Phosphatidylinositol 4,5-biphosphate (PtdIns(4,5)P 2 ), an important element in eukaryotic signal transduction, is synthesized either by phosphatidylinositol-4-phosphate 5-kinase (PtdIns(4)P 5K) from phosphatidylinositol 4-phosphate (PtdIns(4)P) or by phosphatidylinositol-5-phosphate 4-kinase (PtdIns(5)P 4K) from phosphatidylinositol 5-phosphate (PtdIns(5)P). Two Saccharomyces cerevisiae genes, MSS4 and FAB1, are homologous to mammalian PtdIns(4)P 5Ks and PtdIns(5)P 4Ks. We show here that MSS4 is a functional homolog of mammalian PtdIns(4)P 5K but not of PtdIns(5)P 4K in vivo. We constructed a hemagglutinin epitope-tagged form of Mss4p and found that Mss4p has PtdIns(4)P 5K activity. Immunofluorescent and fractionation studies of the epitope-tagged Mss4p suggest that Mss4p is localized on the plasma membrane, whereas Fab1p is reportedly localized on the vacuolar membrane. A temperature-sensitive mss4-1 mutant was isolated, and its phenotypes at restrictive temperatures were found to include increased cell size, round shape, random distribution of actin patches, and delocalized staining of cell wall chitin. Thus, biochemical and genetic analyses on Mss4p indicated that yeast PtdIns(4)P 5K localized on the plasma membrane is required for actin organization.Phosphatidylinositol 4,5-biphosphate (PtdIns(4,5)P 2 ) 1 has been recognized as an important element in eukaryotic signal transduction. Hydrolysis of PtdIns(4,5)P 2 by phospholipase C produces two second messengers, inositol 1,4,5-triphosphate (IP 3 ) and diacylglycerol. IP 3 mobilizes Ca 2ϩ from intracellular stores, such as the endoplasmic reticulum in animal cells (1) and vacuoles in plants (2) and yeast (3). It is well known that the elevated intracellular Ca 2ϩ stimulates a variety of calciummodulating signaling enzymes, including calmodulin-dependent protein kinases and calcineurin, a type II B phosphoprotein phosphatase (4). Diacylglycerol, on the other hand, activates the conventional isoforms of protein kinase C, which in turn play a critical role in the regulation of a number of cellular functions in mammalian cells (5). In the budding yeast Saccharomyces cerevisiae, a protein kinase C-homologous gene (PKC1) was isolated (6), whose product was shown to function in cell wall integrity and cell cycle progression (7,8). In vitro studies of Pkc1p, however, indicated that Pkc1p is strongly activated by phosphatidylserine in the presence of Rho1p, but not by diacylglycerol (9). The stimulation by phosphatidylserine alone is characteristic of the atypical isoform of protein kinase C, which is stimulated by phosphatidylserine alone. Since the biochemical property of Pkc1p is different from that of the conventional isoforms of mammalian protein kinase C, it remains unclear whether and how diacylglycerol acts as an important second messenger in S. cerevisiae.PtdIns(4,5)P 2 is also known to function as a regulator of actin-binding proteins (10) such as profilin (11), gelsolin (12), and ␣-actinin of vertebrates (13). Recently, profilin was reported to be localized...

Background:  The cell wall has an important role in maintaining cell shape. In the budding yeast Saccharomyces cerevisiae, the major filamentous component of the cell wall responsible for its rigidity is 1,3‐β‐glucan and is synthesized by 1,3‐β‐glucan synthase (GS), localized on the plasma membrane. Results:  Observations of green fluorescent protein (GFP)‐conjugated Fks1p, a catalytic subunit of GS, revealed that it is co‐localized with cortical actin patches and moves on the cell surface at the sites of cell wall remodelling. Mutants with impaired actin patch movement show immobility of Fks1p‐GFP spots, indicating that actin patch motility is required for the movement of Fks1p. Cells with immobilized Fks1p exhibit defective cell wall structure and function. The cell wall thickness of the mutants becomes irregular, eventually leading to cell lysis. Conclusion:  We propose that GS movement is necessary for proper cell wall remodelling.

