Hélian Boucherie

High cAMP levels antagonize the reprogramming of gene expression that occurs at the diauxic shift in Saccharomyces cerevisiae.

In order to analyse the involvement of the cAMP pathway in the regulation of gene expression in Saccharomyces cerevisiae, we have examined the effect of cAMP on protein synthesis by using two-dimensional gel electrophoresis. cAMP had only a minor effect on the protein pattern of cells growing exponentially on glucose. However, it interfered with the changes in gene expression normally occurring upon glucose exhaustion in yeast cultures, maintaining a protein pattern typical of cells growing on glucose. This effect was accompanied by a delay before growth recovery on ethanol. We propose a model in which the cAMP-signalling pathway has a role in the maintenance of gene expression, rather than in the determination of a specific programme. A decrease of cAMP would then be required for metabolic transitions such as the diauxic phase.

Structure and expression of a yeast gene encoding the small heat-shock protein Hsp26

The nucleotide sequence of the Saccharomyces cerevisiae gene encoding a small heat-shock protein (Hsp26) has been determined. It reveals a 213-amino acid protein (27 kDa) that contains no methionine (Met) residues. Radiolabelling studies demonstrate the N-terminal Met residue is cleaved post-translationally. The Hsp26 amino acid sequence shows significant homology with both a range of eukaryotic small Hsps and with vertebrate alpha-crystallins. Particularly highly conserved among these proteins is a hydrophobic tetrapeptide sequence Gly-Val-Leu-Thr. These findings are discussed in relation to the structure and function of small Hsps.

Protoplasmic incompatibility and female organ formation in Podospora anserina: Properties of mutations abolishing both processes

SummaryA genetic block, effective only with the addition of mutations in two genes, suppresses both the incompatibility process in strain confrontation (“barrage”) and the formation of female organs. Investigations on this block lead us to propose that it operates at the ribosomal level and prevents the translation of the messenger of a proteolytic enzyme, a postulated early protein in protoperithecia differenciation.

Polypeptide synthesis during protoplasmic incompatibility in the fungus Podospora anserina

In Podospora anserina, self-lysis resulting from the combination of the R and V incompatibility genes is accompanied by the appearance, in lysing cells, of specific enzyme activities, among which is a laccase exoenzyme, and by a quenching of ribonucleic acid synthesis. Present results show that the occurrence of the laccase is the result of de novo synthesis. By means of two-dimensional gel electrophoresis it was shown that the onset of self-lysis is accompanied by the immediate shut-off of more than 60% of the pre-existing normal polypeptide synthesis and the occurrence of at least 20 new polypeptides. The synthesis of these new polypeptides is active for several hours after the cessation of RNA synthesis, concurrently with the synthesis of about 30 normal polypeptides which is maintained. These modifications of protein synthesis are not accompanied by a concomitant variation in the level of polysomes. It is deduced that incompatibility genes are involved in the control of both transcription and translation.

The molecular mechanism of protoplasmic incompatibility and its relationship to the formation of protoperithecia in Podospora anserina.

In Podospora anserina, protoplasmic incompatibility due to interactions between non-allelic genes was suppressed by the effect of mutations in two modifier genes, mod-I and mod-2. It is shown that mod-I and mod-2 are involved in the production of three specific proteins, a phenoloxidase and two previously identified proteases (Bégueret & Bernet 1973 a) which are associated with the phenomenon of protoplasmic disintegration. These enzymes, whose messengers are normallly latent during vegetative growth, appear at this stage of the life cycle only as a consequence of incompatible gene interactions. The mode-I and mod-2 genes and each of the five incompatibility loci involved in non-allelic incompatibility systems also participate in the formation of the protoperithecia. This pleiotropic effect suggests that protoplasmic incompatibility is a deviation in the normal physiological processes of protoperithecial formation.

Cloning and sequencing of the GMP synthetase-encoding gene of Saccharomyces cerevisiae

We have localised, within a Saccharomyces cerevisiae genomic fragment, the GUA1 gene whose amplification leads to the accumulation of several polypeptides on the two-dimensional (2-D) map of yeast proteins. Comparison of the sequence of the putative GUA1 protein with a data library shows a strong similarity with Escherichia coli, Bacillus subtilis and Dictyostelium discoideum GMP synthetases (GMPS) and other glutamine amidotransferases. The fact that disruption of the chromosomal copy of the gene leads to guanine auxotrophy, that the gual::URA3 disruption does not complement an independently obtained gual-3 mutation deficient in GMPS and that GUA1 complements this latter mutation, confirms the identification of the cloned gene as GUA1 encoding the S. cerevisiae GMPS. Finally, using microsequencing, we have identified one of the polypeptides, which is overproduced in response to GUA1 amplification, as corresponding to GUA1.

Intracellular and extracellular phenoloxidases in the fungus Podospora anserina: Effect of a constitutive mutation in a gene involved in their posttranscriptional control

SummaryThree phenoloxidases- and in particular phenoloxidase B, an enzyme found in cell extracts only in the cases of protoplasmic incompatibility and cell lysis where it occurs in association with a protease — were investigated in a wild type strain. Phenoloxidase B was present only in the culture filtrates, while the other two phenoloxidases were found in cell extracts and in the culture medium. Addition of casaminoacids and ammonium acetate to the culture medium produced opposite effects: under the first conditions, phenoloxidase B increased in being present only in the culture filtrates and in the second medium, like a second phenoloxidase (activity C), phenoloxidase B completely disappeared from the culture filtrates.A recessive mutation was isolated in a gene modB that resulted in the production of phenoloxidases B and C in higher amounts. The temperature dependence of the mutation modB allowed us to show that the formation of the phenoloxidases was 5 Fluorouracilresistant and cycloheximide sensitive, suggesting that constitutivity for these enzymes resulted from a derepression in their posttranscriptional control. Because of this derepression, phenoloxidase B not only increased in the culture medium (30x), but was found in cell extracts in association with a protease whose presence is suspected to produce a premature disintegration of the mycelium.The results are discussed from the point of view of the processes capable of regulating the production of exoenzymes and in relationship with the phenomenon of cell death normally affecting mycelia during ageing.

Skp1-Cullin-F-box-dependent Degradation of Aah1p Requires Its Interaction with the F-box Protein Saf1p

When yeast cells enter into quiescence in response to nutrient limitation, the adenine deaminase Aah1p is specifically degraded via a process requiring the F-box protein Saf1p and components of the Skp1-Cullin-F-box complex. In this paper, we show that Saf1p interacts with both Aah1p and Skp1p. Interaction with Skp1p, but not with Aah1p, requires the F-box domain of Saf1p. Based on deletion and point mutations, we further demonstrate that the F-box domain of Saf1p is critical for degradation of Aah1p. We also establish that overexpression of Saf1p in proliferating cells is sufficient to trigger the degradation of Aah1p. Using this property and a two-dimensional protein gel approach, we found that Saf1p has a small number of direct targets. Finally, we isolated and characterized several point mutations in Aah1p, which increase its stability during quiescence. The majority of the mutated residues are located in two distinct exposed regions in the Aah1p three-dimensional model structure. Two hybrid experiments strongly suggest that these domains are directly involved in interaction with Saf1p. Importantly, we obtained a mutation in Aah1p that does not affect the protein interaction with Saf1p but abolishes Aah1p degradation. Because this mutated residue is an exposed lysine in the Aah1p three-dimensional model, we propose that it is likely to be a major ubiquitylation site. All together, our data strongly argue for Saf1p being a bona fide Skp1-Cullin-F-box subunit.