin D. Levin D. Levin D. Levin L. Hartwell (Fred Hutchinson Cancer Research Center) T. Davis (University of Washington, Seattle) T. Davis Vallen et al. (1994) Vallen et al. (1994) Biggins et al. (1996) Biggins et al. (1996) This study This study Mark Winey (University of Colorado, Boulder) Rose and Fink (1987) Rose and Fink (1987) Hollingsworth et al. (1992) Sikorski and Hieter (1989) Sikorski and Hieter (1989) D. O. Morgan (University of California, San Francisco)

(MS5589) strain. MS5589 was transformed with a URA3 YCp50 library (Rose et al. 1987). Of 18,000 transformants examined, 7 were Ts⫹. All seven plasmids rescued the temperature sensitivity of SL6 when reintroduced into yeast. The insert junctions of three plasmids were sequenced and contained two open reading frames in common, YHR029C and MPK1/ SLT2. A plasmid containing only the MPK1/SLT2 gene (p666, kindly provided by Dr. Levin), rescued the temperature sensitivity of MS5589 and the 5-FOA sensitivity of MS5584. To confirm that the MPK1 gene and SL6 locus were allelic, an mpk1⌬ kar1-⌬17 double mutant (MS5886) was generated, shown to be 5-FOA sensitive, and the 5-FOA sensitivity could be suppressed with a LEU2 MPK1 plasmid. To establish linkage, MS5831, a strain containing the SL6 mutation alone, was crossed to a mpk1⌬ strain (MS5933). The resulting diploid strain was Ts⫺, indicating that mpk1⌬ and SL6 fail to complement. Because a homozygous mpk1⌬ diploid cannot sporulate, an MPK1 LEU2 plasmid was introduced into the diploid. Upon sporulation, all spores that did not contain the MPK1 LEU2 plasmid (43/43) were temperature sensitive, indicating that there were no wild-type recombinants. We concluded that MPK1 and SL6 are allelic because they are closely linked (⬍4.7 cM). dsk2⌬ rad23⌬ high-copy suppressor screen: Strain MY3592 was transformed with a YEp24 genomic library (Carlson and Botstein 1982). A total of 21,300 Ura⫹ transformants (17 genome equivalents) were selected on SC-ura medium at 30⬚ and subsequently replica printed to SC-ura at 37⬚. Thirty putative Ts⫹ colonies were picked and retested. Plasmids were recovered from yeast as previously described (Rose et al. 1990) and plasmid linkage of the Ts⫹ phenotype was tested after retransformation into MY3592. The major class of plasmids (11) contained the CDC31 gene. RAD23 was isolated twice. DSK2 was not isolated, presumably because of its toxicity when overexpressed. Two independent secondary screens were used to classify the remaining 17 suppressors. Because rad23⌬, but not dsk2⌬, causes UV sensitivity, two plasmids that conferred UV resistance were deemed specific to rad23⌬ and not studied

further. To identify suppressors that were specific for SPB duplication, the plasmids were tested for their ability to suppress KAR1 and CDC31 mutations. Nine plasmids partially suppressed CDC31 mutations in an allele-specific manner. To identify the suppressing genes, we sequenced the ends of the library inserts using primers from the YEp24 vector. Two of them contained the SLG1 gene and were strong suppressors of both kar1-⌬17 and cdc31. One plasmid contained the PKC1 gene. Microscopy: To examine the G2/M arrest phenotype of the SPB duplication mutants, strains were grown at 23⬚ to early logarithmic phase and one-half of the cultures were shifted to the indicated temperatures for 4-8 hr. To examine the nuclear morphology, 1 ml of each culture was collected by centrifugation and stained with 4⬘,6-diamidino-2-phenylindole (DAPI) essentially as described (Rose et al. 1990). DAPI was obtained from Accurate Biochemicals and Scientific Corp. (Westbury, NY). For cell cycle analysis of mpk1⌬ kar1-⌬17, strain MS5886 was grown on 5-FOA at 23⬚ for several days until small, slow-growing colonies appeared (incubation at lower temperatures failed to give any colonies). As a control, strain MS2373 was also grown on 5-FOA at 23⬚. The colonies arising on the 5-FOA plate were propagated in YPD liquid where the double mutant strain continued to grow very slowly compared to the control strain. For live/dead analysis, strains were grown as described above. FUN-1 dye (Millard et al. 1997), was added to 1 ml of culture to a final concentration of 10 ␮m (Molecular Probes, Eugene, OR). The cultures were incubated at room temperature, in the dark, for 0.5 hr. Cells were examined by differential interference and fluorescence microscopy (Axiophot, Carl Zeiss, Inc., Thornwood, NY). Spc110 Western blot analysis: Affinity-purified Spc110 antibodies were obtained from T. Davis (University of Washington, Seattle, WA) and crude Spc110 antibodies were obtained from Dr. Stirling (University of Dundee, Dundee, United Kingdom). Crude Spc110 antibodies were purified against a GST-

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W. Khalfan et al. TABLE 2 Saccharomyces cerevisiae strains Strain 2032 DL523 HSY11-4D MS1554 MS2083 MS2374 MS2373 MS2376 MS5584 MS5589 MS5820 MS5831 MS5886 MS5888 MS5930 MS5933 MS5938 MS6547 MS6934 MS6937 MS6944 MS6947 MY2259 MY2260 MY3592 MY3752 MY3753 MY3754 MY3755 MY3883 MY3885 MY4193 MY4197 MY4449 MY4455 MY4642 MY4646 MY4916 MY5064 MY5689 MY6297 MY6296 MY6663 MY6739 MY6764 MY6805 MY6809 MY6855 MY6857 MY6859 MY6861 MY6862 MY6871

