Characterization of a Saccharomyces cerevisiae thermosensitive lytic mutant leads to the identification of a new allele of the NUD1 gene

The International Journal of Biochemistry & Cell Biology 36 (2004) 2196–2213

Characterization of a Saccharomyces cerevisiae thermosensitive lytic mutant leads to the identification of a new allele of the NUD1 gene Irina Alexandar a , Pedro San Segundo a , Pencho Venkov b , Francisco del Rey a , Carlos R. Vázquez de Aldana a,∗ a

Departamento de Microbiolog´ıa y Genética, Instituto de Microbiolog´ıa-Bioqu´ımica, Universidad de Salamanca/CSIC, Campus Miguel de Unamuno, 37007 Salamanca, Spain b Institute of Molecular Biology, Bulgarian Academy of Sciences, 1113 Sofia, Bulgaria Received 20 January 2004; received in revised form 1 April 2004; accepted 19 April 2004

Abstract To improve our understanding of the factors involved in the osmotic stability of yeast cells, a search for novel conditional Saccharomyces cerevisiae cell lysis mutants was performed. Ten temperature-sensitive (ts) mutant strains of S. cerevisiae were isolated that lyse at the restrictive temperature on hypotonic, but not on osmotically supported medium. The ten mutants fell into four complementation groups: ts1 to ts4. To clone the wild-type gene corresponding to the ts4 mutation, a strategy aimed at complementing the thermosensitive phenotype—using low-copy and high-copy DNA libraries—was followed, but only two extragenic suppressors were identified. Another approach, in which classic genetic methods were combined with the use of yeast artificial chromosomes and traditional cloning procedures, allowed the identification of the NUD1 gene—which codes for a component of the spindle-pole body—as the wild-type gene corresponding to the ts4 mutation. Cloning and sequencing of the defective allele from the chromosome of the mutant cells resulted in the identification of a point mutation that produces a single amino acid change in the protein: a Gly-to-Glu change at position 585 (the nud1-G585E allele). Further analysis revealed that cells carrying this allele show a thermosensitive growth defect. At the restrictive temperature, the cells arrest with large buds, elongated spindles, and duplicated nuclei. In addition, with longer incubation times they are unable to maintain cellular integrity and lyse. Our results have allowed the identification of the first single amino acid mutation in NUD1, and suggest a link between cell cycle progression and cellular integrity. © 2004 Elsevier Ltd. All rights reserved. Keywords: Saccharomyces cerevisiae; SPB; Cell lysis; MEN; Mitotic exit

1. Introduction The isolation of mutants displaying a thermosensitive lytic phenotype is a general strategy that has ∗ Corresponding author. Tel.: +34-923-294675; fax: +34-923-224876. E-mail address: [email protected] (C.R. V´azquez de Aldana).

been successfully exploited to identify genes controlling cell wall integrity and cell viability. Mutants of this type have been isolated in Saccharomyces cerevisiae (Baymiller & McCullough, 1997; Cabib & Durán, 1975; Cid, Sánchez, & Nombela, 1994; Paravicini et al., 1992; Torres et al., 1991; Venkov, Hadjiolov, Battaner, & Schlessinger, 1974), Candida albicans (Payton & de Tiani, 1990), Schizosaccha-

1357-2725/$ – see front matter © 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.biocel.2004.04.008

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romyces pombe (Ribas, Diaz, Durán, & Pérez, 1991) and Aspergillus nidulans (Borgia & Dodge, 1992). One characteristic shared by all these mutant strains is the loss of viability when they are cultured at the non-permisissive temperature (usually 37 ◦ C), resulting in the release of intracellular contents to the surrounding medium. The broad variety of lytic mutants isolated reflects the complexity of functions involved in regulating cell wall integrity and cell viability. A preliminary study of the nature of the primary defect present in the mutants can be evaluated by testing whether the addition of osmotic stabilizers to the culture medium is able to complement the lytic phenotype. In some mutants, the defect in lysis is complemented by the addition of an osmotic support to the culture medium, suggesting that the primary phenotypic defect must be directly or indirectly related to the cell wall. This is the case of slt2 mutants, which were isolated on the basis of their lytic phenotype (Torres et al., 1991). Slt2p/Mpk1p is a component of the Pkc1p cell integrity pathway, which has been implicated in numerous cellular processes, including the promotion of bud emergence at the G1/S transition (Levin, Fields, Kunisawa, Bishop, & Thorner, 1990; Mazzoni, Zarov, Rambourg, & Mann, 1993; Zarzov, Mazzoni, & Mann, 1996). Activation of this pathway is particularly important in the response to several external stresses, including high temperature, low osmolarity, and cell wall disruption (Heinisch, Lorberg, Schmitz, & Jacoby, 1999). Mutations in different components of this pathway render the cells prone to lysis at elevated temperatures due to a weakening of the cell wall, a phenotype that can be overcome by growth on media with an osmotic support (Levin, Bowers, Chen, Kamada, & Watanabe, 1994). The study of lytic mutants is also a good approach for analysing the mechanisms that coordinate cell integrity with other cellular events during the cell cycle. Thus, in a screening for temperature-sensitive lytic mutants, Cabib and Durán (1975) isolated the lyt1 mutant. At the restrictive temperature, strains bearing this mutation display cell lysis, defects in polarity, and a cdc-like phenotype (Molero et al., 1993). This mutation was later identified as a thermosensitive allele of CDC15 (Jiménez et al., 1998), which encodes a protein kinase that functions in late steps of the cell cycle and is part of the mitotic exit network (MEN),

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a signalling network required for exit from mitosis (for reviews, see Bardin & Amon, 2001; McCollum & Gould, 2001). Mitotic cell division requires the coordination of a number of cellular events to achieve the efficient inheritance of a complete set of chromosomes by each of the two progeny cells. In S. cerevisiae, commitment to a new round of cell division occurs at G1 at a key regulatory point known as START. Bud growth and spindle formation by duplication of the spindle pole body (SPB) are both initiated at START. Bud formation and growth require the addition of new plasma membrane and cell wall at specific points of the cell surface, and this process is coordinated with other events of the cell cycle, such as nuclear division and segregation, in order to ensure similar contents in both mother and daughter cells. Microtubules are organised around the SPB to form the spindle, to which replicated chromosomes are attached prior to segregation into the mother and daughter cells during mitosis. Failure in either bud growth or spindle formation ultimately results in arrest of the cell cycle late in the G2 phase through the action of checkpoints, which monitor both cellular morphogenesis and mitotic spindle formation (Gardner & Burke, 2000; Lew, 2000). In budding yeast, the SPB is a three layer structure embedded in the nuclear envelope. It contains an inner plaque that nucleates the nuclear microtubules and an outer plaque that organizes the cytoplasmic microtubules (Byers & Goetsch, 1975). Many of the components of the SPB have now been identified and localized within this structure (Adams & Kilmartin, 1999; Wigge et al., 1998). Compelling evidence is recently accumulating to suggest that SPB function and duplication is directly coordinated with cell morphology and cell integrity. Thus, mutations in SPC110, which encodes an essential protein located at the nuclear side of the SPB, result in cells that are prone to lysis due to defects in maintaining cell integrity (Stirling & Stark, 2000). Similar results have been found for other components of this structure, such as Cdc31p. This protein is the yeast homolog of centrin, a highly conserved calcium-binding protein of the calmodulin superfamily that is essential for SPB duplication. Mutations in CDC31 block the earliest stage of SPB duplication, i.e., the formation of the satellite precursor (Baum, Furlong, & Byers, 1986; Byers, 1981). In addition to SPB duplication, Cdc31p also plays a role in mor-

