Marc Crouzet

Functional characterization of the Bag7, Lrg1 and Rgd2 RhoGAP proteins from Saccharomyces cerevisiae

Rho proteins are down‐regulated in vivo by specific GTPase activating proteins (RhoGAP). We have functionally studied three Saccharomyces cerevisiae putative RhoGAP. By first identifying Rho partners with a systematic two‐hybrid approach and then using an in vitro assay, we have demonstrated that the Bag7 protein stimulated the GTPase activity of the Rho1 protein, Lrg1p acted on the Cdc42 and Rho2 GTPases and we showed that Rgd2p has a GAP activity on both Cdc42p and Rho5p. In addition, we brought the first evidence for the existence of a sixth functional Rho in yeast, the Cdc42/Rac‐like GTPase Rho5.

The yeast Rgd1p is a GTPase activating protein of the Rho3 and Rho4 proteins

The RGD1 gene, identified during sequencing of the Saccharomyces cerevisiae genome, encodes a protein with a Rho‐GTPase activating protein (GAP) domain at the carboxy‐terminal end. The Rgd1 protein showed two‐hybrid interactions with the activated forms of Rho2p, Rho3p and Rho4p. Using in vitro assays, we demonstrated that Rgd1p stimulated the GTPase activity of both Rho3p and Rho4p; no stimulation was observed on Rho2p. In addition, the rho3Δrgd1Δ double mutant exhibited a dramatic growth defect compared to the single mutants, suggesting that Rgd1p has a GAP activity in vivo. The present study allowed the identification of the first GAP of Rho3p and Rho4p.

Functional assessment of the yeast Rvs161 and Rvs167 protein domains

Mutations in RVS161 and RVS167 yeast genes induce identical phenotypes associated to actin cytoskeleton disorders. The whole Rvs161 protein is similar to the amino‐terminal part of Rvs167p, thus defining a RVS domain. In addition to this domain, Rvs167p contains a central glycine‐proline‐alanine rich domain and a SH3 domain. To assess the function of these different domains we have expressed recombinant Rvs proteins in rvs mutant strains. Phenotype analysis has shown that the RVS and SH3 domains are necessary for phenotypical complementation, whereas the GPA domain is not. Moreover, we have demonstrated that the RVS domains from Rvs161p and Rvs167p have distinct roles, and that the SH3 domain needs the specific RVS domain of Rvs167p to function. These results suggest that Rvs161p and Rvs167p play distinct roles, while acting together in a common function.

Cloning of the Multicopy Suppressor Gene SUR7: Evidence for a Functional Relationship between the Yeast Actin-binding Protein Rvs167 and a Putative Membranous Protein

The rvs161 and rvs167 mutant cells exhibit several identical phenotypes including sensitivity to several different growth conditions and morphological defects such as alteration of the actin cytoskeleton and budding patterns. The selection of genes that, when overexpressed, are able to suppress the reduced viability upon carbon starvation of the rvs167 mutant strain, has allowed the cloning of the SUR7 gene (Accession Number Z46729x11).

Evidence for the genetic interaction between the actin‐binding protein Vrp1 and the RhoGAP Rgd1 mediated through Rho3p and Rho4p in Saccharomyces cerevisiae

The non‐essential RGD1 gene from Saccharomyces cerevisiae encodes a protein that has been characterized in vitro as a Rho GTPase activating protein (RhoGAP) for the Rho3 and Rho4 proteins. Rgd1p, which displays a conserved FCH–coiled coil–RhoGAP domain organization, showed a patch‐like distribution in the cell, including a localization in growing buds. Using a genetic screen, we found that rgd1Δ and vrp1Δ mutations exhibited a synthetic lethality, thus revealing an interaction between these genes. The VRP1 product is an actin and myosin interacting protein involved in polarized growth. Using mutant forms of both Rho3 and Rho4 proteins, we provide evidence for the involvement of these two GTPases in RGD1–VRP1 co‐lethality. In addition, these results strongly argue in favour of Rho3p and Rho4p being the targets of Rgd1p RhoGAP activity in vivo. Genetic relationships between either VRP1 or RGD1 and actin cytoskeleton‐linked genes were also studied. These and other well‐established data support the idea that Vrp1, Las17, Rvs167 proteins belong to the same complex. This protein structure might act with myosins in various actin cytoskeleton‐based activities, in co‐operation with a Rho3p/Rho4p signalling pathway that is negatively regulated by Rgd1p GAP activity.

New evidence of a mitochondrial genetic background paradox: impact of the J haplogroup on the A3243G mutation.

BackgroundThe A3243G mutation in the tRNALeu gene (UUR), is one of the most common pathogenic mitochondrial DNA (mtDNA) mutations in France, and is associated with highly variable and heterogeneous disease phenotypes. To define the relationships between the A3243G mutation and mtDNA backgrounds, we determined the haplogroup affiliation of 142 unrelated French patients – diagnosed as carriers of the A3243G mutation – by control-region sequencing and RFLP survey of their mtDNAs.ResultsThe analysis revealed 111 different haplotypes encompassing all European haplogroups, indicating that the 3243 site might be a mutational hot spot. However, contrary to previous findings, we observed a statistically significant underepresentation of the A3243G mutation on haplogroup J in patients (p = 0.01, OR = 0.26, C.I. 95%: 0.08–0.83), suggesting that might be due to a strong negative selection at the embryo or germ line stages.ConclusionThus, our study supports the existence of mutational hotspot on mtDNA and a haplogroup J paradox, a haplogroup that may increase the expression of mtDNA pathogenic mutations, but also be beneficial in certain environmental contexts.