Genotype

Source

MATa ura3 omnc cdc7-1 TRP⫹ MATa ura3-52 trp1-1 his4 can1R pkc1⌬::LEU2 [pkc1ts Ycp50] MATa ura3-1 ade2-1oc can1-100 leu2-3, 112 trp1-1 ade3⌬ spc110-220 MATa ura3-52 leu2-3, 112 ade2-101 his3-⌬200 MATa ura3-52 leu2-3, 112 trp1⌬1 kar1-⌬17 MAT␣ ura3-52 leu2-3, 112 trp1⌬1 ade2-101 kar1-⌬17 MATa ura3-52 leu2-3, 112 trp1⌬1 kar1⌬17 [MR76] MAT␣ ura3-52 leu2-3, 112 ade2-101 kar1⌬17 [MR76] MAT␣ ura3-52 leu2-3, 112 ade2-101 kar1⌬17 mpk1 (SL6) [MR76] MAT␣ ura3-52 leu2-3, 112 ade2-101 kar1⌬17 mpk1 (SL6) [MR326] MAT␣ ura3-52 leu2-3, 112 ade2-101 his3-⌬200 kar1⌬17 [MR76] MAT␣ ura3-52 leu2-3, 112 ade2-101 mpk1 (SL6) MATa ura3-52 leu2-3, 112 trp1⌬1 kar1⌬17 mpk1⌬::TRP1 [MR76] MAT␣ ura3-52 leu2-3, 112 ade2-101 hsi3-⌬200 kar1⌬17 bck1⌬::HIS3 [MR76] MATa ura3-52 leu2-3, 112 trp1⌬1 mpk1⌬::TRP1 MATa ura3-52 leu2-3, 112 mpk1⌬::TRP1 MAT␣ ura3-52 leu2-3, 112 ade2-101 his3-⌬200 bck1⌬::HIS3 MATa ura3-52 leu2-3, 112 ade2-101 his3-⌬200 mpk1⌬::HIS3 MATa ura3-52 ade2-101 his3-⌬200 leu2-3, 112 swi4⌬::HIS3 MATa ura3-52 ade2-101 his3-⌬200 leu2-3, 112 rlml⌬::HIS3 MAT␣ ura3-52 ade2-101 his3-⌬200 leu2-3, 112 swi4⌬::HIS3 kar1⌬17 [MR76] MAT␣ ura3-52 ade2-101 his3-⌬200 leu2-3, 112 rlm1⌬::HIS3 kar1⌬17 [MR76] MAT␣ ura3 leu2-3, 112 his7 trp1 cdc31-1 MAT␣ ura3 leu2-3, 112 his7 trp1 ade2 cyh2R cdc31-2 MAT␣ ura3-52 leu2 ade2 his3 lys2 dsk2⌬::LEU2 rad23⌬ MATa ura3-52 leu2-3, 112 ade2-101 kar1-⌬17 [MR2223] MATa ura3-52 leu2-3, 112 ade2-101 kar1-⌬17 [MR2032] MATa ura3-52 leu2-3, 112 ade2-101 kar1-⌬17 [MR2745] MATa ura3-52 leu2-3, 112 ade-101 kar1-⌬17 [pRS416] MATa ura3-52 ade2-101 leu2-3, 112 his3-⌬200 cdc31-1 [MR2012] MATa ura3-52 ade2-101 leu2-3, 112 cdc31-1 MAT␣ ura3-52 leu2 ade2 his3-⌬200 dsk2⌬::LEU2 rad23⌬ MAT␣ ura3-52 ade2 leu2 his3-⌬200 dsk2⌬::LEU2 rad23⌬::URA3 MATa ura3-52 leu2 ade2 his3-⌬200 dsk⌬::LEU2 rad23⌬::URA3 MAT␣ ura3-52 leu2 his3-⌬200 trp1-⌬63 lys2-801 slg1⌬::LEU2 MAT␣ ura3-52 leu2-3, 112 his3-⌬200 kar1-⌬17 slg1⌬::LEU2 MATa ura3-52 leu2-3, 112 his3-⌬200 kar1-⌬17 slg1⌬::LEU2 MAT␣ ura3-52 leu2-3, 112 lys1-801 ade2 his3-⌬200 dsk2⌬::LEU2 rad23⌬::URA3 slg1⌬::LEU2 MAT␣ ura3-52 leu2 ade2-101 his3-⌬200 dsk2⌬::LEU2 rad23⌬ [MR2757] MAT␣ ura3-52 leu2-3, 112 his3-⌬200 dsk2⌬::LEU2 rad23⌬ slg1⌬::LEU2 MATa ura3-52 leu2-3, 112 his3-⌬200 ade2-101 dsk2⌬::LEU2 rad23⌬ pkc1ts MATa ura3-52 trp1-1 his4 ade2-101 leu2 pkc1⌬::LEU2 dsk2⌬::LEU2 rad23⌬::URA3 [pkc1ts Ycp50] MATa ura3-52 ade2-101 leu2-3, 112 his7 trp1⌬1 cdc13-1 MAT␣ ura3-52 his3-⌬200 leu2-3, 112 mps2-1 [MPS2 URA3] MATa ura3-1/52 ade2-101/1oc can1-100? leu2-3, 112 his3-⌬200 spc110-220 [pJG109] MATa ura3 trp his kar1⌬17 pkc1⌬::LEU2 [pkc1ts Ycp50] MAT␣ ura3-52 leu2 ade2-101 his3-⌬200 dsk2⌬::LEU2 rad23⌬ mpk1⌬::HIS3 [MR2757] MATa ura3-52 ade2-101 leu2-3, 112 his3-⌬200 cdc31-1 MATa ura3-52 ade2-101 leu2-3 112 his3-⌬200 cdc31-1 bck1⌬::HIS3 MATa ura3-52 ade2-101 leu2-3, 112 his3-⌬200 cdc31-1 mpk1⌬::HIS3 MAT␣ ura3-52 leu2 ade2-101 his3-⌬200 dsk2⌬::LEU2 rad23⌬ bck1⌬::HIS3 MATa ura3-52 ade2-101 leu2-3, 112 trp1-⌬63 cdc31-1 slg1⌬::LEU2 MAT␣ ura3-52 leu2 lys2-801 GAL2⫹ his3-⌬200 trp1⌬63 spc110-220 slg1⌬::LEU2

a b c

d d

e

b

(continued)

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TABLE 2 (Continued) Strain MY6877 MY6880 MY6903 MY6974 SLJ127 V.370

Genotype MATa ura3-1/52 ade2-101/1oc can1-100? leu2-3, 112 his3-⌬200 spc110-220 bck1⌬::HIS3 MATa ura3-1/52 ade2-101/1oc can1-100? leu2-3, 112 his-3-⌬200 spc110-220 mpk1⌬::HIS3 MAT␣ ura3-52 his3-⌬200 leu2-3, 112 mpk1⌬::HIS3 mps2-1 [MPS2 URA3] MATa ura3-52 ade2-101 leu2-3, 112 his3-⌬200 cdc31-1swi4⌬::HIS3 [MR2012] MATa ura3-1 ade2-1 can1-100 leu2-3, 112 his3-11 trp1-1 cdc15-2 MAT␣ ura3-52 leu2-3, 112 his3-⌬200 mps2-1

Source

f g

a

John Diffley, Imperial Cancer Research Fund, United Kingdom. D. E. Levin, Johns Hopkins University, Baltimore. c T. Davis, University of Washington, Seattle. d Breck Byers, University of Washington, Seattle. e Virginia Zakian, Princeton University, Princeton. f D. O. Morgan, University of California, San Francisco. g Mark Winey, University of Colorado, Boulder. b

Spc110p fusion (Stirling et al. 1994). Protein extracts were prepared by trichloroacetic acid (TCA) precipitation (Wright et al. 1989). Protein extracts were separated on a 6.5% SDS-PAGE as described (Laemmli 1970). A 1:1000 dilution of affinity-purified Spc110p antibodies was used for Western blotting. RESULTS

Overexpression of members of the PKC1/MPK1 pathway suppresses SPB duplication mutants: Mutations in CDC31, KAR1, DSK2, and RAD23 result in defects at an early step during SPB duplication. Specifically, cdc31, kar1-⌬17, and dsk2⌬ rad23⌬ mutants arrest in G2/M as large-budded cells with a monopolar spindle and a single SPB (Byers 1981; Vallen et al. 1992; Biggins et al. 1996). All combinations of dsk2⌬, rad23⌬, and kar1⌬17 mutants can be suppressed by high-copy CDC31, suggesting that Cdc31p is downstream in this pathway. To identify other genes that interact with the CDC31-related network of SPB duplication genes, we screened for highcopy suppressors of the temperature sensitivity of dsk2⌬ rad23⌬ (see materials and methods). As expected, the most frequent high-copy suppressor was CDC31. In addition, we identified SLG1/WSC1 and PKC1, as partial suppressors of the growth defect of dsk2⌬ rad23⌬ at restrictive temperatures (Figure 1A). SLG1/WSC1 encodes a plasma membrane protein that signals to Pkc1p via Rho1p (Verna et al. 1997; Jacoby et al. 1998). Pkc1p activates the MAP kinase module consisting of Bck1p, Mkk1p/Mkk2p, and Mpk1p (Lee and Levin 1992; Irie et al. 1993; Lee et al. 1993). To determine whether the suppression by high-copy SLG1 and PKC1 was relevant to SPB duplication, we examined their ability to suppress mutations in SPB components that show genetic interactions with dsk2⌬ rad23⌬. As shown in Figure 1, B and C, high-copy SLG1 and PKC1 also partially suppressed kar1-⌬17 and cdc31-2. Two other alleles of CDC31, cdc31-1 and cd31-5, were not suppressed by any of the plasmids at any tempera-