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phogenesis and cell integrity via its interaction with the protein kinase Kic1p (Sullivan, Biggins, & Rose, 1998). Mutations in the kinase domain of Kic1p and certain cdc31 alleles result in abnormal bud morphology and cell lysis, suggesting that Cdc31p and Kic1p may also play a role in this process. Furthermore, overexpression of PKC1 or SLG1/WSC1 (which encodes a plasma membrane protein that sends signals to the Pkc1p pathway) has been shown to partially rescue the temperature sensitivity of several cdc31 alleles and other SPB duplication mutants (such as kar1, rad23 or dsk2), suggesting that the Pkc1p pathway might also be involved in the regulation of SPB duplication (Khalfan, Ivanovska, & Rose, 2000). Here, we describe the isolation of new thermosensitive lytic mutants and the molecular characterization of one of them as a new allele of NUD1. This gene codes for a component of the outer plaque of the SPB (Adams & Kilmartin, 1999; Wigge et al., 1998), which also participates in regulating mitotic exit through the MEN (Gruneberg, Campbell, Simpson, Grindlay, & Schiebel, 2000). Our results have allowed the identification of a single amino acid change in this protein (the nud1-G585E allele), and demonstrate that this mutation leads to cell cycle arrest and, with prolonged incubation times, to cell lysis. 2. Materials and methods 2.1. Strains, media and growth conditions Escherichia coli strains DH5␣, DH10B and XL1-Blue were used as hosts for the maintenance and propagation of plasmids. Bacterial cultures were grown in LB medium (Sambrook, Fritsch, & Maniatis, 1989). When required, media were supplemented with ampicillin (100 ␮g/ml). The yeast strains used in this study are listed in Table 1. Yeast cells were routinely grown in either YEPD or synthetic complete (SC) medium. Ura+ or Trp+ selections were carried out on SC medium without uracil or tryptophan, respectively. When required, glucose was replaced by a mixture of galactose (2%) and raffinose (1%) as carbon sources. Standard yeast media were used to sporulate diploids and to score nutritional markers in auxotrophic strains (Sherman, Fink, & Hicks, 1986).

2.2. Mutagenesis and selection procedures For mutagenesis, exponentially growing S. cerevisiae AH22 cells were suspended in 100 mM phosphate buffer, pH 7.5, at a concentration of 1×108 cells per ml; to 3 ml of this suspension, 1.15 ml of ethyl methane sulfonate (EMS) was added and, after shaking at 30 ◦ C for 60 min in a water bath, the mutagen was inactivated by the addition of excess 5% sodium thiosulphate. The cells were washed, plated on YEPD, and incubated for two days at 25 ◦ C. Assessment of cell lysis at the non-permissive temperature was accomplished by screening for the release of an intracellular enzyme, i.e. alkaline phosphatase. This was carried out using the method described previously (Cabib & Durán, 1975), consisting of overlaying the plates with an assay solution containing 0.5 M glycine–NaOH buffer (pH 9.7), 0.5% agar, and 10 mM sodium p-nitrophenylphosphate. In this test, colonies containing lysed cells were able to hydrolyse the chromogenic phosphatase substrate, and acquired a yellow colour with a yellow halo gradually spreading out from them. Flow cytometry was used to monitor the viability of mutant strains subjected to restrictive conditions essentially as described by de la Fuente et al. (1992). Aliquots from the desired cultures were adjusted to a final concentration of 20 ␮g of propidium iodide per ml, and analyzed on a FACSort flow cytometer (Becton Dickinson) to discriminate viable and non-viable cells. Viable cells gave only background staining, whereas non-viable cells (lysed) cells were intensely stained by propidium iodide, as indicated by a shift of the peak to more intense fluorescence. 2.3. Plasmids, cosmids, and YACs The plasmids generated in the present work are listed in Table 2. They were constructed using the YEplac195 (Gietz & Sugino, 1988), pRS316 (Sikorski & Hieter, 1989), pRS416 or pRS426 vectors (Christianson, Sikorski, Dante, Shero, & Hieter, 1992). Plasmid p30.8.1.2, carrying the wild-type allele of the NUD1 gene, was kindly provided by Dr. J. Kilmartin (MRC, Cambridge). This plasmid, referred to here as pNUD1, contains a DNA fragment (from 1330 bp before the NUD1 ATG to 1514 bp after the stop codon) cloned in the pRS316 vector between the

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Table 1 S. cerevisiae strains used in this study Strain B-7588 B-7170 B-7171 B-7589 B-7590 B-7591 B-7173 B-7174 B-7175 B-7593 B-7178 B-7595 B-7255 B-7596 B-7180 B-7598 Y01595 Y01634 Y01654 Y01677 Y01681 Y11671 Y11672 Y11673 Y11674 AH22 TD29 W303-1A A3617C F137 1-4B 6-4D EF-1B FF-5A FFT-1A FFT-1B FW-7B WIN303 WIN304 KCY2 YPH499 IAY520 IAY521 IAY522

Genotype

Origin chrI::URA3+

[cir0 ]

