In Saccharomyces cerevisiae Ras proteins control the activity of the adenylate cyclase, coded by CDC35/CYR1 gene(1) . cAMP is a pleiotropic signaling molecule that regulates through pKA activation, carbohydrate and nitrogen metabolism, transcriptional repression of several heat shock genes(2, 3, 4, 5) , and cellular processes such growth(6, 7) , resting state, and sporulation. The activation of Ras, transition from the inactive GDP complex to the active GTP complex, is triggered by guanine nucleotide exchange factors (GEF). ()Two such GEF have been identified in S. cerevisiae, Cdc25p and Sdc25p(8, 9, 10) . Cdc25p appears to be the determinant GEF for ras activation during growth. Although several observations suggest that cAMP is produced in response to nutritional variations such as glucose addition to glucose starved cells, little is known about the molecular basis of nutrient sensing leading to Ras activation. Since the variation of cAMP in response to glucose is lost in cells containing the RAS2 mutation (11) which bypass the need for GEF, Cdc25p is thought to be a key element in the control of ras activity. Indeed, the homologous GEFs found in higher multicellular organisms such as Drosophila and mammals have been shown to relay extracellular signalization(12) .

The GEF activity of Cdc25p could be regulated either by a change in activity, by a quantitative change in protein content and/or a change in subcellular protein localization. The cellular content of Cdc25p is known to be quite low, the product of the non amplified gene has been very hard to detect even with good antibodies. Moreover, it has been shown that Cdc25p can be completely sequestered by a dominant negative mutant form of Ras2p (RAS2). If the cellular level of Cdc25p is maintained at a limiting amount within the cell by a fast destruction rate, its control becomes obvious target for regulation of the Ras-cAMP signaling pathway. The identification of several common motifs between B cyclins and Cdc25p, one of which is a cyclin destruction box, point to such a mechanism(13) . To further assess the possibility that Cdc25p could be regulated in quantity rather then by an activation-inactivation mechanism, we have determined the degradation rate of this protein. We have discovered that Cdc25p is very unstable and contains a CDB motif involved in its degradation. In contrast to the cyclin B CDB motif, the Cdc25p CDB motif does not appear to be cell cycle regulated.

MATERIALS AND METHODS

Strains, Transformation, and Cells Growth

The S. cerevisiae strains used in this work are listed in Table 1. For yeast cells transformation, the modified lithium acetate method (16) was used. Yeasts were grown on minimal medium (0, 17% Yeast Nitrogen Base, 0, 5% ammonium sulfate, 2% glucose or 2% galactose + 3% raffinose) supplemented with 0,5% casein acid hydrolysate and 20 µg/ml adenine, 20 µg/ml tryptophan.

Plasmids

The plasmid pT1 was constructed by cloning a polymerase chain reaction amplified fragment of the CDC25 gene using as upper primer oligonucleotide 213 (5`-ACGCTCGAGCAAGGTGAATATTGGATAG-3`) containing a XhoI site and as lower P2 primer (5`-TAGCCTGCAGCCTAACTGTGTG-3`). A 1553bp fragment (codons 1-491 of CDC25 ORF) was amplified, cut with XhoI and EcoRV and inserted into the pKS vector linearized with the same enzymes resulting in the plasmid pT1.

An EcoRV-PvuII CDC25 gene fragment was inserted into the plasmid pT1 digested by EcoRV. The recombinant plasmid was named pT2. It contains the whole length CDC25 ORF.

To construct the plasmid pTK2, the 5285bp DNA fragment containing CDC25 gene was ligated to the vector pYEDP1/8-2 prepared as follow: the plasmid pYEDP1/8-2 was digested by EcoRI enzyme; protruding ends were filled by the ``Klenow enzyme'' in the presence of dATP and dTTP to get blunt ends, digested by BamHI endonuclease and filled partially by the ``Klenow enzyme'' in the presence of only dATP and dGTP. The insert was obtained from the pasmid pT2 by digestion with XhoI, partial filling by Klenow enzyme in the presence of dCTP and dTTP and SmaI enzyme digestion.

To obtain the pT3, site directed mutagenesis of the pT2 was carried out by the double primer method (19) using Transformer Site Directed Mutagenesis Kit (Clontech). A small, precise deletion of 129bp was generated. Two oligonuceotides were used in mutagenesis: mutagenic primer 075 (5`-GAGACAGTCATCTCTCTACTTTATCAGCGTC-3`- flanking deleted region) and selection primer 422 XbaI > NruI (5`-GATCCACTAGTTCGCGAGCGGCCGCCAC-3`). Mutagenesis was performed according to manufacturer protocol.