ture (data not shown). We also tested whether the downstream components of the PKC1 pathway, BCK1-20 and MPK1 2␮, could suppress dsk2⌬ rad23⌬, kar1-⌬17, and cdc31-2. As shown in Figure 1, BCK1-20 was a suppressor of kar1-⌬17 but not of the other mutations. High-copy MPK1 was a very weak suppressor of kar1-⌬17. All highcopy suppression results are summarized in Figure 1D. These results show that overexpression of multiple components of the PKC1 pathway can suppress mutations in all of the genes that are known to affect the first step in SPB duplication. Strikingly, the upstream components of the PKC1 pathway, PKC1 and SLG1, were consistently the strongest suppressors. Slg1p suppresses the SPB duplication defects through Pkc1p: High-copy SLG1 was a better suppressor of kar1⌬17 than high-copy PKC1. On the other hand both were equally good suppressors of cdc31-2. We therefore wanted to determine whether SLG1 suppresses the SPB defects through PKC1 or via an independent pathway. We tested whether PKC1 lies downstream of SLG1 for suppression of SPB duplication mutants by asking whether high-copy PKC1 can suppress a dsk2⌬ rad23⌬ slg1⌬ triple mutant. The triple mutant showed a stronger temperature-sensitive growth defect than the dsk2⌬ rad23⌬ double mutant. Nevertheless, high-copy PKC1 suppressed both the dsk2⌬ rad23⌬ slg1⌬ triple mutant and the dsk2⌬ rad23⌬ double mutant well (Figure 2A). Therefore, Pkc1p does not require Slg1p to suppress dsk2⌬ rad23⌬ consistent with PKC1 being downstream of SLG1. We next tested whether Slg1p required Pkc1p for suppression. This experiment is complicated by the fact that PKC1 is an essential gene. We therefore determined whether high-copy SLG1 could suppress a dsk2⌬ rad23⌬ pkc1ts triple mutant. Once again, the temperature sensitivity of the dsk2⌬ rad23⌬ pkc1ts mutant was more severe than that of either dsk2⌬ rad23⌬ or pkc1ts. We found that high-copy SLG1 did not suppress the dsk2⌬ rad23⌬ pkc1ts triple mutant at 37⬚ (Figure 2B), consistent with Slg1p being upstream of

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Figure 1.—Overexpression of the PKC1 pathway suppresses the temperature sensitivity of SPB duplication mutants. (A) dsk2⌬ rad23⌬ (MY4193), (B) kar1-⌬17 (MS2083), and (C) cdc31-2 (MY2260), bearing the indicated plasmids were spotted onto SC-ura in four 10-fold serial dilutions and incubated at different temperatures. Shown are plates at 23⬚ and 37⬚ (left to right, A) and 23⬚, 35⬚, and 37⬚ (left to right, B and C). (D). Summary diagram showing suppression of the SPB duplication mutants by plasmids containing PKC1 pathway genes. The PKC1 pathway genes are listed in the order of the genetic pathway, with SLG1 being the most upstream. Solid lines represent partial suppression and broken lines represent very weak suppression. We found that SLG1 and PKC1 overexpression suppressed all four SPB duplication mutants (data for spc110-220 suppression shown in Figure 3). In addition, BCK1-20, and to a lesser extent overexpression of MPK1, suppressed kar1-⌬17. BCK1-20 and MPK1 2␮ did not suppress cdc31-2 or dsk2⌬ rad23⌬ mutants. The ability of BCK1-20 and MPK1 2␮ plasmids to suppress spc110-220 was not tested.

Pkc1p’s essential function. However, we found that, at an intermediate temperature of 32⬚, high-copy SLG1 did suppress the dsk2⌬ rad23⌬ pkc1ts triple mutant as well as DSK2 (Figure 2B). This result can be interpreted in two ways. First, the pkc1ts allele may be partially active

at 32⬚, and high-copy SLG1 may be suppressing by enhancing the partial activity. Alternatively, suppression at 32⬚ may be due to a function of Slg1p that is partially independent of Pkc1p. Overexpression of Pkc1p pathway components does not suppress G2/M-arrested mutants in general: The SPB duplication mutations that showed genetic interactions with the PKC1 pathway cause a G2/M arrest at the restrictive temperature. Therefore, we considered the possibility that the PKC1 pathway may simply be required for maintaining the integrity of large-budded cells at G2/M. If so, activation of the pathway may simply suppress the growth defect of SPB mutants by suppressing the loss of viability of G2/M-arrested cells. To address this possibility, we examined whether high-dosage SLG1 or PKC1 could rescue the temperature sensitivity of a variety of mutants that arrest at G2/M through different mechanisms. We examined cdc13-1, defective for telomere metabolism (Wood and Hartwell 1982; Hartwell and Smith 1985; Garvik et al. 1995), cdc15-2, defective in APC activation in M phase ( Jaspersen et al. 1998), and cdc7-1, defective for DNA replication (Njagi and Kilbey 1982; Sclafani et al. 1988). Moreover, to address whether SPB mutants may generally interact with the PKC1 pathway, we tested two other SPB duplication mutants not known to be linked to Cdc31p, spc110-220 (Sundberg et al. 1996) and mps2-1 (Winey et al. 1991). We found that cdc13-1, mps2-1, cdc152, or cdc7-1 strains were not suppressed by 2␮ SLG1 or 2␮ PKC1 (Figure 3). Therefore, we conclude that overexpression of PKC1 pathway genes do not generally suppress mutants that arrest in G2/M. Another trivial possibility is that overexpression of PKC1 pathway components suppresses SPB duplication mutants by extending the length of G1 and thereby allowing extra time for SPB duplication to occur. To test this possibility, we monitored cell cycle progression in strains either overexpressing or deleted for PKC1 pathway components after release from various synchronizations. We found that in all cases the cells proceeded through the cell cycle with equivalent kinetics (Ivanovska and Rose 2000, and data not shown). Therefore, the PKC1 pathway does not suppress the SPB duplication mutants by altering progression through G1. SPC110 is genetically linked to the CDC31 network of SPB duplication genes: Strikingly, the spc110-220 mutant strain was strongly suppressed by high-copy PKC1 and SLG1 (Figure 3). This observation raised the possibility that Spc110p may have functions related to those of Kar1p, Cdc31p, or Dsk2p and Rad23p. Spc110p is a component of the inner SPB plaque that binds calmodulin (Geiser et al. 1993; Kilmartin et al. 1993). The spc110-220 mutation leads to defective SPB assembly at the restrictive temperature ostensibly because of reduced interaction between Spc110p and calmodulin (Sundberg et al. 1996). Although binding of Cdc31p to Spc110p has been observed in vitro (Geier et al. 1996),

PKC1 Pathway and SPB Duplication

1549 Figure 2.—(A) PKC1 overexpression suppresses dsk2⌬ rad23⌬ slg1⌬. The suppression of dsk2⌬ rad23⌬ was examined in the presence (top) or absence (bottom) of SLG1. dsk2⌬ rad23⌬ (MY4193) and dsk2⌬ rad23⌬ slg1⌬ (MY5689) strains were transformed with the indicated plasmids and spotted as in Figure 1. The dsk2⌬ rad23⌬ slg1⌬ grew more poorly than dsk2⌬ rad23⌬ at both 30⬚ and 37⬚. PKC1 overexpression suppressed the temperature sensitivity of both strains, suggesting that SLG1 was not required for suppression. (B) SLG1 overexpression partially suppressed dsk2⌬ rad23⌬ pkc1ts. To determine whether suppression of dsk2⌬ rad23⌬ by SLG1 overexpression requires PKC1, we constructed a dsk2⌬ rad23⌬ pkc1ts (MY6297) and transformed it with the indicated plasmids. While SLG1 overexpression did not suppress dsk2⌬ rad23⌬ pkc1ts at 37⬚, it partially suppressed at 32⬚.