MATa ura3-52 leu2-3,112 trp1-289 met2 MATa ura3-52 leu2-3,112 trp1-289 his3-1 met2 CyhR chrII::URA3+ [cir0 ] MATa ura3-52 leu2-3,112 trp1-289 his3-1 met2 CyhR chrIII::URA3+ [cir0 ] MATa ura3-52 leu2-3,112 trp1-289 his3-1 met2 CyhR chrIV::URA3+ [cir0 ] MATa ura3-52 leu2-3,112 trp1-289 his3-1 met2 CyhR chrV::URA3+ [cir0 ] MATa ura3-52 leu2-3,112 trp1-289 his3-1 met2 CyhR chrVI::URA3+ [cir0 ] MATa ura3-52 leu2-3,112 trp1-289 his3-1 met2 CyhR chrVII::URA3+ [cir0 ] MATa ura3-52 leu2-3,112 trp1-289 his3-1 met2 CyhR chrVIII::URA3+ [cir0 ] MATa ura3-52 leu2-3,112 trp1-289 his3-1 met2 CyhR chrIX::URA3+ [cir0 ] MATa ura3-52 leu2-3,112 trp1-289 his3-1 met2 CyhR chrX::URA3+ [cir0 ] MATa ura3-52 leu2-3,112 trp1-289 his3-1 met2 CyhR chrXI::URA3+ [cir0 ] MATa ura3-52 leu2-3,112 trp1-289 met2 CyhR chrXII::URA3+ [cir0 ] MATa ura3-52 leu2-3,112 trp1-289 his3-1 met2 CyhR chrXIII::URA3+ [cir0 ] MATa ura3-52 leu2-3,112 trp1-289 his3-1 met2 CyhR chrXIV::URA3+ [cir0 ] MATa ura3-52 leu2-3,112 trp1-289 his3-1 met2 CyhR chrXV::URA3+ [cir0 ] MATa ura3-52 leu2-3,112 trp1-289 met2 CyhR chrXVI::URA3+ [cir0 ] MATa his31 leu20 ura30 bud7::kanMX4 MATa his31 leu20 ura30 tea1::kanMX4 MATa his31 leu20 ura30 grd19::kanMX4 MATa his31 leu20 ura30 YOR380w::kanMX4 MATa his31 leu20 ura30 fre5::kanMX4 MATα his31 leu20 ura30 ald4::kanMX4 MATα his31 leu20 ura30 gdh1::kanMX4 MATα his3∆1 leu2∆0 ura3∆0 YOR376w::kanMX4 MATα his31 leu20 ura30 atf1::kanMX4 MATa leu2-3,112 his4-519 MATα ura3-52 ade2 MATa ura3-1 leu2-3,112 his3-11 trp1-1 ade2-1 can1-100 MATa his3-532 MATa leu2-3 pha2 prt1 arg8 MATα leu2 his4 ts4 MATα ura3 ade2 ts4 MATa ura3 leu2 ts4 MATα ura3 his3 ts4 MATα ura3 ade2 ts4 MATa ura3 ade2 leu2 ts4 MATa ura3 his3 leu2 ade2-1 trp1-1 can1 ts4 MATa ura3-1 leu2-3,112 his3-11 trp1-1 ade2-1 can1-100 nud1::kanMX4 p341(nud1-G585E) MATa ura3-1 leu2-3,112 his3-11 trp1-1 ade2-1 can1-100 nud1::kanMX4 pNUD1(NUD1) MATa ura3-52 lys2-801 ade2-101 trp1-63 his3-200 leu2-1::pKC8(LEU2-nud1-2) nud1::kanMX4 MATa ura3-52 lys2-801 ade2-101 trp1-63 his3-200 leu2-1 MATa ura3-1 leu2-3,112 his3-11 trp1-1(TRP1 nud1-44) ade2-1 can1-100 nud1::Sphis5 MATa ura3-1 leu2-3,112 his3-11 trp1-1 (TRP1 nud1-52) ade2-1 can1-100 nud1::Sphis5 MATa ura3-1 leu2-3,112 his3-11 trp1-1 (TRP1 NUD1) ade2-1 can1-100 nud1::Sphis5

XhoI and XbaI sites of the polylinker. The plasmid is toxic for E. coli cells and elicits a severe slowing of colony growth (J. Kilmartin, personal comunication). Plasmid pF-1 was kindly provided by Dr. Sandra Ufano (University of Salamanca) and carries a BamHI insert containing the MRS6, GPE1 and NDD1 genes,

F. Sherman F. Sherman F. Sherman F. Sherman F. Sherman F. Sherman F. Sherman F. Sherman F. Sherman F. Sherman F. Sherman F. Sherman F. Sherman F. Sherman F. Sherman F. Sherman EUROSCARF EUROSCARF EUROSCARF EUROSCARF EUROSCARF EUROSCARF EUROSCARF EUROSCARF EUROSCARF F. del Rey F. del Rey F. del Rey F. del Rey M. Tamame This work This work This work This work This work This work This work This work This work E. Schiebel E. Schiebel J. Kilmartin J. Kilmartin J. Kilmartin

cloned in the high-copy vector YEp24 (Botstein et al., 1979). The cloning vector pYAC4c (Burke, Carle, & Olson, 1987) was kindly provided by Dr. Jose Luis Revuelta (University of Salamanca). Plasmids pYES-TEM1, pGAL1-LTE1, pGAL1-SPO12, pGAL1-CDC5 and pGAL1-CDC14 have been de-

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Table 2 Plasmids used in this study Plasmid

Description

pISA1 pISA2 pISA3 pISA4 pISA6 pISA7 pISA8 pEF1 pEF2 pEF3 pEF4 PEZ1 PEZ2 PEZ3 PEZ4 PEZ5 pF-1 pNUD1 p341 pYES-TEM1

YEplac195 with a PstI-SmaI fragment containing RPS27B, ECM12 and YHR022c YEplac195 with a PstI/SalI fragment containing MYO1 YEplac195 with a PstI/BamHI fragment containing the ECM12 and YHR022c genes YEplac195 with a BamHI/SmaI fragment containing the RPS27B gene pRS426 with a SpeI/SmaI fragment containing the gene RPS27B (obtained by PCR) pRS426 with a SpeI/SalI fragment containing the gene ECM12 (obtained by PCR) pRS426 with a SpeI/SalI fragment containing the gene YHR022c (obtained by PCR) pRS416 with a ClaI fragment containing YDR296w, SUR2 and ATP5 pRS416 with a PstI/PvuII fragment containing DPL1, YDR295c and YDR296w pRS416 with a PvuII insert containing the SSD1 gene pRS426 with a SpeI/SalI insert containing YDR295c, YDR296w, SUR2 and ATP5 pRS416 with a NotI/XhoI insert containing GDS1, YOR356w and GRD19 pRS416 with a XhoI/SalI insert containing HAP5, YOR359w, PDE2, PRT1 and PRE10 pRS416 with a SacI insert containing YOR356w, GRD19 and HAP5 pRS416 with a SacII fragment containing YOR366w, SCP1, RAD17, RPS12 and MRS6 pRS316 with a SnaBI/SacI insert containing PIP2, YOR364w, YOR365c and YOR366w YEp24 with a BamHI insert containing the MRS6, GPE1 and NDD1 genes (kindly provided by S. Ufano) pRS316 with a XhoI/XbaI insert containing the NUD1 gene (kindly provided by J. Kilmartin) pRS316 with a XhoI/XbaI insert containing the mutant gene nud1-G585E (obtained by gap-repair) pYES2 with a BamHI–SalI insert containing TEM1 under the control of the GAL1 promoter (kindly provided by E. Schiebel) p414-GAL1 plasmid with a 4.3 kb BamHI–XhoI insert containing the LTE1 gene (kindly provided by E. Schiebel) p414-GAL1 plasmid with a BclI–XhoI insert containing the SPO12 gene (kindly provided by E. Schiebel) pRS315 with a 2.6 Kb BamHI/XhoI insert containing the CDC14 gene (kindly provided by E. Schiebel) pRS304 carrying the CDC15 gene under the control of the GAL1 promoter (kindly provided by E. Schiebel) YIp5 containing a GAL1-SIC1 construct (kindly provided by A. Bueno)

pGAL-LTE1 pGAL1-SPO12 pCDC14 PGAL1-CDC15 pGAL1-SIC1

scribed previously (Gruneberg, Campbell, Simpson, Grindlay, & Schiebel, 2000) and were kindly provided by Dr. E. Schiebel (The Paterson Institute for Cancer Research, Manchester, UK). Plasmid pGAL1-SIC1 was kindly provided by Dr. Avelino Bueno (Centro de Investigación del Cáncer, Salamanca). Cosmids pEOA343, pEOA360, pEOA387 and pEOA434 containing DNA inserts of the distal region of chromosome XV were kindly provided by Dr. B. Dujon (Tettelin, Thierry, Goffeau, & Dujon, 1998). They were constructed by cloning Sau3A DNA fragments from chromosome XV at the BamHI site of the cosmid vector pWE15 (Thierry, Gaillon, Galibert, & Dujon, 1995). 2.4. Genetic methods Mating, sporulation, tetrad dissections, and tetrad analyses were performed as described previously (Sherman, Fink, & Hicks, 1986). Chromosomal as-

signment of the ts4 mutation was achieved following the 2 ␮m mapping method (Wakem & Sherman, 1990). Genetic map distances in centimorgans (cM) were calculated using the equation derived by Perkins (1949): Xp = 100(T + 6NPD)/2(PD + NPD + T). Because of the underestimation of map distances over 35 cM using the Perkins equation, when necessary Xp was converted into the more accurate estimation given by Xe (Ma & Mortimer, 1983), according to the formula Xe = (80.7Xp − 0.883Xp2 )/(83.3 − Xp ). 2.5. Manipulation of nucleic acids DNA manipulations were accomplished using standard techniques (Sambrook, Fritsch, & Maniatis, 1989). To introduce DNA into yeast cells, a modification of the lithium acetate procedure was followed (Gietz, Schiestl, Willems, & Woods, 1995). DNA sequencing was performed on an ABI PRISM 377 Sequencer (Applied Biosystems, Inc.), using specific

