François Doignon

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.

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).

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.

Secretory pathway-dependent localization of the Saccharomyces cerevisiae Rho GTPase-activating protein Rgd1p at growth sites.

ABSTRACT Establishment and maintenance of cell polarity in eukaryotes depends upon the regulation of Rho GTPases. In Saccharomyces cerevisiae, the Rho GTPase activating protein (RhoGAP) Rgd1p stimulates the GTPase activities of Rho3p and Rho4p, which are involved in bud growth and cytokinesis, respectively. Consistent with the distribution of Rho3p and Rho4p, Rgd1p is found mostly in areas of polarized growth during cell cycle progression. Rgd1p was mislocalized in mutants specifically altered for Golgi apparatus-based phosphatidylinositol 4-P [PtdIns(4)P] synthesis and for PtdIns(4,5)P2 production at the plasma membrane. Analysis of Rgd1p distribution in different membrane-trafficking mutants suggested that Rgd1p was delivered to growth sites via the secretory pathway. Rgd1p may associate with post-Golgi vesicles by binding to PtdIns(4)P and then be transported by secretory vesicles to the plasma membrane. In agreement, we show that Rgd1p coimmunoprecipitated and localized with markers specific to secretory vesicles and cofractionated with a plasma membrane marker. Moreover, in vivo imaging revealed that Rgd1p was transported in an anterograde manner from the mother cell to the daughter cell in a vectoral manner. Our data indicate that secretory vesicles are involved in the delivery of RhoGAP Rgd1p to the bud tip and bud neck.

The Saccharomyces cerevisiae RhoGAP Rgd1 is phosphorylated by the Aurora B like kinase Ipl1.

Polarized growth of the yeast Saccharomyces cerevisiae depends on different biological processes and requires several signaling pathways. Signaling is mediated through a set of proteins, which include Rho3p and Rho4p GTPases. Although these two proteins are involved in the control of distinct aspects of polarized growth in yeast, they have a common regulator: the Rgd1 RhoGAP protein. Here we demonstrate that Rgd1p is phosphorylated by the Aurora B like kinase Ipl1 and we observe that loss of Ipl1 function leads to a new Rgd1p distribution in a small part of the cell population.

The yeast Rgd1p which is a GTPase activating protein of Rho3p and Rho4p, interacts with Sig1p and Mid2p two putative sensors for cell integrity signaling

pl3OCas is an adaptor protein involved in protein kinase signal transduction cascades. It is tyrosine phosphorylated upon stimulation of different receptors, especially integrins. Although its precise function is unclear, a role in cell motility and actin cytoskeletton regulation has been demonstrated. In motile NfH3T3 cells, immunofluorescence staining of pl3OCas reveals a new vesicular structure found in pseudopodial cell extensions and lamellipodia. Although pl3oca.s was previously described as a focal contact associated protein, we show that this vesicular structure differs clearly from paxillin-labeled adherence structures, although being in their very close vicinity. Furthermore, this vesicular structure does not appear related to the actin cytoskeleton but rather to a subset of microtubules found in pseudopodial area. While the majority of pl3CCas vesicules accumulate in this area, a subset is aligned with microtubules radiating from the central area of the cell towards the leading edge, suggesting vesicular trafficking. A protein known to interact with pl3OCas, Crk-L, could also be found in vesicular structures very reminiscent of pl3OCas’s one. However, another Cas binding partner, FAK, is rather distributed to focal contacts, suggesting a dual intracellular localization for these proteins. Jn osteoclasts, pl3OCas associates with podosomes and participates in their formation. We confirm this podosomal localization but we also show pl3GCa.s vesicules aligned with microtubules, as in NfH3T3, providing a direct evidence for dual localization of Cas. These microtubules end in the podosomal area. It was previously reported that microtubules regulate podosomes in osteocksk, and more generally adherence structures in fibroblask. Our results suggest that microtubules could target pl3OCas-loaded vesicules to the podosomes, henceforth regulating their formation. We previously reported that another adaptor molecule, c-Cbl, is associated with Golgiderived vesicles. Together, these data suggest that vesicles targeted to areas of microfilamenk remodeling could carry actin-regulating proteins on their cytoplasmic face.