no evidence for in vivo interaction has been reported. To test whether spc110-220 shares characteristics with kar1-⌬17 and dsk2⌬ rad23⌬, we asked whether spc110220 could be suppressed by high-copy CDC31 or CDC3116, known suppressors of dsk2⌬ rad23⌬ and kar1-⌬17 (Vallen et al. 1994; Biggins et al. 1996); see Figure 4. We found that high-copy CDC31, but not CDC31-16, partially rescued spc110-220 (Figure 4). In contrast, mps2-1, an SPB mutant that is not suppressed by overexpression of the Pkc1p pathway, was also not suppressed by any of the plasmids. Therefore, we concluded that high-copy CDC31 suppressed spc110-220 specifically. Because CDC31-16 did not suppress spc110-220, suppres-

sion of spc110-220 must occur by a mechanism different from that of kar1-⌬17. These results suggest that Cdc31p also interacts with Spc110p in vivo, consistent with the in vitro data. In summary, increased dosage of Pkc1p and Slg1p suppressed all four mutations that can be suppressed by high-copy Cdc31p. These findings suggest that the PKC1 pathway interacts with one or several proteins in the Cdc31p SPB assembly network. Mechanism of high-dosage suppression: We next wanted to investigate how high-dosage SLG1 and PKC1 suppressed the growth defect of the SPB mutants. The simplest model is that the PKC1 pathway contributes to SPB function. However, the PKC1 pathway has a well-

Figure 3.—High-copy SLG1 or PKC1 is not a general suppressor of G2/Marrested mutants. The spc110220 mutant was strongly suppressed by overexpression of SLG1 or PKC1. However, none of the other mutants were suppressed by these plasmids. Temperature-sensitive mutant strains spc110-220 (HSY11-4d), cdc13-1 (MY6663), mps2-1 (v.370), cdc15-2 (SLJ127), cdc7-1 (2032) were transformed with corresponding wild-type plasmids, vector (pRS426), SLG1 2␮ (MR4090), or PKC1 2␮ (p10). Transformants were spotted as in Figure 1. (Top) Plates grown at the permissive temperatures (23⬚); (bottom) plates grown at restrictive temperatures (28⬚ for cdc13-1, 30⬚ for cdc7-1, and 37⬚ for spc110-220, mps2-1, and cdc15-2).

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W. Khalfan et al. Figure 4.—High-dosage Cdc31p specifically suppresses the temperature sensitivity of spc110-220. The spc110-220 mutant was suppressed by high-copy CDC31, but not by singlecopy CDC31 or CDC31-16. In contrast, kar1⌬17 was suppressed by high-copy CDC31 and CDC31-16, while mps2-1 was not suppressed by any plasmid. Strains kar1-⌬17 (MS2083), spc110-220 (HSY11-4d), and mps2-1 (v.370) were transformed with vector (pRS426), CDC31 CEN (MR2012), CDC31 2␮ (MR2032), corresponding wild-type plasmid, or CDC3116 CEN (MR2223). Ura⫹ transformants were spotted as in Figure 1 and incubated for 2 days at the permissive temperature (30⬚) shown at the bottom and restrictive temperatures (37⬚ for kar1-⌬17 and spc110-220 and 35⬚ for mps2-1), shown at the top.

established role in cell-wall integrity. Therefore, there is a formal possibility that genes in the CDC31 network play an additional role in cell-wall integrity and that these SPB mutants suffer cell-wall defects that can be suppressed by SLG1 and PKC1. The compromised cell walls in PKC1 pathway mutants cause cell lysis and death. We therefore tested whether the kar1-⌬17 and dsk2⌬ rad23⌬ mutants exhibited cell lysis, using FUN-1, a fluorescent viability probe (Millard et al. 1997). The majority of pkc1ts mutant cells (98%) were dead after 6 hr at the nonpermissive temperature of 37⬚ (Table 3). In contrast, under similar conditions, we found only a small percentage of dead cells in the dsk2⌬ rad23⌬ and kar1⌬17 strains at the restrictive temperature (8 and 16% respectively) and these were not affected by high-copy SLG1 or high-copy PKC1 (Table 3). These results indicate that decreased lysis/cell death is not likely to be the mechanism by which SLG1 and PKC1 suppress the SPB duplication mutants. TABLE 3 SPB duplication mutations do not cause a significant cell lysis defect Strain

Plasmid

Temperature

% Dead

pkc1ts dsk2⌬ rad23⌬

— DSK2 vector SLG1 2␮ PKC1 2␮ KAR1 vector SLG1 2␮ PKC1 2␮

37⬚

98 7 8 7 5 5 16 10 16

kar1-⌬17

Mutant strains dsk2⌬ rad23⌬ (MY4193) and kar1⌬-17 (MS2083) transformed with indicated plasmids and a pkc1ts strain (DL523) were grown at 23⬚ to early log phase and then transferred to 37⬚ for 6 hrs. Death was assayed using the FUN-1 dye (see materials and methods). This experiment was repeated several times with similar results.

We next tested whether high-dosage SLG1 and PKC1 suppressed the G2/M arrest of kar1-⌬17, dsk2⌬ rad23⌬, and spc110-220 mutants, caused by the failure in SPB duplication. The G2/M arrest is demonstrated by largebudded cells with a single nucleus, indicating failure of the duplicated DNA to segregate. If high-dosage SLG1 and PKC1 suppressed the SPB duplication defects of the mutants, we would expect a decrease in the percentage of G2/M-arrested cells. Indeed, SLG1 and PKC1 overexpression partially suppressed the G2/M arrest defect of kar1-⌬17 at 35⬚ (from 44 to 30%) and SLG1 overexpression partially suppressed at 37⬚ (from 40 to 23%) (Table 4), consistent with the partial suppression of the temperature sensitivity of kar1-⌬17. Similarly, the G2/M defect of dsk2⌬ rad23⌬ was partially suppressed by high-copy SLG1 at 37⬚ (from 71 to 44%; Table 4). Moreover, high-copy PKC1 suppressed the G2/M arrest phenotype of spc110-220 (from 76 to 36%). Therefore, SLG1 and PKC1 partially suppressed the G2/M arrest of kar1-⌬17, dsk2⌬ rad23⌬, and spc110-220 mutants, implying that they suppress the SPB duplication defects. Our findings support the idea that SLG1 and PKC1 provide a positive function for SPB duplication. Synthetic lethal interactions between SPB duplication mutants and multiple members of the PKC1 pathway: If the Pkc1p pathway does regulate SPB duplication, then PKC1 pathway mutations should be partially compromised for SPB duplication. If so, their function should be revealed by synthetic lethal interactions with the relevant SPB mutants. In an independent genetic screen, we identified mutations in MPK1, the PKC1 pathway MAP kinase, as being synthetically lethal with kar1⌬17 (see materials and methods). In Figure 5A, we demonstrate synthetic lethality by the inability of a strain to segregate a URA3-based plasmid containing a wildtype copy of one of the genes mutated on the chromosome. kar1-⌬17 and mpk1⌬ all grew on 5-FOA, a wildtype control, at 23⬚. In contrast, the mpk1⌬ kar1-⌬17 double mutant strain was extremely sensitive to 5-FOA because of a requirement for the KAR1 URA3 plasmid

PKC1 Pathway and SPB Duplication

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TABLE 4 Activation of PKC1 pathway partially suppresses the G2/M arrest of SPB duplication mutants

Strain

Temperature

Plasmid

kar1-⌬17

35⬚

[vector] [SLG1 2␮] [PKC1 2␮] [KAR1 CEN]

30 33 42 39

22 27 18 31

44 30 30 9

4 10 10 21

kar1-⌬17

37⬚

[vector] [SLG1 2␮] [PKC1 2␮] [KAR1 CEN]

35 40 22 45

23 24 33 16

40 23 37 24

3 13 8 15

dsk2⌬ rad23⌬

37⬚

[vector] [SLG1 2␮] [PKC1 2␮] [DSK2 CEN]

16 27 15 52

7 19 10 15

71 44 73 20

6 10 2 13

spc110-220

37⬚

[vector] [PKC1 2␮]