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oligonucleotide primers. Amplification of specific DNA fragments was achieved by Polymerase Chain Reaction (PCR) with specific oligonucleotide primers and different commercial kits or polymerases. 2.6. Indirect immunofluorescence Staining of microtubules was performed by indirect immunofluorescence as previously described (Bähler & Pringle, 1998), using the anti-tubulin antibody TAT-1. DNA was visualized after staining with 4,6-diamidino-2-phenylindole (DAPI). Samples were viewed and photographed using a Leica DMXRA microscope equipped with Nomarski optics and epifluorescence. Pictures were taken with a Photometrics Sensys CCD camera. 3. Results 3.1. Selection of osmotically remediable S. cerevisiae cell lysis mutants In order to improve our understanding of the factors involved in the osmotic stability of yeast cells, we attempted to isolate novel conditional S. cerevisiae cell lysis mutants. For this purpose, strain AH22 was mutagenized with EMS and suitable dilutions were spread onto YEPD plates, each plate receiving approximately 150 survivors. Cells were grown for two days at 25 ◦ C, transferred to 37 ◦ C overnight, and then overlaid with an alkaline phosphatase assay solution to test for cell lysis. Since alkaline phosphatase is an intracellular enzyme, cells that were permeable after incubation at the restrictive temperature were able to use the chromogenic substrate and the colonies acquired a yellow colour, whereas cells resistant to lysis at 37 ◦ C remained white. Out of approximately 20,000 colonies screened, 32 colonies turned yellow after the overlay assay, indicating that they represented potential mutants with defects in cell integrity under restrictive conditions. In order to ascertain whether these strains were defective in plasma membrane assembly or maintenance or, by contrast, they had defects in the assembly of the cell wall or cytoskeletal organization, the ability to grow at the restrictive temperature in the presence of a osmotic stabilizer (1 M sorbitol) was tested. Of the 32 mutants tested, 10 were able to form

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single colonies on the osmotically supported medium, and when they were outcrossed with strain TD29, the cell lysis phenotype, the temperature sensitivity, and the osmotic sensitivity cosegregated, indicating that all three characteristics were due to a mutation in a single gene. Complementation analyses showed that all mutations were recessive and defined four complementation groups: ts1 (four alleles), ts2 (two alleles), ts3 (two alleles) and ts4 (two alleles). Out of the four complementation groups, we chose ts4 in order to further characterize the mutation present in the two mutants obtained (mutants 1-4B and 6-4D), since the lytic phenotype was more severe in this group and the complementation by sorbitol was complete. The viability of the ts4 strain at the non-permissive temperature and the ability of the osmotic support to compensate the mutant phenotype were analysed by growth on liquid medium. Strain ts4 grew normally at 28 ◦ C on liquid medium, but when shifted to the restrictive temperature the cultures did not even double twice in optical density (data not shown). Addition of 1 M sorbitol to the medium allowed the cells to grow, although with a longer doubling time than that seen for the control wild-type strain (3.8 and 2.5 h, respectively; data not shown). 3.2. Complementation of the thermosensitive ts4 mutation using DNA libraries identifies two extragenic suppressors In an attempt to clone the gene responsible for the thermosensitive mutation present in ts4 mutants, strains EF-1B and FF-5A (derived from the 6-4D ts4 mutant) were transformed with low-copy and high-copy number gene libraries (based on YCp50 and YEp24 plasmids, respectively). Transformants were initially selected at the permissive temperature (28 ◦ C) and then replicated on SC-Ura and incubated at the restrictive temperature (37 ◦ C). Out of a total of about 950,000 transformants screened, 5 were consistently able to grow at the restrictive temperature: 4 of them were isolated from the YEp24-based library, and the fifth was from the centromeric gene bank. Plasmid DNA was isolated from the each of the five transformants and sequenced. Two different DNA regions of the yeast genome were present in the plasmids recovered. One of them (p13) was present in all four plasmids isolated from the YEp24 library and

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Fig. 1. Physical map and gene arrangement of the complementing DNA fragments. (A) A fragment from chromosome VIII containing the RPS27B, ECM12 and YHR022c genes and part of the MYO1 coding region was isolated from a YEp24 library for its ability to complement the thermosensitive growth defect of ts4 mutants. Different DNA fragments were subcloned (plasmids pISA1 to pISA8) and tested for complementation (right column). (B) A YCp50-based plasmid carrying a region of the right arm of chromosome IV (containing the SRP101, SSD1, DPL1, YDR295c, YDR296w SUR2 and ATP5 genes and part of the BFR2 ORF) complements the ts4 mutation. Subclones pEF1 to pEF4 were constructed to identify the complementing gene, and tested for their ability to complement the thermosensitive phenotype (right column).

the second one (p50-A) was from the YCp50 library. The insert present in the high-copy vector YEp24 matched the right arm of chromosome VIII between coordinates 146,700 to 154,500 and contained three different genes (RPS27B, ECM12 and YHR022c) and part of the MYO1 coding region (Fig. 1A). In turn, the DNA fragment able to complement the ts4 mutation contained in the single-copy vector YCp50 belonged to the right arm of chromosome IV (coordinates 1,044,470 to 1,061,029) and carried seven genes (SRP101, SSD1, DPL1, YDR295c, YDR296w SUR2 and ATP5) and part of the BFR2 ORF (Fig. 1B). In order to ascertain which of the genes present in each DNA fragment was able to complement the ts4 mu-