Saccharomyces cerevisiae is a multifunctional molecular switch involved in establishment of cell morphogenesis. We systematically characterized isolated temperature-sensitive mutations in the RHO1 gene and identified two groups of rho1 mutations (rho1A and rho1B) possessing distinct functional defects. Biochemical and cytological analyses demonstrated that mutant cells of the rho1A and rho1B groups have defects in activation of the Rho1p effectors Pkc1p kinase and 1,3-␤-glucan synthase, respectively. Heteroallelic diploid strains with rho1A and rho1B mutations were able to grow even at the restrictive temperature of the corresponding homoallelic diploid strains, showing intragenic complementation. The ability to activate both of the essential Rho1p effector proteins was restored in the heteroallelic diploid. Thus, each of the complementing rho1 mutation groups abolishes a distinct function of Rho1p, activation of Pkc1p kinase or 1,3-␤-glucan synthase activity.After establishment of cell polarity, morphogenesis of plant and fungal cells is determined by organization of the intracellular cytoskeleton and construction of the extracellular cell wall. A Rho-type small GTP-binding protein (Rho1p) in the budding yeast Saccharomyces cerevisiae has been shown to play a pivotal role in cell morphogenesis by regulating its effector proteins. Rho1p binds and activates Fks1p and Fks2p, two closely related catalytic subunits of 1,3-␤-glucan synthase (GS), 1 thereby directly controlling cell wall synthesis (1, 2). Rho1p also binds and activates Pkc1p, a yeast homolog of mammalian protein kinase C. Through the mitogen-activated protein kinase (MAPK) cascade, Pkc1p regulates organization of the actin cytoskeleton and transcription of several genes involved in cell wall integrity (3-6). Other Rho1p-interacting proteins include Bni1p, Skn7p, and Sec3p (7-10). Gene disruption analyses revealed that among the five Rho1p effector proteins, Fks1/2p and Pkc1p are the most important in yeast cell growth. ⌬fks1⌬fks2 and ⌬pkc1 are both lethal in complete medium (11, 12), whereas ⌬sec3 shows slow growth in synthetic medium (13), and ⌬bni1 and ⌬skn7 display normal growth (7, 15). Thus, Rho1p controls cell morphogenesis by regulating the activities of two essential effector proteins important for cell wall synthesis and actin cytoskeleton organization.Several conditional lethal mutations (high temperature-sensitive mutations) in the RHO1 gene (rho1-2, rho1-3, rho1-4, rho1-5) have been isolated in our laboratory and characterized for elucidation of Rho1p function. Biochemical analyses of the rho1 mutants greatly contributed to the understanding of the essential pathways downstream of Rho1p (2, 5, 6). However, the rho1 mutants did not always exhibit a single unique phenotype. Helliwell et al. (6) reported that actin morphologies differ among the rho1 mutants: rho1-3 and rho1-4 display normal polarized actin patches, whereas rho1-2 and rho1-5 possess delocalized actin patches. In this study, we investigated what kind of phenotypic differences ...

The 6b gene of Agrobacterium tumefaciens AKElO (AK-6b) induces crown gall tumors on certain plants but so far there have been no reports of the gene being able to induce tumors on culture medium. We cloned T-DNA segments containing the 6b gene but lacking the auxin and cytokinin biosynthesis genes from A. fumefaciens AKEl O. Tobacco (Nicotiana tabacum) leaf discs infected with A. tumefaciens LBA4404 carrying the clones produced shooty calli on hormone-free Murashige-Skoog medium. The relevant T-DNA segment was integrated into plant DNA as determined by Southern hybridization. Some of these immature shoots spontaneously developed into mature shoots, of which severa1 leaves displayed morphological abnormalities. When leaf discs of these mature plants were placed onto the same medium numerous shoots developed from the wounding sites, indicating that the transgenic plants possessed a high regenerative potential. Northern blot and reverse transcriptase-polymerase chain reaction analyses showed a large accumulation of the AK-6b transcripts in the shooty calli, but only a limited degree in mature plants, demonstrating that AK-66 expression is regulated in plants and essential for the early stages of regeneration. Cytokinin levels in the shooty calli were comparable to those in normal shoots, suggesting that shoot regeneration is not mediated by the modulation of cytokinin content.

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