First characterization of the gene RGD1 in the yeast Saccharomyces cerevisiae

We identified the ORF YBR260c during systematic sequencing of one region of chromosome II of Saccharomyces cerevisiae. This ORF encodes a putative protein of 666 aa, of which the C-terminal part of the deduced amino acid sequence resembles human and yeast Rho/Rac GTPase activating proteins (GAP). An initial study is reported in the paper. This gene was expressed in haploid and diploid cells and was called RGD1 for related GAP domain 1. Inactivation of RGD1 was carried out and phenotypic analysis of the mutant strain revealed only a slight viability defect when cells grown in minimal medium were close to stationary phase. Northern and western analyses showed that the RGD1 transcript and the corresponding protein were still abundant in cells cultivated in YNB during the stationary phase. No functional link seems to exist with the highly conserved GTPase Cdc42 involved in cytoskeletal polarization and cell polarity.

RGD1 genetically interacts with MID2 and SLG1, encoding two putative sensors for cell integrity signalling in Saccharomyces cerevisiae.

The RGD1 gene was identified during systematic genome sequencing of Saccharomyces cerevisiae. To further understand Rgd1p function, we set up a synthetic lethal screen for genes interacting with RGD1. Study of one lethal mutant made it possible to identify the SLG1 and MID2 genes. The gene SLG1/HCS77/WSC1 was mutated in the original synthetic lethal strain, whereas MID2/SMS1 acted as a monocopy suppressor. The SLG1 gene has been described to be an upstream component in the yeast PKC pathway and encodes a putative cell surface sensor for the activation of cell integrity signalling. First identified by viability loss of shmoos after pheromone exposure, and since found in different genetic screens, MID2 was recently reported as also encoding an upstream activator of the PKC pathway. The RGD1 gene showed genetic interactions with both sensors of cell integrity pathway. The rgd1 slg1 synthetic lethality was rescued by osmotic stabilization, as expected for mutants altered in cell wall integrity. The slight viability defect of rgd1 in minimal medium, which was exacerbated by mid2, was not osmoremediated. As for mutants altered in PKC pathway, the accumulation of small‐budded dead cells in slg1, rgd1 and mid2 after heat shock was prevented by 1u2009M sorbitol. In addition, the rgd1 strain also displayed dead shmoos after pheromone treatment, like mid2. Taken together, the present results indicate close functional links between RGD1, MID2 and SLG1 and suggest that RGD1 and MID2 interact in a cell integrity signalling functionally linked to the PKC pathway. Copyright

Functional interactions between the VRP1–LAS17 and RHO3–RHO4 genes involved in actin cytoskeleton organization in Saccharomyces cerevisiae

Abstract. The RGD1 gene from Saccharomyces cerevisiae, which encodes a GTPase-activating protein for the Rho3 and Rho4 small G proteins, exhibits synthetic lethality with the VRP1 and LAS17 genes. Their products are proline-rich proteins that interact with both actin and myosins to ensure polarized growth. By testing functional links, we found that the VRP1 and LAS17 genes are potent suppressors of the rho3Δ mutation. In particular, they restore the polarization of actin patches in rho3Δ cells. Moreover, the vrp1Δ and las17Δ mutations were found to display a similar pattern of genetic interactions with specific actin-linked genes. These mutations also increase the sensitivity to activated forms of both Rho3p and Rho4p. These data support our working model, in which the VRP1 and LAS17 genes define a cellular complex that works in concert with the RHO3–RHO4 signaling pathway in yeast polarized growth. In addition, other observations lead us to propose that Rvs167p may act as a linking protein between the two cellular elements.

FIRST CHARACTERIZATION OF THE PHOSPHONOACETALDEHYDE HYDROLASE GENE OF PSEUDOMONAS AERUGINOSA

The phnX gene encoding the phosphonoacetaldehyde hydrolase (phosphonatase) from the Gram-negative bacterium Pseudomonas aeruginosa A237 has been cloned and its sequence determined. The open reading frame consists of 825 nucleotides specifying a protein of 275 amino acid residues corresponding to a predicted molecular weight of 29929. The deduced amino acid sequence of PhnX did not share significant amino acid sequence similarity with any other polypeptide. Expression of the phosphonoacetaldehyde hydrolase coding sequence in Escherichia coli under control of the E. coli tac promoter resulted in the production of enzymatically active protein with an affinity constant similar to that of the phosphonoacetaldehyde hydrolase purified from P. aeruginosa A237. This is the first nucleic sequence report of the phosphonoacetaldehyde hydrolase, an enzyme involved in the carbon-phosphorus bond cleavage.