tation, different DNA fragments were tested for their ability to promote growth of the mutant strain EF-1B at 37 ◦ C. As shown in Fig. 1A, for the genes isolated from the high-copy vector YEp24, plasmids pISA1, pISA4 and pISA6 conferred a growth rate similar to that of the wild-type strain, while plasmids pISA2, pISA3, pISA7 and pISA8 were unable to complement the mutation. The common gene present in all complementing plasmids was RPS27B, which codes for a ribosomal protein of the small subunit and is nearly identical (99% identity) to the RPS27A gene located on chromosome XI. With regard to the insert present in the plasmid isolated from the YCp50 library (p50-A), the subcloning strategy indicated that only plasmid pEF3, carrying the SSD1 gene, was able to restore the wild-type phenotype (Fig. 1B). The SSD1 gene has been described as a single-copy or high-copy suppressor of multiple mutations, including mutations in SIT4, RPC31 and BCK1 (Costigan, Gehrung, & Snyder, 1992; Stettler et al., 1993; Sutton, Immanuel, & Arndt, 1991). These results indicated that the RPS27B and the SSD1 genes were able to fully complement the ts4 mutant phenotype, suggesting that one of them could correspond to the wild-type gene. However, genetic analysis indicated that neither of the two cloned genes corresponded to the wild-type gene responsible of the ts4 mutation (data not shown), and that they were extragenic suppressors of the mutation. 3.3. The ts4 mutation maps to chromosome XV, tightly linked to the FRE5 gene To identify the gene responsible for the ts4 mutant phenotype, a genetic approach was used. First, the 2 ␮m mapping method described by Wakem and Sherman (1990) was followed to identify the chromosome on which the ts4 mutation was located. Strain FFT-1A (cir+ ura3 ade2 ts4) was crossed with each of the 16 cir mapping strains (see Table 1 for genotypes) and the resulting diploids were grown in YEPD medium for several generations to promote aneuploidization before being plated on YEPD. Several hundred colonies from each diploid were then replica-plated on YEPD medium and tested for their ability to grow at the restrictive temperature. Although at low frequency, thermosensitive colonies were only found in diploids derived from the parental

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strain harbouring the 2 ␮m plasmid DNA integrated on chromosome XV, suggesting that the ts4 mutation is present in this chromosome. The ts4 mutation was subsequently mapped more precisely by conventional tetrad analysis. These studies included strains with reference markers at different positions on chromosome XV (arg8, ade2 and his3), and strains in which a selectable marker (the kanMX4 cassette, Wach, Brachat, Pohlmann, & Philippsen, 1994) had been used to replace the coding sequence of some genes (BUD7, TEA1, GRD19 and FRE5) located on this chromosome. Diploids constructed by crossing these strains with a ts4 mutant strain were sporulated and the tetrads were analysed for segregation of the thermosensitive phenotype and the different markers. From the results obtained, summarized in Table 3, a significant linkage was detected between the ts4 mutation and the reference markers tea1::kanMX4, grd19::kanMX4 and fre5::kanMX4, indicating a common positioning of the four loci on the right arm of chromosome XV. The segregation ratios revealed a close linkage between ts4 and FRE5: a majority of parental ditypes (34) and zero non-parental ditypes was found among the 37 tetrads analysed, suggesting a map distance of approximately 4 cM. In sum, by using a genetic approach the ts4 mutation was mapped to the terminal segment of the right arm of chromosome XV, close to the telomere, at about 4 cM from the FRE5 gene.

Table 3 Meiotic linkage analysis of chromosome XV markers Interval

ts4-ARG8 ts4-ADE2 ts4-HIS3 ts4-BUD7 ts4-TEA1 ts4-GRD19 ts4-FRE5

Crossa

IA-1 IA-1 IA-2 IA-2 IA-3 IA-4 IA-5 IA-6

Ascus type

Total

Map distance (cM)

PD

NPD

T

16 28

22 18

56 109

94 155

Unlinked Unlinkedb

13 6 14 14 34

7 1 1 0 0

41 14 10 9 3

61 21 25 23 37

Unlinkedb 51.6 32.0 19.5 4.0

a Parent strains for each cross were as follows (see Table 1 for genotypes). IA-1: FFT-1A × F137; IA-2: FFT-1A × A3617C; IA-3: FFT-1A × Y01595; IA-4: FFT-1A × Y01634; IA-5: FFT-1A × Y01654; IA-6: FFT-1A × Y01681. b Chi-square analysis indicated that the excess of parental ditypes over non-parental ditypes was not statistically significant.

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3.4. The ts4 mutation is a new mutant allele of the NUD1 gene Final identification of the gene responsible for the thermosensitive phenotype was achieved by a mixed approach, combining the use of YACs and the subcloning of different DNA fragments (obtained from cosmids) with a genetic strategy based on the construction of diploids with strains containing null mutations in different genes of the region. Since several different cosmids containing overlapping regions of the terminal portion of chromosome XV are available as a result of the genome sequencing project (Tettelin, Thierry, Goffeau, & Dujon, 1998), these were used to test for complementation. For this purpose, the different DNA inserts present in cosmids pEOA387, pEOA360, pEOA343 and pEOA434 (Fig. 2) were independently transferred to YACs (Burke, Carle, & Olson, 1987) and introduced into the FW-7B mutant strain. When the different yeast strains carrying the artificial chromosomes were tested for growth at the restrictive temperature, only those containing the YAC with the insert from cosmid pEOA343 were able to grow at 37 ◦ C. This was taken as an indication that one of the genes in the YOR359w–YOR379c interval was responsible for complementation. Furthermore, the lack of complementation in strains carrying the DNA fragments derived from pEOA360 and pEOA434, which largely overlap with pEOA343 (Fig. 2), reduced the possibility to the four genes (NUD1, ALD4, GDH1 and YOR376w) present in the region between NDD1 (the last gene whose ORF is completely included in pEOA360) and ATF1 (the first gene present in pEOA434). To test which of them complemented the ts4 mutation, the region present in cosmid pEOA343 was subcloned in smaller fragments into conventional plasmids, and the ability of each of them to complement the thermosensitive defect was tested (Fig. 2). Plasmids pEZ1 to pEZ5, pNUD1 and pF-1 were introduced into strain FW-7B and the transformants were grown at 37 ◦ C. The results of this complementation test showed that only pNUD1 was able to complement the mutant phenotype. Additional confirmation of this result was obtained by crossing strains carrying marked alleles of some of the genes with the mutant strains FFT-1A or FFT-1B and testing the resulting diploids for complementation. Wild-type growth

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Fig. 2. Physical map of the distal region of chromosome XV. The genes present between coordinates 1,000,000 to 1,095,000 (indicated in kb at the left of each lane) are marked in black (in the Watson strand) or grey (in the Crick strand) rectangles. Solid lines below the chromosome show the regions contained in each cosmid. DNA regions subcloned to test for their ability to complement the thermosensitive ts4 mutation are indicated by dashed lines. Genes that were tested by genetic crossing with marked strains are indicated by triangles.

was detected for diploids constructed by crossing the ts4 mutant with marked versions of the ALD4, GDH1, YOR376w, ATF1 and YOR380w genes (Fig. 2), confirming that none of them was the wild-type corresponding to the ts4 mutation. Thus, all these results indicate that the ts4 mutation is indeed a new thermosensitive allele of the NUD1 gene. 3.5. The ts4 mutant allele is a single amino acid change in a conserved region In order to identify the mutation responsible for the thermosensitive phenotype of strain FW-7B, the gap-repair technique was used (Rothstein, 1991). Plasmid DNA (p341) was isolated from several independent transformants and the complete coding region

of the mutated NUD1 gene was sequenced. A single base-pair substitution was detected in four of the independent plasmids sequenced; namely, a change of G to A at position 1754, resulting in a single amino acid mutation (a Gly-to-Glu change at position 585, which henceforth will be referred to as the nud1-G585E allele) in the third leucine-rich repeat (LRR) present in Nud1p (Fig. 3A). The same point mutation was found to be present in the original mutants (1-4B and 6-4D), as shown by PCR amplification and sequencing of the mutant alleles harboured by these two strains. To confirm that this single amino acid change was indeed sufficient to confer the mutant phenotype, strain FW-7B was transformed with plasmids pNUD1 and p341, containing the NUD1 or the nud1-G585E alleles, respectively. The transformants were grown in se-

