Cdc50p, a transmembrane protein localized to the late endosome, is required for polarized cell growth in yeast. Genetic studies suggest that CDC50 performs a function similar to DRS2, which encodes a P-type ATPase of the aminophospholipid translocase (APT) subfamily. At low temperatures, drs2Delta mutant cells exhibited depolarization of cortical actin patches and mislocalization of polarity regulators, such as Bni1p and Gic1p, in a manner similar to the cdc50Delta mutant. Both Cdc50p and Drs2p were localized to the trans-Golgi network and late endosome. Cdc50p was coimmunoprecipitated with Drs2p from membrane protein extracts. In cdc50Delta mutant cells, Drs2p resided on the endoplasmic reticulum (ER), whereas Cdc50p was found on the ER membrane in drs2Delta cells, suggesting that the association on the ER membrane is required for transport of the Cdc50p-Drs2p complex to the trans-Golgi network. Lem3/Ros3p, a homolog of Cdc50p, was coimmunoprecipitated with another APT, Dnf1p; Lem3p was required for exit of Dnf1p out of the ER. Both Cdc50p-Drs2p and Lem3p-Dnf1p were confined to the plasma membrane upon blockade of endocytosis, suggesting that these proteins cycle between the exocytic and endocytic pathways, likely performing redundant functions. Thus, phospholipid asymmetry plays an important role in the establishment of cell polarity; the Cdc50p/Lem3p family likely constitute potential subunits specific to unique P-type ATPases of the APT subfamily.

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The RHO1 gene in Saccharomyces cerevisiae encodes a homolog of the mammalian RhoA small GTP‐binding protein, which is implicated in various actin cytoskeleton‐dependent cell functions. In yeast, Rho1p is involved in bud formation. A yeast strain in which RHO1 is replaced with RhoA shows a recessive temperature‐sensitive growth phenotype. A dominant suppressor mutant was isolated from this strain. Molecular cloning of the suppressor gene revealed that the mutation occurred at the pseuodosubstrate site of PKC1, a yeast homolog of mammalian protein kinase C. Two‐hybrid analysis demonstrated that GTP‐Rho1p, but not GDP‐Rho1p, interacted with the region of Pkc1p containing the pseudosubstrate site and the C1 domain. MKK1 and MPK1 encode MAP kinase kinase and MAP kinase homologs, respectively, and function downstream of PKC1. A dominant active MKK1–6 mutation or overexpression of MPK1 suppressed the temperature sensitivity of the RhoA mutant. The dominant activating mutation of PKC1 suppressed the temperature sensitivity of the RhoA mutant. The dominant activating mutation of PKC1 suppressed the temperature sensitivity of two effector mutants of RHO1, rho1(F44Y) and rho1(E451), but not that of rho1(V43T). These results indicate that there are at least two signaling pathways regulated by Rho1p and that one of the downstream targets is Pkc1p, leading to the activation of the MAP kinase cascade.

The RHO1 gene encodes a homologue of mammalian remains to be clarified whether these target proteins of RhoA small G-protein in the yeast Saccharomyces Rho are involved in the reorganization of actin cytocerevisiae. Rho1p is required for bud formation and is skeleton. Very recently, one of these proteins, Rho kinase, localized at a bud tip or a cytokinesis site. We have recently shown that Bni1p is a potential target of has been shown to inhibit the myosin phosphatase activity Rho1p. Bni1p shares the FH1 and FH2 domains with (Kimura et al., 1996), although its physiological significproteins involved in cytokinesis or establishment of ance remains to be clarified. cell polarity. In S.cerevisiae, there is an open reading Cells of the budding yeast Saccharomyces cerevisiae frame (YIL159W) which encodes another protein grow by budding for cell division, and the actin cytohaving the FH1 and FH2 domains and we have named skeleton plays a pivotal role in the budding process this gene BNR1 (BNI1 Related). Bnr1p interacts with (Drubin, 1991 Johnson and Pringle, 1990). mutant shows a severe temperature-sensitive growth RHO1 is a homologue of the mammalian RhoA gene and phenotype. Cells of the bni1 bnr1 mutant arrested we have shown that the rho1 mutants are deficient in at the restrictive temperature are deficient in bud the budding process (Yamochi et al., 1994). Moreover, emergence, exhibit a random distribution of cortical immunofluorescence microscopic studies indicate that actin patches and often become multinucleate. TheseRho1p is localized at the growth site with cortical actin phenotypes are similar to those of the mutant of patches, including the presumptive budding site, the bud PFY1, which encodes profilin, an actin-binding protein.tip and the cytokinesis site (Yamochi et al., 1994). These Moreover, yeast two-hybrid and biochemical studies results suggest that RHO1 regulates the processes of bud demonstrate that Bni1p and Bnr1p interact directly formation. Concerning the downstream targets of Rho1p, with profilin at the FH1 domains. These results indicate we have shown that one of them is a homologue of that Bni1p and Bnr1p are potential targets of the Rho mammalian protein kinase C, Pkc1p (Nonaka et al., 1995), family members, interact with profilin and regulate which regulates cell wall integrity through the activation the reorganization of actin cytoskeleton.of the MAP kinase cascade (Levin and Errede, 1995). We Keywords: actin cytoskeleton/profilin/Rho have also shown that another target of Rho1p is 1,3-β-glucan synthase (Drgonová et al., 1996;Qadota et al., 1996), which is involved in cell wall synthesis. Very recently, we have identified BNI1 as a third potential target

