The previously described strains we used are W303-1B, W3031B/D (Nasmyth, 1985), C13ABYS86 (Heinemeyer et al., 1991), T141 (MATa, ura3-52, his3-11,15, leu2-3,112, lys2, ssa1::LEU2), MW123 (MAT a, ura3, his3, leu2, lys2, Δtrp1, ssa1::HIS3, ssa2::LEU2 (Werner-Washburne et al., 1987), ΔECU82a (MATa, ade2-1, ura3-1, his3-11,15, leu2-3,112, trp1-1, hsc82::LEU2, hsp82::LEU2).
The following strains have been constructed for the present work. OL565 (MATa, ura3, his3, leu2, lys2, trp1, ssa1::HIS3, ssa2::LEU2) was derived from a cross between MW123 and W303-1B/D. To construct the CDC25 deleted strains: (i) the RAS2ile152 allele (Camonis and Jacquet, 1988) contained within a Cla1–BamH1 fragment was cloned into the integration plasmid pRS304 (Sikorski and Hieter, 1989) to produce pGR113, this plasmid linearized with Pst1 was introduced into the diploid yeast strain FY1679 (Baudin et al., 1993) to give the trp+ OL549 strain; (ii) the CDC25 gene was replaced by the HIS3 gene amplified with two oligonucleotides corresponding to the 35 nucleotides upstream of the CDC25 ATG (at −26) and downstream of the stop codon (+48), respectively, extended by the HIS3 primers (Baudin et al., 1993); (iii) the resulting strain OL550 was sporulated and asci dissected. Among the progeny His+,Trp+ cells were recovered and analysed by PCR to confirm gene replacement. Two of these strains were used in this report: OL550–11A (MATα, ura3-52, his3-Δ200, leu2-Δ1, trp1-Δ63, cdc25::HIS3, RAS2-RAS2ile152::TRP1) and OL550-11B (MATa, ura3-52, his3-Δ200, leu2-Δ1, trp1-Δ63, cdc25::HIS3 RAS2-RAS2ile152:: TRP1).
The Δssa1, Δssa2 mutant containing the cdc25::HIS3, RAS2ile152 was obtained from a cross between the strain OL550-11B and OL565. One strain among the progeny, OL576-3C, was found to contain (i) the double ssa1 and ssa2 deletion, as judged from HIS3 and LEU2 segregation in the tetrad and the associated phenotypes: slow growth and thermosensitivity; (ii) the CDC25::HIS3 replacement from PCR analysis; and (iii) the presence of RAS2ile152 from TRP1 segregation and growth rescue of the cdc25 deletion.
The OL568-1C strain was derived from the diploid strain W303–1B by insertion of three tandem repeats encoding the influenza virus haemagglutinin epitope (HA) at the 5′ end of the CDC25 gene, between codons 4 and 5, using the method of Schneider et al. (1995). The 3xHA-URA3-3xHA cassette of the pMPy-3xHA plasmid was amplified by PCR, using ol25NHA up and ol25NHA down and was transferred to the W303-1B strain. Ura+ transformants with the CDC25 gene replaced were checked by PCR and plated onto 5-fluoro-orotic acid to select ura− cells. After sporulation and tetrad analysis, the haploid OL568-1C strain (ade2-1, ura3-1, his3-11, leu2-3, 112, trp1-1, CDC25:3xHA) was recovered. As checked by PCR, it contained three tandem repeats of the HA epitope. The MGL20-4C strain (ura3, lys2, trp1, ssa1::HIS3, ssa2::LEU2, CDC25:x3HA) was obtained from a cross between OL568-1C and OL565.
Plasmids used in this study are listed in Table 2. pTK3 was constructed by insertion of a three HA tag coding sequence within the Nhe1 site of pTK2, allowing expression of the full CDC25 gene under the GAL1-10 promoter (Kaplon and Jacquet, 1995). pEG-FS4 was obtained by insertion of a PCR fragment containing the last 148 codons of CDC25 in the SmaI–SalI sites of the pEG(KG) (Mitchell et al., 1993); this fragment was amplified with olYF1 and olYF2 on the plasmid pYFS4 with ol1690 and ol2218 and inserted into YeF1 (Cullin and Minvielle-Sebastia, 1994) by Not1 and EcoR1.
. Plasmids and oligonucleotides used.
The SSA1 gene was cloned by gap repair into the plasmid pGRSSA1 using the intergenic flip-flop procedure (Mallet and Jacquet, 1996). The SSA1 promoter sequence (from −579 to −108 of ATG) and terminator sequence (from +50 to +585 of the stop codon) were amplified using oligonucleotides ol600, ol1053 and ol3155, ol3657 respectively. The second PCR has been primed with ol600 and ol3657 and the two amplified fragments. The resulting fragment was cut with XhoI, and NheI was cloned into XhoI–SpeI sites of pRS314 (Sikorski and Hieter, 1989). The Sma1 linearized plasmid was transformed into W303 on medium lacking tryptophan. The plasmid pGRSSA1 complements the thermosensitivity and the reduced rate of growth of a Δssa1, Δssa2 mutant.
pGR103, expressing the PDE2 gene, was obtained by cloning a 3 kb BamHI–BglII fragment obtained from pW4 (Wilson and Tatchell, 1988) into the BamHI site of YEp352 (Hill et al., 1986).
Whole-cell protein extract, subcellular fractionation, GST fusion proteins purification and immunoprecipitation
Crude cell extracts and membrane fractions were prepared as previously described (Garreau et al., 1990) with the addition of 20 mM sodium molybdate in the lysis buffer. Solubilization of complete Cdc25p or fused protein was performed as described by Gross et al. (1992b) with 2 mM EDTA pH 12. GST fusion proteins were further purified by incubation of the neutralized supernatant with glutathione–agarose beads for 1 h at 4°C. After extensive washing in washing buffer (PBS, 0.5% Triton X-100, 0.25 M KCl), bound proteins were recovered by boiling the beads in 2× Laemmli sample buffer and subjected to SDS–PAGE. For GST control, the supernatant of the first centrifugation was directly incubated with glutathione–agarose beads.
Immunoprecipitation was performed by incubation of the neutralized supernatant from 50 ml of culture, at 4°C, 1 h with the antibody and then 1 h in the presence of protein A–Sepharose. Bound proteins were recovered after washing by boiling in 2× Laemmli sample buffer and subjected to SDS–PAGE. Immunoreactive bands were detected by goat anti-rabbit or anti-mouse IgG serum, alkaline phosphatase conjugate (ProMega) or peroxidase conjugate (Amersham).
Glycogen, trehalose, β-galactosidase and protein determinations
Glycogen and trehalose were measured as described (Parrou and François, 1997). β-Galactosidase was measured from cell extracts with the Ozyme Galacto-Light plus chemiluminescent reporter assay kit as recommended by the manufacturer. Protein concentrations of extracts were measured according to the method of Bradford.
Digestion and separation of peptides were performed by the Laboratoire de Microséquençage des Protéines of the Institut Pasteur (Paris) as described in Kawasaki and Suzuki (1990) with slight modifications: proteins separated on SDS polyacrylamide gel were stained with 0.03% Amido Black; the buffer used for digestion was 0.1 M Tris-HCl pH = 8.8, 0.03% SDS, an Aquapore AX300 (Brownlee) column was used to remove SDS and the peptides were separated on a C18 Vydac 218TP52 column. Amino acid sequences were determined with an Applied Biosystems Procise ABI 498 (PROCISE-HT).