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constructed a set of strains in which the only copy of the NUD1 gene (wild-type or mutant allele) was plasmid-borne. Because the NUD1 gene is essential for viability, null-mutations cannot be obtained. Thus, a wild-type strain (W303-1A) was transformed with plasmid pNUD1 (wild-type NUD1 gene) or p341 (nud1-G585E allele), and the chromosomal copy of NUD1 was deleted in these transformants by using a cassette carrying the kanMX4 marker, generating strains WIN304 (nud1, NUD1 plasmid-borne) and WIN303 (nud1, nud1-G585E plasmid-borne). The gene present in plasmid p341 is functional at the permissive temperature since the strains are viable in the absence of the chromosomal copy of NUD1. In addition, the WIN303 mutant strain had a phenotype similar to that of the original ts4 mutant; i.e., it was unable to grow at 37 ◦ C in the absence of an osmotic support, although this phenotype was recovered when the plates contained 1 M sorbitol (data not shown). 3.6. The nud1-G585E allele arrests cells late in anaphase

Fig. 3. The ts4 mutation is a new allele of the NUD1 gene. The mutation present in the ts4 mutant was recovered from the chromosome by the gap repair technique. (A) The NUD1 gene is represented as a white rectangle, grey boxes indicating the position of the leucine-rich repeats (LRR) characteristic of this protein. The nucleotide and protein sequences of the wild-type gene (NUD1) and of the nud1-G585E allele (nud1), which results in the thermosensitive ts4 mutation, are shown. (B) Growth of transformants at the restrictive temperature. The mutant strain FW-7B transformed with plasmids containing the wild-type NUD1 gene (pNUD1), the nud1-G585E allele (p341), or vector alone (pRS316) were streaked on YEPD plates and incubated at 37 ◦ C for 48 h.

lective media at the restrictive temperature and, as expected, the cells carrying the wild-type allele grew at the restrictive temperature while cells harbouring the nud1-G585E allele were unable to do so, suggesting that the plasmid-borne allele is unable to complement the chromosomal mutation (Fig. 3B). The results obtained suggested that the allele present in the p341 plasmid was not functional at 37 ◦ C, but failed to show whether it was functional at the permissive temperature since the chromosomal allele was also present. To confirm this point, we

To further characterize the phenotype of cells containing the nud1-G585E allele, the morphology of the cells was analysed during growth at the restrictive temperature. Most of the cells carrying the conditional allele arrested at 37 ◦ C as large-budded cells, with only a minor percentage of cells containing elongated buds, a phenotype similar to that found for cells containing the nud1-2 allele (Gruneberg, Campbell, Simpson, Grindlay, & Schiebel, 2000). Anti-tubulin immunofluorescence revealed that the phenotype of nud1-G585E cells was also similar to those containing the nud1-2 allele after 6 h of incubation at the restrictive temperature. Most of the large-budded cells (around 70%) had a long spindle and separated nuclei, while the remaining 30% of the cells displayed a failure in nuclear migration, with misoriented spindles that were retained in the mother cell (Fig. 4A). Interestingly, after 12 h of incubation at the restrictive temperature (when a large proportion of the cells show lysis, see below), the spindle had been almost completely disassembled in most of the large-budded cells (90% of the cells) (Fig. 4B). Mutant cells at the restrictive temperature were also stained with Calcofluor White (a dye that stains chitin, which is most abundant in the division septum that separates mother and daughter cells) and this revealed

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Fig. 4. Analysis of the phenotype of nud1-G585E cells. Wild-type (WIN304) and nud1-G585E mutants (WIN303) cells were grown to mid-log phase and then shifted to 37 ◦ C. Cells were stained with anti-tubulin antibody (TAT-1) and DAPI to visualize the DNA. Images of differential interference contrast (DIC), nuclear localization (DAPI), and microtubules (anti-tubulin) are shown. Left panels correspond to cells incubated for 6 h at the restrictive temperature, and right ones to cells grown for 12 h at the same temperature. Mother-localized spindles are indicated by arrowheads. Scale bars represent 4 ␮m.

that in mutant cells Calcofluor fluorescence was not only restricted to the neck region but was also spread all over the cell wall (Fig. 5), suggesting a defect in the cell wall. This is similar to what has been described for other mutants with defects in cell wall assembly, such as fks1 or gas1 (Garc´ıa-Rodr´ıguez et al., 2000; Valdivieso, Ferrario, Vai, Duran, & Popolo, 2000). It has been shown that Nud1p shows genetic interactions with the MEN pathway, and that the defect in nud1-2 cells can (at least partially) be over-

come by overexpression of some components of this pathway (Gruneberg, Campbell, Simpson, Grindlay, & Schiebel, 2000). To test whether the nud1-G585E allele showed similar interactions, strain FW-7B was transformed with plasmids carrying several genes under the control of the GAL1 promoter and the transformants were tested for growth at the restrictive temperature. The results, summarized in Table 4, indicated that nud1-G585E displays a similar set of genetic interactions to the nud1-2 allele, since the growth defect

Table 4 Suppression of nud1-G585E by components of the MEN Allele

nud1-G585E nud1-2

Growth at the restrictive temperature Vector

NUD1

GAL1-LTE1

GAL1-TEM1

GAL1-CDC5

GAL1-SPO12

CDC14

GAL1-SIC1

GAL1-CDC15

− −

++ ++

– −

– −

+/− +

+ ++

+/− +/−

++ ++

+/− +

nud1-2 (strain KCY2) or nud1-G858E (strain FW-7B) were transformed with the indicated plasmids. Transformants were streaked onto selective plates containing galactose as carbon source and incubated 5–7 days at the restrictive temperature (37 ◦ C). No growth defects were observed at the permissive temperature (28◦ C). (−) No growth; (+/−) very weak growth; (+) weak growth; (++) good growth.

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Fig. 5. nud1-G585E cells show abnormal chitin distribution. Wild-type (WIN304) and nud1-G585E mutants (WIN303) cells were grown to mid-log phase, shifted for 5 h to 37 ◦ C, and then stained with Calcofluor White. Pictures of differential interference contrast (DIC) and calcofluor-stained cells are shown. While staining in NUD1 cells is mainly restricted to the bud neck region (asterisk) and bud scars (arrowheads), nud1-G585E mutants show intense fluorescence around the cell, including the daughter cell that it is faintly stained in wild-type cells. Scale bars represent 4 ␮m.

was almost completely recovered by overexpression of the SIC1 inhibitor, but only partially by overexpression of SPO12, CDC14 and CDC5. 3.7. The nud1 mutant alleles cause cell lysis at restrictive temperature To confirm that the nud1-G585E allele also produced defects in cell integrity (the phenotype used to isolate the ts4 mutant), several experiments were performed. The growth of wild-type and mutants cells at the restrictive temperature was analysed in the presence and absence of osmotic stabilizers. As can be seen in Fig. 6A, strains containing the nud1-G585E allele were only able to grow at 37 ◦ C in the presence of sorbitol. This phenotype was also analysed in liquid medium. Under these conditions, the growth of both strains at the permissive temperature was indistinguishable, indicating that no defect can be detected un-

