Abstract. The Rho small GTP-binding protein family regulates various actomyosin-dependent cell functions, such as cell morphology, locomotion, cytokinesis, membrane ruffling, and smooth muscle contraction. In the yeast Saccharomyces cerevisiae, there is a homologue of mammalian RhoA, RH01, which is essential for vegetative growth of yeast cells. To explore the function of the RH01 gene, we isolated a recessive temperature-sensitive mutation of Rtt01, rho1-104. The rho1-104 mutation caused amino acid substitutions of Asp 72 to Asn and Cys 164 to Tyr of Rholp. Strains bearing the rho1-104 mutation accumulated tiny-or small-budded cells in which cortical actin patches were clustered to buds at the restrictive temperature. Cell lysis and cell death were also seen with the rho1-104 mutant. Indirect immunofluorescence microscopic study demonstrated that Rholp was concentrated to the periphery of the cells where cortical actin patches were clustered, including the site of bud emergence, the tip of the growing buds, and the motherbud neck region of cells prior to cytokinesis. Indirect immunofluorescence study with cells overexpressing RH01 suggested that the Rholp-binding site was saturable. A mutant Rholp with an amino acid substitution at the lipid modification site remained in the cytoplasm. These results suggest that Rhol small GTP-binding protein binds to a specific site at the growth region of cells, where Rholp exerts its function in controlling cell growth. THE yeast Saccharomyces cerevisiae grows by budding for cell division (Drubin, 1991;Nelson, 1992). This polarized cell growth is initiated by signals from the cell surface that result in realignment of the cytoskeleton and the biosynthetic machinery toward a targeting patch at the bud site. Membrane protein transport to the cell surface is mediated by vesicles, which become selectively targeted to the bud site. Bud site assembly and growth are also coupled to reorganization of the actin cytoskeleton. Disruption of the single actin gene in S. cerevisiae results in abnormal cell growth and intraceUular accumulation of vesicles (Novick and Botstein, 1985). Moreover, there is a strong correlation between occurrence of active growth at the bud tip and clustering of cortical actin patches at the same tip. Cortical actin patches are concentrated at the site of bud emergenceThe present address of W. Yamochi is Department of Internal Medicine (lst Division), Kobe University School of Medicine, Kobe 650, Japan.The present address of K. Tanaka, H. Nonaka, and Y. Takai is Department of Molecular Biology and Biochemistry, Osaka University Medical School, 2-2 Yamada-oka, Suita 565, Japan.The present address of T. Musha is Division of Cardiovascular Pharmacology, Eisai Co., Ltd., 1-3-5 Tokaidai, Tsulmba, Ibaraki 300-26, Japan.Address all correspondence to Y. Takai, Department of Molecular Biology and Biochemistry, Osaka University Medical School, 2-2 Yamada-oka, Suita 565, Japan.on unbudded cells and in small and medium size buds in budding cells, whereas actin fibers are g...

Abstract-The precise regulation of cell growth in the vascular wall maintains vascular integrity, and its disruption leads to cardiovascular disorders including atherosclerosis and restenosis. Vascular endothelial growth factor (VEGF) is a specific mitogen for endothelial cells, and endothelin-1 (ET-1) is known to stimulate the proliferation of smooth muscle cells. The aim of this study was to explore a potential interaction between VEGF and ET-1 on each expression in vascular cells. VEGF enhanced preproET-1 mRNA expression and ET-1 secretion in bovine aortic endothelial cells (BAECs

The small GTP-binding proteins of the Rho family, consisting of the Rho, Rac, and Cdc42 subfamilies, are implicated in various cell functions, such as cell shape change, cell motility and cytokinesis, through reorganization of actin cytoskeleton. Rho GDI is a general regulator which forms a complex with the GDP-bound inactive form of the Rho family members and inhibits their activation. We have purified Rho GDI from the yeast Saccharomyces cerevisiae, cloned its gene, and named it RDII (Rho GD). In this study, we have further characterized yeast Rho GDI. Rho GDI was found in the cytosol by immunoblot and immunofluorescence microscopic analyses. Rho1p and Cdc42p were co-immunoprecipitated with Rho GDI from the cytosol. This immunoprecipitated Rho1p was mainly bound to GDP. In the disruption mutant of Rho GDI, which did not show any apparent phenotype, both Rho1p and Cdc42p were also present in the cytosol. These results indicate that yeast Rho GDI possesses properties similar to those of mammalian Rho GDI, and that there is a cytosolic factor which functionally substitutes for Rho GDI in yeast.

Hemodynamic forces on vasculature profoundly influence atherogenesis. We examined the effect of stretch force on the oxidation of low-density lipoprotein (LDL) by rat aortic smooth muscle cells (RASM) and superoxide production. Stretch force was imposed on RASM cultured on deformable dishes by stretching the dishes. Incubation of native LDL with static RASM for 24 h resulted in LDL oxidation as indicated by increases in thiobarbituric acid-reacting substances from 9.5 ± 2.3 to 24.5 ± 2.3 nmol malondialdehyde/mg. Stretch force on RASM augmented cell-mediated LDL oxidation to 149.3 ± 17.1% concomitantly with increase in superoxide production. LDL oxidation was inhibited by superoxide dismutase or depletion of the metal ion in the culture medium, indicating that it was a metal ion-dependent and superoxide-mediated process. The enhancement of LDL oxidation by stretch force was inhibited by diphenyliodonium, indicating the involvement of the NADH/NADPH oxidase system. Our findings suggest that the increased oxidant stress induced by stretch force is one of the potential mechanisms whereby hypertension facilitates atherosclerosis.

No abstract