5 18 43

16 34 32

76 36 0

3 12 25

Wild type

SPB mutant strains were transformed with the indicated plasmids and grown at 23⬚ in SC-ura and then shifted to 35⬚ or 37⬚. Cell cycle distribution was analyzed after 6 hr at 35⬚ or 4 hr at 37⬚ for kar1-⌬17 (MS2083), after 8 hr at 37⬚ for dsk2⌬ rad23⌬ (MY4193), and after 6 hr at 37⬚ for spc110-220 (HSY11-4d) and wild type (MS1554). Cell cycle distribution was analyzed as described in materials and methods. More than 100 cells were counted for each strain. Numbers in table represent percentages. The experiment was repeated several times with reproducible results. Boldface type indicates the aberrant G2/M class of cells. Underlined numbers indicate instances where the G2/M class is significantly reduced relative to the vector control.

for survival. Thus, the mpk1⌬ kar1-⌬17 double mutant combination is synthetically lethal. We next asked whether the synthetic lethality observed between mpk1 and kar1-⌬17 could be extended to other SPB components and members of the PKC1 pathway. First, to test whether other SPB mutants showed synthetic lethal interactions with mpk1⌬, we deleted MPK1 in the dsk2⌬ rad23⌬, cdc31-1, and spc110220 mutant strains containing the appropriate wild-type gene on URA3 plasmids. The mpk1⌬ was synthetically lethal with dsk2⌬ rad23⌬ and spc110-220 mutations (Figure 5A). The cdc31-1 mpk1⌬ double mutant showed a greatly exacerbated growth defect at temperatures permissive for either single mutant (Figure 5B). In contrast, the mps2-1 mpk1⌬ double mutant did not show a synthetic lethal phenotype (Figure 5A) confirming that the synthetic lethal interactions are restricted to the CDC31 network of SPB duplication genes. In summary, all the CDC31-related SPB mutants analyzed showed either synthetic lethality or synthetic growth defects when MPK1 was deleted. Next, we tested whether mutations in other components of the PKC1 pathway showed synthetic lethal interactions with the SPB duplication mutants. All the double mutant combinations tested showed synthetic lethality or severely exacerbated growth defects (Figure 5, A and B). In particular, the bck1⌬ kar1-⌬17 double mutant was

synthetically lethal, while the other combinations had restrictive temperatures lower than either parent. Figure 5C summarizes all of the exacerbated growth defects and synthetic lethal interactions observed. On the basis of both high-dosage suppression and synthetic lethal genetic interactions, we conclude that the PKC1 pathway provides a function that is crucial for survival of the SPB duplication mutants. Characterization of the double mutants: The genetic interactions presented thus far do not distinguish whether the synthetic defects affect SPB duplication per se. Although this seems most likely, an alternative is that mutations in SPB components could have secondary defects in cell integrity that are exacerbated in the absence of the PKC1 pathway. To assess the nature of the synthetic defects directly, we analyzed the phenotypes of the double mutant strains. This analysis is technically compromised by the fact that the terminal phenotype associated with a defect in SPB duplication (arrest at G2/M with a large bud and an unduplicated SPB) requires an extended period of bud growth. Bud growth is extremely sensitive to defects in the PKC1 pathway causing cells to lyse before the large bud stage. Thus, at the restrictive temperature, most double mutants arrested as a mixture of small-budded and large-budded cells and so were not informative as to whether there was an increase in the SPB duplication defect (data not

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Figure 5.—Synthetic lethality and synthetic growth defects between SPB mutants and mutations in the PKC1 pathway. (A) mpk1⌬ is synthetically lethal with kar1-⌬17, dsk2⌬ rad23⌬, and spc110-220, but not with mps2-1. In addition, bck1⌬ is synthetically lethal with kar1-⌬17. kar1-⌬17 mpk1⌬: (I) kar1-⌬17 mpk1⌬ (MS5886), (II) mpk1⌬ (MS5930), (III) kar1-⌬17 (MS2373), and (IV) wild type (MS1554). kar1-⌬17 bck1⌬: (I) kar1-⌬17 bck1⌬ (MS5888), (II) bck1⌬ (MS5938), (III) kar1-⌬17 (MS5820), and (IV) wild type (MS1554). dsk2⌬ rad23⌬ mpk1⌬: (I) dsk2⌬ rad23⌬ mpk1⌬ (MY6809), (II) mpk1⌬ (MS6547), (III) dsk2⌬ rad23⌬ (MY5064), and (IV) wild type (MS1554). spc110-220 mpk1⌬: (I) spc110-220 mpk1⌬ (MY6880), (II) mpk1⌬ (MS6547), (III) spc110-220 (MY6764), and (IV) wild type (MS6556). mps2-1 mpk1⌬: (I) mps2-1 mpk1⌬ (MY6903), (II) mpk1⌬ (MS6547), (III) mps2-1 (MY6739), and (IV) wild type (MS1554). Strains growing on SC-ura contain a URA3 plasmid bearing the appropriate SPB duplication gene. (B) Exacerbation of growth defects of SPB mutants in the absence of PKC1 pathway members. In each case, we observed that the double or triple mutants had lower restrictive temperatures than their parents. Strains were spotted on synthetic complete or YPD at permissive temperature of 23⬚ or 30⬚ (left) and restrictive temperature (right). Shown from top to bottom: slg1⌬ (MY4455), kar1-⌬17 (MS2083), slg1⌬ kar1-⌬17 (MY4646), dsk2⌬ rad23⌬ (MY4193), slg1⌬ dsk2⌬ rad23⌬ (MY4916), cdc31-1 (MY2259), slg1⌬ cdc31-1 (MY6862), pkc1ts (DL523), kar1-⌬17 (MS2083), pkc1ts kar1-⌬17 (MY6805), dsk2⌬ rad23⌬ (MY4193), pkc1ts dsk2⌬ rad23⌬ (MY6296), bck1⌬ (MS 5938), dsk2⌬ rad23⌬ (MY4193), bck1⌬ dsk2⌬ rad23⌬ (MY6861), cdc31-1 (MY6855), bck1⌬ cdc31-1 (MY6857), mpk1⌬ (MS6547), cdc31-1 (MY6855), and mpk1⌬ cdc31-1 (MY6859). (C) Summary diagram showing synthetic lethal interactions (solid line) or synthetic growth defects (broken line) between PKC1 pathway and SPB duplication mutants. PKC1 pathway mutations are listed in order of the genetic pathway, with slg1⌬ being the most upstream mutation in the pathway. Note that the growth phenotype of pkc1ts cdc31-1 and pkc1ts spc110-220 double mutants were not tested.

shown). Synchronizing the cultures in G1 with ␣-factor, followed by release from arrest, did not alleviate the problem (data not shown). However, mpk1⌬ exhibited a strong synthetic lethality with kar1-⌬17 at a temperature where the single mutant did not lyse (Table 5). A slow-

growing mpk1⌬ kar1-⌬17 mutant propagated at 23⬚ showed an increase in G2/M-arrested cells (53%) relative to the kar1-⌬17 single mutant (33%) without showing an elevated frequency of dead cells (Table 5). Thus, this double mutant showed an increase in G2/M arrest,

PKC1 Pathway and SPB Duplication

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TABLE 5 The kar1-⌬17 mpk1⌬ shows an increase in large-budded, G2/M-arrested cells

Strain Wild type kar1-⌬17 mpk1⌬ kar1-⌬17 mpk1⌬

Temperature 23⬚

% dead 60 25 63 33

36 42 34 14

0 33 1 53

4 0 2 0

1 3 16 5

Analysis of the cell cycle distribution was performed as described in materials and methods. More than 100 cells were counted for each strain. The numbers represent percentages of total number of cells counted. The experiment was repeated several times with reproducible results. Boldface indicates significant enhancement of the aberrant G2/M class relative to the single mutants.