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der these conditions (data not shown). However, when the strains were transferred to the restrictive temperature, results similar to those found for the original ts4 mutant were observed. While the wild-type strain WIN304 grew normally in the absence of sorbitol in the medium, growth of the mutant strain WIN303 (nud1-G585E) ceased after 6 h of incubation in the absence of osmotic support (Fig. 6B). In the presence of sorbitol, the growth of both strains was fairly similar, indicating that the defect can be overcome by osmotic support. The viability of the WIN303 strain was further analysed by flow cytometry analysis. The results obtained (shown in Fig. 6C) indicated that the nud1-G585E allele also affected cell viability at 37 ◦ C since a large fraction of the cells (52%) were permeable to propidium iodide and showed intense staining after 12 h of incubation at the restrictive temperature in comparison with the isogenic WIN304 strain. Similar to the growth experiments, the addition of sorbitol to the culture medium resulted in a complementation of the defect, since the number of cells permeable to the fluorescent compound was similar to that of the wild-type control strain (0.9% of lysed cells versus 0.6%, data not shown). Thus, cells carrying the nud1-G585E allele do have defects in maintaining cell integrity, resulting in cell lysis during growth at restrictive temperature. To test whether other mutant alleles of the NUD1 gene isolated previously had a similar cell lysis phenotype, viability was measured by FACS analysis in strains carrying the nud1-2 (Gruneberg, Campbell, Simpson, Grindlay, & Schiebel, 2000), nud1-44 or nud1-52 (Adams & Kilmartin, 1999) alleles. In all three strains, cell integrity was also compromised to a level similar to that found for the nud1-G858E allele (Fig. 7), with a percentage of lysed cells ranging from 50% (nud1-52) to 90% (nud1-44 and nud1-2). In contrast to the results found for cells carrying the nud1-G585E allele, the addition of sorbitol to cultures of cells containing the other three alleles did not result in the complementation of the lysis defect (Fig. 7), since the number of cells permeable to the fluororogenic compound was similar with and without the osmotic stabilizer. This is in good agreement with the fact that cells carrying these three mutant alleles (nud1-2, nud1-44 or nud1-52) are unable to grow on plates containing sorbitol at the restrictive temperature (data not shown). Thus, the inability to

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Fig. 6. The nud1-G585E allele is responsible for the thermosensitive defect of ts4 cells. (A) WIN304 (NUD1) and WIN303 (nud1-G585E) strains were streaked onto YEPD or YEPD supplemented with sorbitol and the plates were incubated for 1–2 days at 37 ◦ C. (B) Growth of the S. cerevisiae WIN303 strain in liquid medium (YEPD) at the non-permissive temperature (37 ◦ C) in the presence (black squares) or in the absence (white squares) of an osmotic stabilizer (1 M sorbitol). The isogenic wild-type strain WIN304 used as control was grown in the absence of the osmotic stabilizer (black circles) or in the presence of 1 M sorbitol (white circles). Growth was measured by optical density (OD) determinations at 600 nm. (C) Flow cytometry analysis. Strains WIN303 and WIN304 were grown in YEPD medium at 28 ◦ C, shifted to 37 ◦ C, and incubated in YEPD medium without osmotic support or with 1 M sorbitol. After 24 h at the restrictive temperature, aliquots were taken from the cultures, adjusted to 20 ␮g of propidium iodide, and analyzed on a FACSort. Grey areas, cells incubated in medium without sorbitol; black lines, fluorescent signals of cells incubated in medium supplemented with sorbitol; Cells rescued by the osmotic stabilizer show less-intense fluorescent staining (left peaks) than lysed cells (right peaks). Permeable cells peak at a position indicating more intense staining with propidium iodide than intact cells.

maintain cell integrity during growth at restrictive temperature is shared by strains carrying different NUD1 mutant alleles, but the complementation of this defect by sorbitol is specific to the nud1-G585E allele.

4. Discussion Lytic mutants can be used to identify genes that are essential for cell growth and viability. In order to gain

a better understanding of the factors involved in the stability of yeast cells, we undertook the isolation of new mutants with defects in this process. A collection of temperature-sensitive S. cerevisiae mutant strains was screened at the restrictive temperature using a plate overlay assay, alkaline phosphatase activity being detected. The colonies that showed permeability (or lysis) at the restrictive temperature were reanalysed on plates containing 1 M sorbitol as an osmotic stabilizer. Only the cell lysis mutants that could be rescued on the stabilized medium were chosen for further genetic

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Fig. 7. nud1 mutants lyse at the restrictive temperature. Strains WIN303 (nud1-G585E), KCY2 (nud1-2), IAY520 (nud1-44) and IAY521 (nud1-52) were grown in YEPD medium at 28 ◦ C and shifted to 37 ◦ C in YEPD medium with and without sorbitol 1 M. After 12 h at the restrictive temperature, aliquots were taken from the cultures, adjusted to 20 ␮g of propidium iodide, and analysed on a FACSort. Black areas show the fluorescent signals of the different mutant cells grown in medium without sorbitol (middle panels). Grey areas show the fluorescent signals of the same strains grown in the presence of sorbitol (bottom panels). The fluorescence of the corresponding wild-type parental strain (WIN304, YPH499 and IAY522) grown under the same conditions in the absence of sorbitol is shown as white areas in the top panels.

analysis. With this approach, 10 mutants were isolated, falling into four complementation groups. We decided to analyse the mutants in the ts4 complementation group in further detail because the defect seemed to have been successfully rescued by the presence of sorbitol. The conventional approach to complementing the thermosensitive phenotype using gene libraries had failed completely, and in several different attempts only two suppressors were identified. The reason for this failure was understood when the wild-type gene was identified as the NUD1 gene. This protien is toxic for E. coli cells and elicits a very slow growth phenotype, and it may therefore be under-represented in the gene banks used. Two different extragenic suppressors were isolated during the cloning approach: the RPS27B and the SSD1 genes. The SSD1 gene was originally characterized as a gene able to suppress the sit4 mutation, which is defective in a protein phosphatase subunit

(Sutton, Immanuel, & Arndt, 1991). In other cases, SSD1 has been isolated as a single-copy suppressor of mutations in several unrelated genes, such as RPC31, which encodes a subunit of RNA polymerase III (Stettler et al., 1993), PDE2, encoding cyclic AMP phosphodiesterase (Wilson et al., 1991); BCK1, encoding a mitogen-activated protein kinase kinase kinase (Costigan, Gehrung, & Snyder, 1992), and MPK1, encoding a mitogen-activated protein kinase (Lee et al., 1993). Thus, it seems that SSD1 is involved in many systems, including the suppression of a thermosensitive mutation in the NUD1 gene. Sutton and co-workers (Sutton, Immanuel, & Arndt, 1991) reported the presence of two different alleles of this gene, one designated ssd1-d (dead) and the other SSD1-V (viable). Ssd1p was identified as a 160 kDa protein able to associate with RNA (preferentially polyA+ and single-stranded) (Stettler et al., 1993; Uesono, Toh-e, & Kikuchi, 1997). Because