The pTK3 with pYEDP1/8-2 vector digested by BamHI enzyme, partially filled by the ``Klenow enzyme'' in the presence of only dATP and dGTP and cut by XmaI endonuclease. The 5154bp CDC25 fragment (containing the 129 bp deletion) was inserted into this vector. This DNA fragment was obtained from the plasmid pT3 by XhoI digestion, partial filling by the ``Klenow enzyme'' in the presence of dCTP and dTTP and XmaI digestion.

The plasmid pSE3/4 was constructed by cloning of the 387bp SalI2 - ClaI2 fragment (isolated from the plasmid 20V3/4) between the sites SalI and SmaI of the multicloning site of the vector Yep358R(20) . ClaI protruding ends of the insert were filled by the ``Klenow fragment'' of DNA polymerase I. Yep358R is a yeast/E. coli shuttle vector containing the yeast 2µ replication origin and the yeast URA3 marker to fuse yeast promoters with coding sequences to E. coli lacZ gene. The plasmid pSE3/4 contains codons 1 to 25 of the CDC25 coding sequence.

The plasmid pLG669Z contains the first codon of CYC1 fused to lacZ gene and the fused gene is preceded by about 1100 nucleotides of DNA that naturally precedes the CYC1 gene(21) .

Other plasmids were used in this work: the plasmid pGRS which contains COOH-terminal part of CDC25 - codons 877-1589 (construction based on the vector pYEDP1/60-2(17) , modified by G. Renault) and pGal25.6-1 (22, 23) which is a centromeric plasmid containing the CDC25 ORF under GAL1 promoter control.

Mini Muduction

Protein Extraction, Gel Electrophoresis, and Immunoblotting

Immunodetection

The antibodies A323 were raised against the COOH-terminal part of the Cdc25p (codons 877 to 1589 of the CDC25 gene) expressed in the strain E. coli 2097-2 from the plasmid pJM1039-25 (30) . Specific antibodies were purified from immune serum on nitrocellulose blots(31) . The Cdc25p immunoreactive bands were visualized by goat anti-rabbit IgG serum phosphatase alkaline conjugate (ProMega Biotech) and 5-bromo-4-chloro-3-indolyl-phosphate/nitro blue tetrazolium (BCIP/NBT) as substrates.

Quantification of the dye produced by alkaline phosphatase linked to anti-rabbit serum was done using the video analysis system including PerfectImage program (CIRX, France). For immunodetection of entire or truncated form of Cdc25p, the experiment was performed in duplicate.

Immunofluorescence Microscopy

-Galactosidase Assay

-Galactosidase activity was assayed in yeast cells permeabilized with chloroform as described previously (35, 36) except that SDS was omitted. -galactosidase activity was measured by the rate of orthonitrophenolgalactoside hydrolysis followed at A. One unit of -galactosidase is defined as the amount of enzyme that produces 1 nmole of ONP/min at 28°, at pH 7.

The yeast cultures have been arrested in G1, S and M phase to study a degradation of Cdc25p395--galactosidase hybrid protein at different phases of a cell cycle. The arrest of the strain CMY519 in G1 phase has been obtained by use of alpha factor at a final concentration of 1,75 mM. After 3 h of growth in the presence of alpha factor, cycloheximide was added (400 µg/ml) and -galactosidase activity of culture samples was assayed each 20 min after addition of protein synthesis inhibitor.

To obtain cultures arrested in phase S and M, the thermosensitive mutants of the cell cycle S. cerevisiae CMY616 and CMY620 were used. Cells were growth in 37° during 3 h. Thereafter cultures were supplemented with cycloheximide to block protein synthesis. -galactosidase activity of the culture was measured each 20 min.

Two independent experiments were performed in every case.

RESULTS

Cdc25p180 kDa Is an Unstable Polypeptide

As shown in Fig. 1A, the immunodectable amount of the 180 kDa polypeptide decreases rapidly when glucose is added to galactose growing cells. Densitometric analysis (Fig. 1B) allows the determination of a half-life of 20 ± 5 min. This experiment has been performed with antibodies reacting against either the amino-terminal (A6) or the carboxyl-terminal moiety of Cdc25p (A323), in both cases a similar half-life was observed. During the time course of glucose repression, two smaller polypeptides at a M = 100 kDa and M = 80 kDa were released and appear to be more stable than the complete 180 kDa polypeptide. These bands correspond to degradation products containing the carboxyl-terminal part of Cdc25p as they only react with the carboxyl-terminal specific antibodies. A similar experiment has been performed with a different plasmid, pTK2. This plasmid contains the ATG of the CDC25 ORF fused to the GAL10/CYC1 promoter and uses a 2µ origin of replication to give higher expression. The results obtained using pTK2 are similar to those found using pGal25.6-1 which is a centromeric plasmid (data not shown).