consistent with the hypothesis that the PKC1 pathway plays a positive role in SPB duplication. Pkc1p pathway activity is required for proper Cdc31p function in vivo: The genetic interactions suggest that Pkc1p pathway activity is required for optimal function of SPB duplication genes. All of the genes suppressed by PKC1 overexpression can also be suppressed by highcopy CDC31. Therefore, we next asked whether the PKC1 pathway is required for Cdc31p to suppress the SPB mutations. Specifically, we tested whether highcopy CDC31 and the dominant alleles CDC31-16 and DSK2-1 could suppress kar1-⌬17 in the absence of the Pkc1p pathway component Slg1p. The dominant alleles CDC31-16 and DSK2-1, but not high-copy CDC31, can suppress a complete deletion of KAR1 (Vallen et al. 1994) and suppress kar1-⌬17 as well as KAR1 (Figure 6, top). Previous analysis has failed to uncover any muta-

Figure 6.—The ability of high-copy CDC31 to suppress kar1⌬17 is compromised in the absence of SLG1. Tenfold serial dilutions of MS2373, MY3752-3755 (top) and MY4642 transformed with indicated plasmids (bottom) were plated on SCura plates and grown for 2 days at the indicated temperatures. The top shows that kar1-⌬17 is temperature sensitive and that CDC31-16 and DSK2-1 strongly suppressed the temperature sensitivity whereas CDC31 overexpression was a partial suppressor. The bottom shows that in the absence of SLG1, the suppressing ability of DSK2-1 and CDC31-16 are partially compromised, and that of high-copy CDC31 is fully compromised.

tions that compromise the ability of CDC31-16 and DSK2-1 to suppress kar1-⌬17 (W. Khalfan and M. Rose, unpublished observations; Biggins et al. 1996). As shown in Figure 6, CDC31-16 or DSK2-1 plasmids did not suppress the slg1⌬ kar1-⌬17 as well as KAR1 at 37⬚. Moreover, high-copy CDC31 completely failed to suppress the double deletion even at 30⬚ (Figure 6, bottom). These results can be interpreted in two ways. First, Slg1p may be required for optimal function of Cdc31p. By this scheme the PKC1 pathway would lie upstream or in a parallel pathway leading to activation of Cdc31p. Alternatively, Cdc31p itself may be required to activate the PKC1 pathway. In this scenario, the PKC1 pathway would lie downstream of Cdc31p, and mutations in cdc31 and the other SPB duplication genes would be predicted to alter PKC1 pathway activity. We tested this second idea by measuring PKC1 pathway activity in the SPB duplication mutants in a number of ways. First, as a measure of MKK1 and MKK2 activity, we tested whether MPK1 was phosphorylated in a kar1-⌬17 strain upon mild heat shock treatment (Kamada et al. 1995; Verna et al. 1997). We found that Mpk1p’s ability to be phosphorylated was not compromised in the kar1-⌬17 strain (data not shown). We also examined the in vitro kinase activity of Pkc1p immunoprecipitated from a kar1-⌬17 strain, using myelin basic protein (MBP) as the substrate (Watanabe et al. 1994). Again, we found no evidence that Pkc1p kinase activity was affected, at least in vitro (data not shown). Finally, using a LacZ-reporter assay system, we examined the transcriptional activity of Rlm1p, a MADS-box transcription factor whose activity depends on Mpk1p phosphorylation (Watanabe et al. 1997). Although Rlm1p transcriptional activity was indeed lower in the kar1-⌬17 strain, this was a general effect of G2/M arrest, since another G2/M mutant, cdc13, also showed reduced levels of Rlm1p transcriptional activity. Taken together, it seems unlikely that the CDC31 pathway activates the PKC1 pathway. We therefore interpret the inability of high-copy CDC31 to suppress the kar1-⌬17 slg1⌬ double mutant (Figure 6) as being due to reduced activity or levels of Cdc31p in

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Figure 7.—Spc110p phosphorylation is defective in pkc1ts and mpk1⌬ mutants. Protein extracts were prepared from asynchronously growing, ␣-factor-arrested, or nocodazole-arrested wild-type strain (MS1554) at 30⬚ and asynchronously growing pkc1ts (DL523) and MPK1⌬ strains (MS5930) at 30⬚ or 37⬚ (see materials and methods). For ␣-factor arrest, MS1554 was treated with 10 ␮g/ml ␣-factor for 3 hr at 30⬚. For nocodazole arrest, MS1554 was treated with 5 ␮g/ml nocodazole for 3 hr at 30⬚. The top arrow marks the p120 form of Spc110p and the bottom arrow marks the p112 form. The p120 form is absent in the pkc1ts mutant at restrictive temperature (37⬚) and is also greatly diminished in the mpk1⌬ mutant at 30⬚ and 37⬚.

the slg1⌬ mutant. The observation that CDC31-16 and DSK2-1, but not high-copy CDC31, could partially suppress the kar1-⌬17 slg1⌬ double mutant at 37⬚ suggests that the dominant suppressors may act by a different mechanism that is partially independent of the PKC1 pathway. In support of the idea that they act differently, CDC31 2␮, but not CDC31-16, suppressed the temperature-sensitive growth defect of spc110-220 (Figure 4). Phosphorylation of Spc110p is defective in pkc1ts and mpk1⌬ mutants: We next sought to explore the molecular mechanism by which the PKC1 pathway affects SPB duplication. One possibility is that the PKC1 pathway phosphorylates an SPB component. On the basis of the genetic interactions, possible candidates include Kar1p, Cdc31p, and Spc110p. We first examined Kar1p in the different PKC1 pathway mutants and found no change in the mobility of various Kar1p forms (data not shown). We next tested Spc110p, which exists as at least two distinct phosphorylated forms of molecular weights 112 kD and 120 kD. The 120-kD form (p120) arises from additional serine/threonine phosphorylation of the 112-kD form and is cell cycle regulated, predominating in small-budded cells with duplicated DNA and short

spindles (Friedman et al. 1996; Stirling and Stark 1996). The SPB duplication gene Mps1p is one of the kinases responsible for Spc110p phosphorylation (M. Winey and T. Davis, unpublished communication). On the basis of the time of the appearance of the p120 form and analysis of cdc mutants, the phosphorylation that produces the p120 form does not appear to be required for SPB duplication (Friedman et al. 1996). We reasoned that the phosphorylation state of Spc110p could serve as a useful marker for earlier event(s) in SPB duplication. We therefore examined the phosphorylation of Spc110p in asynchronous wild-type, pkc1ts, and mpk1⌬ cultures grown in YPD at 30⬚ and shifted to 37⬚ for 3 hr. An asynchronous wild-type culture grown at 37⬚ contained both p120 and p112 forms, ␣-factor-arrested wild-type strain contained the faster migrating p112 form, and nocodazole-arrested wild-type cells contained the p120 form (Figure 7 and Friedman et al. 1996; Stirling and Stark 1996). In contrast to the wild-type strain, the p120 form was absent in the pkc1ts mutant at 37⬚. Similarly, an mpk1⌬ culture showed reduced levels of p120 at permissive temperature (30⬚) and drastically reduced levels of p120 at 37⬚. The disappearance of the p120 form in pkc1ts and mpk1⌬ strains is not likely to be due to the accumulation of cells at a stage in the cell cycle prior to Spc110p phosphorylation. First, although the pkc1ts mutant accumulates small-budded cells, the small-budded cells have already initiated DNA replication and formed short spindles (Levin and Bartlett-Heubusch 1992; Ivanovska and Rose 2000). Therefore, on the basis of the pkc1ts mutant phenotype, we would not expect a block in the production of the p120 form due to cell cycle arrest. Second, in the case of the mpk1⌬ mutant, the strain did not arrest at any particular stage of the cell cycle at the restrictive temperatures. We directly examined the cell cycle distribution of pkc1ts and mpk1⌬ strains from aliquots taken at the time of protein extract preparation. As shown in Table 6, while there were differences in their cell cycle distribution compared to wild type, the distribution was not correlated with the presence or absence of the p120 form. These results suggest that

TABLE 6 Cell cycle distribution of pkc1ts and mpk1⌬ strains

Strain Wild type pkc1ts mpk1⌬

Temperature 37⬚ 30⬚ 37⬚ 30⬚ 37⬚

43 51 23 31 32

37 39 47 35 27

20 8 29 34 41

0 2 1 0 0

Wild type (MS1554), pkc1ts (DL523), and mpk1⌬ (MS5930) strains were grown at 23⬚ in YPD and then shifted to the indicated temperatures for three hours. Cell cycle analysis was performed as described in materials and methods. The experiment was repeated several times with reproducible results.