The enzyme that catalyzes the synthesis of the major structural component of the yeast cell wall, beta(1-->3)-D-glucan synthase (also known as 1,3-beta-glucan synthase), requires a guanosine triphosphate (GTP) binding protein for activity. The GTP binding protein was identified as Rho1p. The rho1 mutants were defective in GTP stimulation of glucan synthase, and the defect was corrected by addition of purified or recombinant Rho1p. A protein missing in purified preparations from a rho1 strain was identified as Rho1p. Rho1p also regulates protein kinase C, which controls a mitogen-activated protein kinase cascade. Experiments with a dominant positive PKC1 gene showed that the two effects of Rho1p are independent of each other. The colocalization of Rho1p with actin patches at the site of bud emergence and the role of Rho1p in cell wall synthesis emphasize the importance of Rho1p in polarized growth and morphogenesis.

The RHO1 gene encodes a homolog of mammalian RhoA small GTP binding protein in the yeast Saccharomyces cerevisiae. Rho1p is localized at the growth sites, including the bud tip and the cytokinesis site, and is required for bud formation. We have recently shown that Pkc1p, a yeast homolog of mammalian protein kinase C, and glucan synthase are targets of Rho1p. Using the two‐hybrid screening system, we cloned a gene encoding a protein which interacted with the GTP‐bound form of Rho1p. This gene was identified as BNI1, known to be implicated in cytokinesis or establishment of cell polarity in S.cerevisiae. Bni1p shares homologous domains (FH1 and FH2 domains) with proteins involved in cytokinesis or establishment of cell polarity, including formin of mouse, capu and dia of Drosophila and FigA of Aspergillus. A temperature‐sensitive mutation in which the RHO1 gene was replaced by the mammalian RhoA gene showed a synthetically lethal interaction with the bni1 mutation and the RhoA bni1 mutant accumulated cells with a deficiency in cytokinesis. Furthermore, this synthetic lethality was caused by the incapability of RhoA to activate Pkc1p, but not glucan synthase. These results suggest that Rho1p regulates cytoskeletal reorganization at least through Bni1p and Pkc1p.

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Phospholipid translocases (PLTs) have been implicated in the generation of phospholipid asymmetry in membrane bilayers. In budding yeast, putative PLTs are encoded by the DRS2 gene family of type 4 P-type ATPases. The homologous proteins Cdc50p, Lem3p, and Crf1p are potential noncatalytic subunits of Drs2p, Dnf1p and Dnf2p, and Dnf3p, respectively; these putative heteromeric PLTs share an essential function for cell growth. We constructed temperature-sensitive mutants of CDC50 in the lem3Delta crf1Delta background (cdc50-ts mutants). Screening for multicopy suppressors of cdc50-ts identified YPT31/32, two genes that encode Rab family small GTPases that are involved in both the exocytic and endocytic recycling pathways. The cdc50-ts mutants did not exhibit major defects in the exocytic pathways, but they did exhibit those in endocytic recycling; large membranous structures containing the vesicle-soluble N-ethylmaleimide-sensitive factor attachment protein receptor Snc1p intracellularly accumulated in these mutants. Genetic results suggested that the YPT31/32 effector RCY1 and CDC50 function in the same signaling pathway, and simultaneous overexpression of CDC50, DRS2, and GFP-SNC1 restored growth as well as the plasma membrane localization of GFP-Snc1p in the rcy1Delta mutant. In addition, Rcy1p coimmunoprecipitated with Cdc50p-Drs2p. We propose that the Ypt31p/32p-Rcy1p pathway regulates putative phospholipid translocases to promote formation of vesicles destined for the trans-Golgi network from early endosomes.