Figure 1: Time course of immunodetection of Cdc25p180 kDa. The yeast strain C13ABYS86 transformed by the plasmid pGal25.6-1 was grown on minimum medium (yeast nitrogen base) containing 2% galactose and 3% raffinose as carbon sources. In the exponential growth phase, 40% glucose was added (to a final concentration of 2%). Samples were taken at time 0 (before glucose addition), 15, 30, 45, 60, and 90 min after glucose addition. Yeast cells were collected, proteins were extracted, and analyzed by immunoblotting. Each slot was loaded with 40 µg of protein. Immunodetection was performed with A323 polyclonal antibody. A, lanes 1-6 represent 0, 15, 30, 45, 60, and 90 min, respectively, after glucose addition. B represents a densitometric analysis of the immunoblot. All immunodetection experients were performed in duplicate and similar results were obtained.

Stability of the C-terminal Region of Cdc25p

Figure 2: Time course of immunodetection of carboxyl-terminal moiety of the Cdc25p. The yeast strain C13ABYS86 transformed by the plasmid pGRS was grown as described in Fig. 1. 40% glucose was added to obtain a final concentration of 2%. Samples were taken at time 0 (before glucose addition), 15, 30, 60, 90, and 120 min after glucose addition. Yeasts cells were processed as described above. Protein extracts were analyzed by immunoblotting. 30 µg of protein was loaded in each slot. With A323 antibody a 76 kDa band was detected. A, lanes 1-6 represent 0, 15, 30, 60, 90, and 120 min, respectively, after glucose addition. Detected bands (carboxyl-terminal part of Cdc25p) are intense compared to the Cdc25p180 kDa (complete Cdc25p polypeptide). B represents a densitometric analysis of the immunoblot.

Involvement of a Cyclin Destruction Box (CDB)

To test the potential role of this element we have deleted 43 codons (132 to 174) encompassing the CDB and some surrounding amino acids from the complete ORF of CDC25 in pTK2 resulting in pTK3.

Stability of the product of the deleted gene Cdc25p175 kDa has been assessed as described above. The results presented in Fig. 3A show that Cdc25p175 kDa is more stable than Cdc25p180 kDa. It shows a slow decrease with half-life in the range 2,5 to 3 h (Fig. 3B).

Figure 3: Time course of immunodetection of Cdc25p175 kDa CDB. The yeast strain C13ABYS86 transformed by the plasmid pTK3 was used in this experiment. The experimental protocol was identical to that described in Fig. 1. 10 µg of protein was loaded in each slot. A, lanes 1-6 represent time 0, 15, 30, 45, 60, and 90 min, respectively, after glucose addition. The densitometric analysis of blots is presented in B.

Immunofluorescence Analysis

Figure 4: Immunofluorescent staining of cells overexpressing Cdc25p180 kDa. Four confocal scanning images of C13ABYS86 cells transformed with the plasmid pTK2 are presented in the figure. Antibody A323 and anti-rabbit fluorescein isothiocyanate-conjugated anti-serum were used, both in 1/100 dilution. Scale bar is 10 µm.

Figure 5: Immunofluorescent staining of cells overexpressing Cdc25p175 kDa. A series of confocal images of cells C13ABYS86 transformed with the plasmid pTK3 is shown in this figure. Antibody A323 was used as a specific antibody and anti-rabbit fluorescein isothiocyanate-conjugated anti-serum as secondary, both in 1/100 dilution. Scale bar is 10 µm.

Destabilizing Role of the CDB

As a control experiment we used the wild type strain CMY250 transformed with the plasmid pLG669Z(21) . This plasmid allows expression of -galactosidase in yeast from the cytochrome C promoter. Enzymatic activity was assayed at different times following the addition of cycloheximide. -galactosidase level remained stable through the experiment (more than 4, 5 h) (Fig. 6A).