PKC1 Pathway and SPB Duplication

Figure 8.—Synthetic lethality of kar1-⌬17 and cdc31-1 with swi4⌬, but not rlm1⌬. The top represents SC-ura plates while the bottom represents 5-FOA plates. (A) (I) kar1-⌬17 swi4⌬ (MS6944), (II) swi4⌬ (MS6934), (III) kar1-⌬17 (MS5820), and (IV) wild type (MS1554) all contain KAR1 URA3 (MR76 or MR77). (B) (I) kar1-⌬17 rlm1⌬ (MS6947), (II) rlm⌬ (MS6937), (III) kar1-⌬17 (MS5820), and (IV) wild type (MS1554) all contain KAR1 URA3 (MR76 or MR77). (C) (I) cdc31-1 swi4⌬ (MY6974), (II) swi4⌬ (MS6934), (III) cdc31-1 (MY3883), and (IV) wild type (MS1554) all contain CDC31 URA3 (MR2012). (D) (I) cdc31-1 rlm1⌬ (II) rlm1⌬ (MS6937), (III) cdc31-1 (MY3883), and (IV) wild type (MS1554) all contain CDC31 URA3 (MR2012).

the lack of phosphorylation is not due to a block in cell cycle progression. Taken together, our results suggest that Pkc1p and/or Mpk1p may directly or indirectly phosphorylate Spc110p. These results may provide a mechanistic basis for the genetic interactions between thePKC1 pathway and SPB duplication genes. Swi4p but not Rlm1p function is required in SPB duplication mutants: Having established that Pkc1p and the MAP kinase module affect SPB duplication, we wanted to identify the downstream effectors of the PKC1 pathway that transduce the signal to the SPB. One wellestablished downstream target of Mpk1p is Rlm1p, a MADS-box family transcription factor whose activity is MPK1 dependent (Watanabe et al. 1995, 1997; Dodou and Treisman 1997). However, unlike mpk1⌬, deletion of RLM1 does not result in cell morphogenesis and/or lysis defects, indicating that there must be additional downstream effectors of MPK1 activity. The transcrip-

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tion factor complex consisting of Swi4p/Swi6p (SBF) has also been suggested to be a second downstream effector of MPK1 (Madden et al. 1997). Swi4p and Swi6p physically associate with Mpk1p, and phosphorylation of Swi4p and Swi6p is dependent on Mpk1p (Madden et al. 1997). These results suggest that Swi4/Swi6 complex may be a target of PKC1 pathway activity. However, SBF activity is also thought to be required to turn on the PKC1 pathway via Cdc28p/Clns (Mazzoni et al. 1993; Marini et al. 1996; Zarzov et al. 1996; Gray et al. 1997). To determine whether Rlm1p and/or Swi4p are important for the survival of the SPB mutants, we generated kar1-⌬17 rlm1⌬ and kar1-⌬17 swi4⌬ double mutants containing KAR1 URA3 plasmids and examined their ability to lose the plasmid on 5-FOA at a range of temperatures. As shown in Figure 8A, we found that the kar1-⌬17 swi4⌬ strain was synthetically lethal, similar to the kar1-⌬17 mpk1⌬ double mutant. In contrast, the kar1-⌬17 rlm1⌬ double mutant did not grow any more poorly than either single mutant alone at a range of temperatures (Figure 8B). Since the cdc31-1 mpk1⌬ double mutant also has a severe growth defect, we examined whether cdc31-1 mutants were sensitive to mutations in either SWI4 or RLM1. Similar to the results obtained with the kar1-⌬17 swi4⌬ double mutant, a cdc31-1 swi4⌬ double mutant was synthetically lethal, whereas a cdc31-1 rlm1⌬ double mutant did not show any synthetic growth defects (Figure 8, C and D). These results argue that Swi4p’s function is crucial for survival of the SPB duplication mutants, kar1-⌬17 and cdc31-1, in activating the PKC1 pathway and/or transmitting a signal downstream of Mpk1p. Clearly however, Rlm1p is not the target of Mpk1p activity that is relevant for SPB duplication. Because Swi4p might mediate its effects through transcriptional regulation, and because Cdc31p function is compromised in the kar1-⌬17 slg1⌬ mutant (Figure 6), one possible explanation for the genetic interactions would be if the PKC1 pathway regulated CDC31 transcription. To address this question, we examined the levels of CDC31 mRNA in asynchronously growing wildtype, pkc1ts, mpk1⌬, and swi4⌬ mutants either at 23⬚ or 37⬚. We found that the levels of CDC31 mRNA were similar in the mutant and wild-type strains (data not shown). We also found the levels of Cdc31p protein in pkc1ts mutant to be comparable to that of wild type at 37⬚. In addition, the levels of Kar1p and Spc110p were wild type in pkc1ts and mpk1⌬ strains (data not shown). Therefore, it is unlikely that the PKC1 pathway regulates the synthesis of Cdc31p, Kar1p, or Spc110p. It remains possible, however, that the expression of an unidentified SPB component is regulated by the PKC1 pathway. DISCUSSION

Genetic interactions with SPB duplication genes suggest a role for the PKC1 pathway in SPB duplication: We identified numerous genetic interactions between SPB duplication genes in the CDC31 pathway and the

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PKC1 signal transduction cascade. Overexpression of SLG1 or PKC1 partially rescued the temperature sensitivity of dsk2⌬ rad23⌬, kar1-⌬17, and cdc31-2. We assume that overexpression leads to activation of the PKC1 pathway. The suppression was specific to SPB duplication defects and was not due to general suppression of G2/M arrest. High-dosage SLG1 and PKC1 did not simply rescue cell-wall-related defects of the SPB mutants, because we did not find significant cell lysis in dsk2⌬ rad23⌬ and kar1-⌬17. It is interesting that the most upstream members of the PKC1 pathway, SLG1 and PKC1, were the strongest suppressors. We present two interpretations of this observation. First, the PKC1 pathway may branch at one or more points and the different components of the PKC1 pathway may suppress the SPB duplication mutants by different mechanisms. Alternatively, consistent with the observations of other researchers, overexpression of upstream components may activate the PKC1 pathway more strongly than overexpression of downstream components, resulting in different levels of suppression of the SPB duplication mutants (Gray et al. 1997). We also observed somewhat different behaviors among the different PKC1 pathway components in our synthetic lethal analyses. The severity of the interactions was the reverse of the suppression, with the most downstream components showing the most severe synthetic phenotypes in combination with SPB duplication mutations. The SPB mutants were particularly sensitive to mutations in MPK1. With the exception of cdc31, all the SPB mutants showed synthetic lethality with mpk1⌬, while mutations in the other PKC1 pathway genes resulted in viable strains with more severe growth defects. In addition, the mpk1⌬ kar1-⌬17 double mutant had an exacerbated G2/M arrest phenotype, suggesting a requirement for the MPK1 gene in SPB duplication. It is possible that Mpk1p is crucial for the survival of SPB mutants because it is targeted and activated by other MAP kinase signaling pathways, especially in the absence of upstream components of the PKC1 pathway. The presence of such cross-talk may help to explain why mutations in upstream components do not exacerbate the growth defect of SPB duplication mutants as severely as mutations in MPK1. The SPB mutants cdc31 and kar1 showed synthetic lethality with swi4⌬ arguing that, like Mpk1p, Swi4p is also crucial for the survival of the SPB duplication mutants. The Swi4p/Swi6p transcription factor complex (SBF) is a proposed target of Mpk1p (Madden et al. 1997). However, SBF is also an activator of the PKC1 pathway via the Cdc28p/Clns (Gray et al. 1997). Therefore, while the synthetic lethal results argue that Swi4p is as crucial as Mpk1p, they do not discriminate whether the upstream or downstream activity of Swi4p is important. The lack of genetic interactions between the SPB mutants and rlm1⌬ clearly rule out the possibility that Rlm1p is the important target of Mpk1p in the SPB

