Hiroshi Qadota

Identification of Yeast Rho1p GTPase as a Regulatory Subunit of 1,3-β-Glucan Synthase

1,3-β-D-Glucan synthase [also known as β(1→3)glucan synthase] is a multi-enzyme complex that catalyzes the synthesis of 1,3-β-linked glucan, a major structural component of the yeast cell wall. Temperature-sensitive mutants in the essential Rho-type guanosine triphosphatase (GTPase), Rho1p, displayed thermolabile glucan synthase activity, which was restored by the addition of recombinant Rho1p. Glucan synthase from mutants expressing constitutively active Rho1p did not require exogenous guanosine triphosphate for activity. Rho1p copurified with β(1→3)glucan synthase and associated with the Fks1p subunit of this complex in vivo. Both proteins were localized predominantly at sites of cell wall remodeling. Therefore, it appears that Rho1p is a regulatory subunit of β(1→3)glucan synthase.

Lack of GTP-bound Rho1p in secretory vesicles of Saccharomyces cerevisiae

Rho1p, an essential Rho-type GTPase in Saccharomyces cerevisiae, activates its effectors in the GTP-bound form. Here, we show that Rho1p in secretory vesicles cannot activate 1,3-β-glucan synthase, a cell wall synthesizing enzyme, during vesicular transport to the plasma membrane. Analyses with an antibody preferentially reacting with the GTP-bound form of Rho1p revealed that Rho1p remains in the inactive form in secretory vesicles. Rom2p, the GDP/GTP exchange factor of Rho1p, is preferentially localized on the plasma membrane even when vesicular transport is blocked. Overexpression of Rom2p results in delocalization of Rom2p and accumulation of 1,3-β-glucan in secretory vesicles. Based on these results, we propose that Rho1p is kept inactive in intracellular secretory organelles, resulting in repression of the activity of the cell wall–synthesizing enzyme within cells.

Prenylation of Rho1p Is Required for Activation of Yeast 1,3-β-Glucan Synthase

One of the essential protein substrates of geranylgeranyl transferase type I in the budding yeastSaccharomyces cerevisiae is a rho-type GTPase, Rho1p, which is a regulatory subunit of 1,3-β-glucan synthase. Previous studies have indicated that modification of Rho1p is significantly reduced in a mutant of the β subunit of geranylgeranyl transferase type I calledcal1-1. Here we present genetic and biochemical evidence showing that modification of Rho1p is required for activity of 1,3-β-glucan synthase. The 1,3-β-glucan synthase activity of thecal1-1 membrane was significantly reduced compared with that of the wild-type membrane. The impaired activity was partly due to the reduced amount of Fks1p, a putative catalytic subunit of 1,3-β-glucan synthase, but also partly due to reduced affinity between unmodified Rho1p and Fks1p. GlutathioneS-transferase (GST)-Rho1 proteins with or without the C-terminal motif required for the modification were purified and used to analyze the interaction. The modified form of GST-Rho1p was specifically able to restore the 1,3-β-glucan synthase of therho1-3 membrane. Gel overlay analysis indicated that an unmodified form of GST-Rho1p fails to interact with Fks1p. These results indicated that the geranylgeranylation of Rho1p is a prerequisite to the assembly and activation of 1,3-β-glucan synthasein vitro. Increased cytoplasmic levels of divalent cations such as Ca2+ restored both Rho1p modification and the 1,3-β-glucan synthase activity of cal1-1, suggesting that cytoplasmic levels of the divalent cations affect geranylgeranyl transferase type I activity in vivo.

Multiple Functional Domains of the Yeast l,3-β-Glucan Synthase Subunit Fks1p Revealed by Quantitative Phenotypic Analysis of Temperature-Sensitive Mutants

The main filamentous structural component of the cell wall of the yeast Saccharomyces cerevisiae is 1,3-β-glucan, which is synthesized by a plasma membrane-localized enzyme called 1,3-β-glucan synthase (GS). Here we analyzed the quantitative cell morphology and biochemical properties of 10 different temperature-sensitive mutants of FKS1, a putative catalytic subunit of GS. To untangle their pleiotropic phenotypes, the mutants were classified into three functional groups. In the first group, mutants fail to synthesize 1,3-β-glucan at the proper subcellular location, although GS activity is normal in vitro. In the second group, mutants have normal 1,3-β-glucan content but are defective in polarized growth and endocytosis. In the third group, mutations in the putative catalytic domain of Fks1p result in a loss of the catalytic activity of GS. The differences among the three groups suggest that Fks1p consists of multiple domains that are required for cell wall construction and cellular morphogenesis.

Molecular cloning and characterization of Drosophila genes encoding small GTPases of the rab and rho families

Abstract We have isolated eight genes from Drosophila, small GTPases. They can be classified into three rab family genes (Drab2, Drab5, Drab11) and five rho family genes (Drac1a, Drac1b, Drac3, Dcdc42, DrhoA). While Drac3 is a novel type of rac gene, others are homologues of known mammalian genes for small GTPases. Northern blot analyses showed that all the genes are expressed throughout all developmental stages from embryo to adult. In situ hybridization to embryos revealed that Drab2, Drac1b, and Drac3 are highly expressed in the nervous system, in the trunk mesoderm, and in the cephalic mesoderm, respectively. Since hemocytes are derived from the cephalic mesoderm, we carried out double stainings using a hemocyte marker – anti-peroxidasin antibody – and Drac3 in situ hybridization. We found that Drac3 is expressed in hemocyte precursor cells. In the Drac3 deficiency embryos, the hemocyte precursor cells start to differentiate normally, but never develop into mature hemocytes, indicating that Drac3 is essential for their maturation. The DrhoA and Dcdc42 genes complemented S. cerevisiae rho1 and cdc42 mutations in the same manner as human rhoA and CDC42, respectively. These results suggest functional similarity between Drosophila and mammalian small GTPase genes.

MUTATIONAL ANALYSIS OF THE BETA -SUBUNIT OF YEAST GERANYLGERANYL TRANSFERASE I

The geneCAL1 (also known asCDC43) ofSaccharomyces cerevisiae encodes theβ subunit of geranylgeranyl transferase I (GGTase I), which modifies several small GTPases. Biochemical analyses of the mutant enzymes encoded bycall-1, andcdc43-2 tocdc43-7, expressed in bacteria, have shown that all of the mutant enzymes possess reduced activity, and that none shows temperature-sensitive enzymatic activities. Nonetheless, all of thecall/cdc43 mutants show temperature-sensitive growth phenotypes. Increase in soluble pools of the small GTPases was observed in the yeast mutant cells at the restrictive temperature in vivo, suggesting that the yeast prenylation pathway itself is temperature sensitive. Thecall-1 mutation, located most proximal to the C-terminus of the protein, differs from the othercdc43 mutations in several respects. An increase in soluble Rholp was observed in thecall-1 strain grown at the restrictive temperature. The temperature-sensitive phenotype ofcall-1 is most efficiently suppressed by overproduction of Rholp. Overproduction of the other essential target, Cdc42p, in contrast, is deleterious incall-1 cells, but not in othercdc43 mutants or the wild-type strains. Thecdc43-5 mutant cells accumulate Cdc42p in soluble pools andcdc43-5 is suppressed by overproduction of Cdc42p. Thus, several phenotypic differences are observed among thecall/cdc43 mutations, possibly due to alterations in substrate specificity caused by the mutations.