Lrg1p Is a Rho1 GTPase-Activating Protein Required for Efficient Cell Fusion in Yeast

Abstract

To identify additional cell fusion genes in Saccharomyces cerevisiae, we performed a high-copy suppressor screen of fus2⌬. Higher dosage of three genes, BEM1, LRG1, and FUS1, partially suppressed the fus2⌬ cell fusion defect. BEM1 and FUS1 were high-copy suppressors of many cell-fusion-defective mutations, whereas LRG1 suppressed only fus2⌬ and rvs161⌬. Lrg1p contains a Rho-GAP homologous region. Complete deletion of LRG1, as well as deletion of the Rho-GAP coding region, caused decreased rates of cell fusion and diploid formation comparable to that of fus2⌬. Furthermore, lrg1⌬ caused a more severe mating defect in combination with other cell fusion mutations. Consistent with an involvement in cell fusion, Lrg1p localized to the tip of the mating projection. Lrg1p-GAP domain strongly and specifically stimulated the GTPase activity of Rho1p, a regulator of ␤(1-3)-glucan synthase in vitro. ␤(1-3)-glucan deposition was increased in lrg1⌬ strains and mislocalized to the tip of the mating projection in fus2⌬ strains. High-copy LRG1 suppressed the mislocalization of ␤(1-3) glucan in fus2⌬ strains. We conclude that Lrg1p is a Rho1p-GAP involved in cell fusion and speculate that it acts to locally inhibit cell wall synthesis to aid in the close apposition of the plasma membranes of mating cells.

Disruption of LRG1 inhibits mother–daughter separation in Saccharomyces cerevisiae

“…Additionally, lrg1/lrg1 diploids do not cluster (Figure 1), and the wall and septum thicknesses of these cells were not statistically different from those of wild-type haploids ( Figure 5). Our results, in conjunction with the recent report that LRG1 localizes to the tip of the mating projection in haploids and is required for efficient cell fusion (Fitch et al, 2004), suggest that matingspecific glucan synthase machinery and regulatory proteins are downregulated in cells that have undergone cell fusion. Since we observed clustering in pseudodiploid lrg1 cells, deactivation of the mating pathway alone is not sufficient to suppress the clustered phenotype.…”

“…Further analysis revealed that Ste12, which regulates expression of genes downstream of the mating and filamentous growth MAPK pathway but is also known to function in the cell integrity pathway (see review by Elion, 2000), was required for cell clustering. LRG1 has previously been identified as a regulator of glucan synthesis (Lorberg et al, 2001;Roumanie et al, 2001;Watanabe et al, 2001;Fitch et al, 2004), and our results suggest that the observed 'clustered' phenotype is a consequence of failed mother-daughter separation due to increased glucan production at the bud site.…”

“…Using Student's t-test, p values <0.005 (%) were obtained for lrg1 fks2 mutants relative to wild-type values for the same cells/cluster size. p values <0.001 ( * ) and <0.005 (#) were obtained for lrg1 rom2 and lrg1 fks1 mutants relative to lrg1 mutants for the same cells/cluster size wall component was examined using the aniline blue staining procedure described by Watanabe et al (2001) with the modifications of Fitch et al (2004). As shown in Figure 10, lrg1 haploids Figure 9.…”

“…Localization staining was performed as described by Watanabe et al (2001) with modifications made by Fitch et al (2004). The cells were grown to mid-log phase, washed twice with PBS and resuspended in water.…”

Mutations in the Saccharomyces cerevisiae Kinase Cbk1p Lead to a Fertility Defect That Can Be Suppressed by the Absence of Brr1p or Mpt5p (Puf5p), Proteins Involved in RNA Metabolism

“…In a similar way, given the known interactions between Mpt5p and elements of the m-MAPK pathway, it is possible that deletion of MPT5 leads to a low-level activation of this pathway. Lrg1p could also constitute an element of this system: the protein is required for efficient cell fusion (Fitch et al 2004) and its expression is inhibited by Mpt5p; thus deletion of MPT5 would lead to increased levels of Lrg1p and improved cell fusion.…”