Figure 6: -Galactosidase activity following cycloheximide addition. The yeast strain CMY250 transformed by either pLG669Z (A), pPI1 2B (B), and pSE3/4 (C) was grown on minimal medium containing glucose, casein acid hydrolysate, adenine, and tryptophan. The culture was divided on two parts. One part was treated with cycloheximide. 5-ml aliquots were taken at 0, 1.5, 3, and 4.5 h after cycloheximide addition. -Galactosidase assays were performed as described under ``Materials and Methods.'' -Galactosidase fusion degradation assays were repeated two times. D represent semilogarithmic plot of -galactosidase activity following cycloheximide addition.

The plasmid pPI1-2b contains a fusion between the 5` part of the CDC25 gene (-313bp to 1185bp) and lacZ gene. As above, -galactosidase activity was assayed every 1,5 h in the strain CMY250 after cycloheximide addition. The enzymatic activity decreases with time indicating that the fusion protein is degraded (Fig. 6B). The half-life of this chimeric protein can be estimated at 90 min.

A construction containing only the 23 first residues of the CDC25 ORF fused to -galactosidase was used in a similar experiment. -galactosidase activity is stable following cycloheximide addition for more than 4,5 h (Fig. 6C).

These results present evidence that a destabilizing element is present in the amino-terminal part of Cdc25p between residues 23 and 395.

Degradation of Fusion Protein during the Cell Cycle

After G1 arrest with alpha factor, -galactosidase activity decreases after cycloheximide addition (Fig. 7A) with an half-life of about 70 min.

Figure 7: Rate of degradation of Cdc25p395- galactosidase during cell cycle arrest. The plasmid pPI1-2B was used to transform the yeast strains CMY519 (A), CMY616-cdc8 thermosensitive mutant (B), and CMY620, cdc15 thermosensitive mutant (C). A, the yeast strain CMY519 transformed with the plasmid pPI1-2b was arrested in G1 by a factor for 3 h. In B and C, strains CMY616 and CMY620 were arrested by temperature shift to 37 °C for 3 h. Then culture was separated into two parts. One part was treated with cycloheximide (CHX). Samples of 5 ml were taken at time 0, 20, 40, 60, and 80 min after cycloheximide addition and -galactosidase activity was determined. Cell cycle experiments were performed in duplicate.

With S phase arrest obtained using a thermosensitive cdc8 mutant (42) kept 3 h at restrictive temperature, we observed after cycloheximide addition a degradation of the fusion protein with a half-life of 70 min (Fig. 7B).

In a similar experiment performed with a thermosensitive cdc15 mutant to obtain cells blocked at mitosis(43) , the -galactosidase activity was found to decrease after cycloheximide addition with a 50 min half-life (Fig. 7C).

DISCUSSION

Cdc25p has been shown to be the main positive regulator of Ras in Saccharomyces cerevisiae. As shown from previously reported experiments, the cellular content of this protein is very low. In this report we demonstrate that the low level of Cdc25p is due to its instability. When the CDC25 ORF is expressed from the GAL1 promoter, the protein product presents a fast decay after glucose repression. If we assume that the immunoreactive protein disappears following a first order kinetic we can estimate the half-life to approximately 20 min. This half-life is much more shorter than a doubling time of yeast cells suggesting that even when overexpressed, Cdc25p does not saturate the system of degradation and it is effectively degraded. Lai et al.(44) have reported that in contrast to the complete Cdc25p the truncated polypeptide containing the carboxyl terminus, codon 877 - 1589, was more abundant. In this report we confirm that the carboxyl-terminal part of Cdc25p expressed from GAL10 promoter is also more abundant and we have shown that it is very stable.

To check a possible role of the amino-terminal part of Cdc25p in the instability of Cdc25p180 kDa, we fused the 395 first codons to -galactosidase. We chose this strategy to measure directly the level of -galactosidase activity. The hybrid polypeptide was much more unstable than -galactosidase alone. Assuming a first order kinetic for protein degradation, the loss of -galactosidase activity following cycloheximide addition gives a half-life of 90 min compared to more than 20 h for -galactosidase itself, therefore the amino-terminal part of Cdc25p contains a destabilizing element. The difference in the degradation rates of Cdc25p180 kDa and the Cdc25p N-terminal/-galactosidase fusion might indicate that only part of the degradation determinants are present in the amino-terminal region of Cdc25p used in this fusion. However, it could also result from a differential accessibility of the active domain of the -galactosidase to proteolytic activity. Different foldings of these two polypeptides could also account for a difference in the rate of degradation. Despite these differences, it can be concluded that Cdc25p is an unstable protein which contains at least one destabilizing element which can be transferred to other proteins.