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the 160 kDa protein is present in strains having the SSD1-V version but cannot be detected in strains carrying the ssd1-d allele, it was proposed that SSD1-V would be the wild-type gene and that ssd1-d would be a defective or mutant allele (Uesono, Toh-e, & Kikuchi, 1997). Thus, the most plausible explanation for the single-copy complementation of the thermosensitive phenotype of ts4 mutants would be that the strain used to clone the gene would contain the ssd1-d allele, while the gene bank used would have been constructed from a strain containing the wild-type allele (SSD1-V). Currently, we have no explanation for the observed suppression of the ts4 mutant phenotype by the ribosomal protein, encoded by the RPS27B gene. Final identification of the mutant gene in the ts4 strain was achieved by using a mixed approach and led to the identification of the NUD1 as the wild-type gene. Nud1p is a component of the outer plaque of the SPB, the structure that controls the assembly of microtubules (Adams & Kilmartin, 1999; Wigge et al., 1998). Although most of the components of the SPB are coiled-coil proteins (Wigge et al., 1998), Nud1p is a LRR protein and has up to nine repeats of the XLX2 LNLSXNXaX2 aX2 aX2 a consensus sequence, where X is any amino acid and a is normally aliphatic. LRR motifs have been found in functionally diverse proteins, and constitute an important superfamily involved in protein-protein interactions or signal transduction pathways (Kobe & Deisenhofer, 1994). Nud1p fulfils two distinct functions during the yeast cell cycle: first, it has a non-essential function, being required for the attachment of cytoplasmic microtubules to the outer plaque of the SPB during the S, G2 and M phases and, second, it is required for cells to exit mitosis. This function is essential for cell cycle progression (Adams & Kilmartin, 1999; Gruneberg, Campbell, Simpson, Grindlay, & Schiebel, 2000). Nud1p is also a component of the mitotic exit network (MEN) and may function as a scaffold for regulating or facilitating the binding of Tem1p to Cdc15p, thereby enhancing the transduction of the signal that triggers the late events of the cell cycle (Gruneberg, Campbell, Simpson, Grindlay, & Schiebel, 2000). Three thermosensitive alleles of NUD1 have previously been isolated and named nud1-2 (Gruneberg, Campbell, Simpson, Grindlay, & Schiebel, 2000), nud1-44 and nud1-52 (Adams & Kilmartin, 1999).

The nud1-2 mutation carries five amino acid changes (Q6E, S224I, G316C, L548H and L683F), only one of which is found in LRR2. The nud1-44 variant contains nine amino acid substitutions (Q96L, E127V, I418V, S419P, V504G, S534C, N600I, N613D and I633F), three of which are located in several LRRs (V504G in LRR-1, N600I in LRR-4 and I633F in LRR-5). The nud1-52 allele contains only two amino acid changes (I353F and Y696N), neither of which is found in LRRs, suggesting that other regions of the protein are also important for function. The allele isolated here (named nud1-G585E) is the first single amino acid change resulting in a thermosensitive phenotype to be reported. This indicates that LRR3 is important for Nud1p function, perhaps for binding to other proteins of the SPB or components of the MEN. It has been shown that nud1-2 cells arrest late in anaphase as large-budded cells (Gruneberg, Campbell, Simpson, Grindlay, & Schiebel, 2000) and that this defect would be due to the inability of the mutant protein to activate MEN components. The data reported here suggest that cells carrying the nud1-G585E allele show similar phenotypes to those of previously described alleles of this gene, since they arrest as large-budded cells with elongated spindles and duplicated nuclei. The protein containing this single amino acid change also shows similar genetic interactions with other components of the MEN to those described for nud1-2 (Gruneberg, Campbell, Simpson, Grindlay, & Schiebel, 2000). In addition, our results indicate that different mutations in NUD1 also affect cell integrity, since cells carrying four different mutant alleles lysed and became permeable to propidium iodide. This result is not surprising, because it has previously been reported that mutations in SPB components or in proteins of the MEN pathway also result in cells that become more prone to cell lysis. This is the case of mutations in SPC110, which encodes a protein located on the nuclear side of the SPB, or in CDC31, the yeast homolog of centrin (Stirling & Stark, 2000; Sullivan, Biggins, & Rose, 1998). Furthermore, a functional interaction between Cdc31p and the components of the Pkc1p cell integrity pathway has been proposed (Khalfan, Ivanovska, & Rose, 2000). These authors suggested that the Pkc1p pathway regulates SPB duplication through phosphorylation of Spc110p, and that this interaction is important

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for coordinating bud emergence with other events in the cell cycle. It has also been reported that mutations in CDC15, a protein kinase that is part of the MEN, result in cell lysis (Jiménez et al., 1998). Our results show that mutations in other components of the SPB, such as Nud1p, also result in a similar cell lysis phenotype. Although Nud1p does not directly interact with Cdc31p or Spc110p, it is possible that the lysis phenotype could be due to the same pathway proposed for cdc31 mutants; i.e., through the protein kinase Kic1p (Sullivan, Biggins, & Rose, 1998). Alternatively, the inability to maintain cellular integrity could be related to the proposed function for Nud1p in activating the MEN, in particular to its interaction with Tem1p and Cdc15p (Gruneberg, Campbell, Simpson, Grindlay, & Schiebel, 2000). Future experiments will address how this single amino acid change in Nud1p affects the protein and its interactions with the cellular integrity pathway. It is interesting to note that the cell cycle arrest that occurs when nud1-G585E mutant strains are grown at the restrictive temperature can be overcome by the presence of sorbitol in the culture medium, the cells being able to grow in both solid and liquid medium, but that this does not occur in the other three nud1 alleles tested. This difference could be due to the fact that the selection method used in our screening was based in the identification of lytic mutants remedied by sorbitol, and suggests that the cell integrity defect may be a secondary defect due to prolonged arrest of the cell cycle at a particular stage but, perhaps not directly related to cell wall integrity. Protection by sorbitol is usually interpreted as compensation of a cell wall defect. However, remedy of a cell wall defect would not explain the complementation of the cell cycle arrest. It is well documented that yeasts have developed mechanisms to become adapted, within certain limits, to high external osmolarity, aimed at maintaining cellular activity. The best-understood osmoresponsive system is the HOG pathway, whose activation enhances the capacity of cells to produce chemically inert osmolytes, such as glycerol, for protection purposes (Hohmann, 2002). In this context, one possibility is that the accumulation of cytoplasmic glycerol, which would occur when the cells are grown in a hyperosmotic medium (1 M sorbitol), would stabilize the mutant Nud1-G585Ep protein and enable it to perform, at least partially, its normal function. The

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other three alleles, which contain several amino acid changes in the sequence, might be more impaired in their function and thus, are not complemented by the presence of sorbitol.

Acknowledgements We are grateful to A. Bueno, B. Dujon, J. Kilmartin, J.L. Revuelta, E. Schiebel, F. Sherman, M. Tamame and S. Ufano for providing strains, plasmids and cosmids. We thank Mar´ıa Sacristán for advice on immunofluorescence, Javier Jiménez for useful comments and suggestions, and Nick Skinner for supervising the English version of the manuscript. This research was supported by Grants from the Comisión Interministerial de Ciencia y Tecnolog´ıa (BIO96-1413-C02-02 and IFD97-1897-C02-02) and from the European Community (CIPA-CT93-0117). I. Alexandar was a recipient of a fellowship from UNESCO-MCBN (fellowship no. 534).

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