duplication mutants. Recently, a genome-wide analysis has identified numerous genes that are transcriptionally upregulated or downregulated in response to MPK1 activity (Jung and Levin 1999). The transcriptional regulation of most of the genes identified in the genome analysis was mediated by Rlm1p, and all genes code for components of the cell-wall biosynthesis machinery (Jung and Levin 1999). Since loss of Mpk1p function results in a much more severe growth defect than the loss of Rlm1p, Mpk1p must have other targets as well. It is possible that other genes that are transcriptionally regulated by Mpk1p were missed in the genome analysis. However, the results of Jung and Levin (1999) raise the interesting possibility that Mpk1p activates its targets post-transcriptionally. Indeed, the transcript levels of CDC31 and protein levels of Cdc31p, Kar1p, and Spc110p are normal in the PKC1 pathway mutants, suggesting that the pathway may regulate SPB component(s) post-translationally. Formally, the PKC1 pathway may function upstream, in parallel, or downstream of the CDC31 pathway. If the PKC1 pathway is downstream, then mutations in the SPB duplication genes may affect the level of PKC1 pathway activity. We tested this hypothesis using several different markers for PKC1 pathway activity and in all cases we found that mutations in SPB duplication genes did not alter the activity of the PKC1 pathway. Therefore, it is unlikely that the CDC31 pathway is upstream of the PKC1 pathway. Less clear is whether the PKC1 functions upstream or in parallel with the CDC31 pathway. We found that Slg1p is required for Cdc31p’s SPB function (Figure 6). This result suggests that the PKC1 pathway affects Cdc31p, either by activating Cdc31p directly or by activating other components with which Cdc31p interacts at the SPB. Given that Spc110p phosphorylation was defective in pkc1ts and mpk1⌬ mutants and that Spc110p functionally interacts with Cdc31p, Spc110p may well be one of the targets of PKC1 pathway activity. However, given the timing of the appearance of p120, it is unlikely that this particular phosphorylation is the relevant regulatory event. The p112 form has not been resolved from the unphosphorylated form of the protein (Friedman et al. 1996). Therefore, it is possible that the p120 appears as a consequence of earlier phosphorylation(s) of Spc110p. In such a scenario, the lack of p120 could be an indication that earlier phosphorylation events relevant to SPB duplication have not occurred in the pkc1ts and mpk1⌬ mutants. While our data suggest that the PKC1 pathway is involved in SPB duplication, pkc1 mutants have not been reported to exhibit defects in SPB duplication. The slg1⌬ strain does have a phenotype that can be taken as including an SPB defect; in the cold, the mutant fails to initiate SPB duplication even though it buds and eventually lyses (Ivanovska and Rose 2000). The possibility of genetic redundancy and the pleiotropic nature

PKC1 Pathway and SPB Duplication

of the mutant phenotype might easily mask an SPB defect in pkc1 mutants. Spc110p may interact with Cdc31p at the SPB: In the course of testing the specificity of the genetic interactions, we found that SLG1 and PKC1 also suppressed the SPB duplication defect of spc110-220, a mutation in a gene not known to be related to genes in the Cdc31p pathway. We also found that overexpression of CDC31 suppressed spc110-220. The point mutation in the spc110-220 allele disrupts binding of Spc110p to Cmd1p, and overexpression of Cmd1p rescues the spc110-220 temperature sensitivity (Geiser et al. 1993; Sundberg et al. 1996). High-copy CDC31 has also been found to partially suppress the temperature sensitivity of a cmd1 allele defective for SPB duplication (Geier et al. 1996). Two hypotheses are consistent with these findings. First, CDC31 might suppress spc110-220 by substituting for its close relative, calmodulin. In this view, Cdc31p and Spc110p would not normally interact in vivo. Consistent with this, Cdc31p and Spc110p have been localized to different regions of the SPB; the major portion of Cdc31p localizes to the half-bridge (Spang et al. 1993) and Spc110p localizes to the region between the central and inner plaques (Rout and Kilmartin 1990). Because Cdc31p and Spc110p would not share functions in vivo, the PKC1 pathway would suppress Cdc31p and Spc110p by different mechanisms. An alternative view, which we favor, is that Spc110p and Cdc31p have interrelated functions that our genetic suppression analysis has uncovered. This interpretation is supported by the observation that, in vitro, an Spc110p peptide containing the calmodulin binding site binds Cdc31p as well as it binds calmodulin (Geier et al. 1996). In this more parsimonious model, the PKC1 pathway would suppress mutations in cdc31 and spc110 by the same mechanism because Spc110p and Cdc31p have shared functions in vivo. One place where Cdc31p and Spc110p might interact is at the junction between the half-bridge and the inner plaque of the SPB. To explain our genetic data, we propose that the PKC1 pathway may signal to the Cdc31p network or to some other target at the SPB to activate SPB duplication. Because we observed that phosphorylation of Spc110p was defective in pkc1ts and mpk1⌬ strains, we suggest that Spc110p may be a direct or indirect target of PKC1 pathway regulation. Does overexpression of PKC1 pathway components suppress SPB duplication mutants by restoring the p120 form of Spc110p? We think that this is unlikely because the p120 phosphorylated form is present in the SPB duplication mutants at the nonpermissive temperature, including spc110-220, kar1⌬17, and cdc31 mutants (Friedman et al. 1996; W. Khalfan, I. Ivanovska and M. D. Rose, unpublished observation). Therefore, the loss of p120 is not likely to be the defect in these mutants. More likely, Spc110p phosphorylation in pkc1 and mpk1 mutants may simply reflect the state of the SPB, revealing that prior event(s) important for

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SPB assembly under the control of the Pkc1p pathway have not been executed properly. Timing of regulation: The kar1-⌬17, cdc31, and dsk2⌬ rad23⌬ mutants are defective for an early step in SPB duplication, the formation of the satellite, which occurs independently of Cdc28p/Clns activation. Hence, if the PKC1 pathway regulates SPB duplication in a positive manner, it may act, at this early stage, in a manner independent of Cdc28p/Clns. Indeed there is substantial evidence to suggest that the PKC1 pathway can function independently of Cdc28p/Clns activity. First, mutations in MPK1/SLT2 and PKC1 have been isolated as enhancers of Start-defective cdc28 mutants (Mazzoni et al. 1993; Marini et al. 1996) suggesting that the PKC1 pathway functions in parallel with Cdc28p. Second, MPK1 activation during vegetative growth is only partially dependent on CDC28; Mpk1p tyrosine phosphorylation is reduced but not eliminated in cdc28 ts mutants, again suggesting that the PKC1 pathway can act partially independently of Cdc28p (Zarzov et al. 1996). Alternatively, the PKC1 pathway may be a transducer of the START/Cdc28p signal to promote the later steps in SPB duplication. Finally, the PKC1 pathway could activate SPB duplication by acting on Cdc28p/Clns. This view is supported by the interaction between MPK1 and the Swi4p/Swi6p complex (SBF), a major transcriptional activator of the G1 cyclins (Madden et al. 1997). Regardless of the details, the PKC1 signal transduction pathway, which also promotes bud emergence, is ideally suited to coordinate two major events at the G1/S transition, SPB duplication and bud emergence. We thank D. Levin, T. Davis, M. Winey, M. Stark, and D. Stirling for strains, antibodies, and useful discussions. We also acknowledge Naz Erdeniz for critical reading of the manuscript. This research was supported by National Institutes of Health grant GM52526 to M.D.R. I.I. and W.K. were supported by fellowships from the New Jersey Commision on Cancer Research.

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