The region fused to -galactosidase contains the amino-terminal extremity of Cdc25p, thus it was possible that the instability of the fusion protein was due the N-end rule(45) . This hypothesis was ruled out by the result obtained with the fusion of the first 23 amino acids of Cdc25p, which does not change the degradation rate of -galactosidase.

The presence of the CDB close to the amino-terminal end of the molecule appears to be the key destabilizing element for Cdc25p. Three conserved sequence elements have been reported to be common between Cdc25p and cyclin B, the CDB being one of them(13) . All three elements are clustered in the amino-terminal part close to a SH3 domain (Fig. 8). The CDB motif has been shown to be responsible for the degradation of sea urchin and yeast mitotic cyclins(40) . This sequence is present at position 149 to 158 from the amino terminus. Deletion spanning from amino acid 132 to 174 leads to a quite stable Cdc25p175 kDa polypeptide with an half-life greater than 90 min. The presence of a CDB has been shown to cause the destabilization of proteins other than cyclins: Galan et al. have recently reported that the yeast uracil permease, which contains a CDB motif, was degraded with a short half-life and that a point mutation replacing a conserved arginine partially restores stability(46) . At least for cyclins the CDB motif appears to be recognized by the ubiquitin proteolytic system. Moreover the CDB of Cdc25p contains several potential phosphorylation sites, some for cAMP dependent protein kinase, and another one for cyclin dependent protein kinase, which is common with cyclins. Phosphorylation, dephosphorylation could regulate the accessibility to the proteolytic system.

Figure 8: Sequence similarity found in amino-terminal part of Cdc25p. A, Cm5 and Cm12 represent, respectively, regions homolog to cyclin motif 5 and cyclin motif 12. The SH3 homology region is placed between Cm5 and Cm12 regions. The putative ``cyclin destruction box'' of Cdc25p (CDB) has been found inside the cyclin motif 12. pKcdk and pKA represent putative target sites for, respectively, cyclin-dependent protein kinase and cAMP-dependent protein kinase. B presents similarity between the cyclin destruction box consensus and putative ``destruction box'' of Cdc25p.

The presence of the CDB has also been reported in Sdc25p, the other exchange factor of Ras in yeast(13) . The product of the SDC25 appears to be quite unstable and it is present at very low level within the cell, even overexpressed (P. Ikonomi and E. Boy-Marcotte unpublished). This is very interesting that in yeast, GEFs (Cdc25p and Sdc25p) are unstable polypeptides. The degradation of these proteins can be an unusual way to regulate the activity of Ras pathway in yeast. In mammalian Sos-Ras system, membrane recruitment of exchange factor seems to be the likely process of regulation of the mitotic signaling pathway(47) . In yeast, function of Ras is not connected to growth factor receptor. The activity of the Ras pathway could be controlled by proteolytic device degrading the exchange factor whose presence in necessary for action of the Ras pathway.

It was attractive to think that the presence in Cdc25p of three motifs common with cyclin B might led to a cell cycle dependent degradation. We have addressed this question with the -galactosidase fusion protein using alpha pheromone arrested cells or cdc mutants for synchronising cells at different stages of the cell cycle. The rate of degradation was followed by the loss of -galactosidase activity after cycloheximide addition. In every case, the decay of -galactosidase activity was similar to that of the non synchronised population. Although variations in the rate of degradation occurring at a different stage of the cell cycle than those examined here, cannot be ruled out, our results strongly argue for a constant degradation process throughout the cell cycle.

Recent results have shown that G2 cyclins degradation in yeast is not limited to a period as short as was originally thought, but extends from mitosis to the next onset of DNA synthesis(48) . Our results indicate that for the Cdc25p, CDB dependent destruction can occurs during the complete cell cycle. Although more subtle variations in degradation rate might have been missed in our experiments it seems that other determinants than those shared by cyclins and Cdc25p are required for the cell cycle dependent destruction. A constitutive destruction system for Cdc25p can lead to a cell cycle dependent activation of Ras only if Cdc25p synthesis occurs at a given period of the cell cycle. Further experiments are required to test this hypothesis.

FOOTNOTES

*

This work was supported by grants from the Ligue Nationale Française contre le Cancer and the Association pour la Recherche contre Le Cancer. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§

Supported by a fellowship from the Conseil Général de l'Essonne.

¶

To whom correspondence should be addressed. Tel.: 33-1-69417963; Fax: 33-1-69417296; JACQUET{at}IGMORS.U-PSUD.FR.

()

The abbreviations used are: GEF, guanine nucleotide exchange factor; CDB, cyclin destruction box; bp, base pair(s); ORF, open reading frame.

ACKNOWLEDGEMENTS

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