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.

linkage region between chitin and ␤(133)-glucan was solubilized and isolated in the form of oligosaccharides, after digestion of yeast cell walls with ␤(133)-glucanase, reduction with borotritide, and subsequent incubation with chitinase. In addition to the oligosaccharides, the solubilized fraction contained tritium-labeled high molecular weight material. We have now investigated the nature of this material and found that it represents areas in which all four structural components of the cell wall, ␤(133)-glucan, ␤(136)-glucan, chitin, and mannoprotein are linked together. Mannoprotein, with a protein moiety about 100 kDa in apparent size, is attached to ␤(136)-glucan through a remnant of a glycosylphosphatidylinositol anchor containing five ␣-linked mannosyl residues. The ␤(136)-glucan has some ␤(133)-linked branches, and it is to these branches that the reducing terminus of chitin chains appears to be attached in a ␤(134) or ␤(132) linkage. Finally, the reducing end of ␤(136)-glucan is connected to the nonreducing terminal glucose of ␤(133)-glucan through a linkage that remains to be established. A fraction of the isolated material has three of the main components but lacks mannoprotein. From these results and previous findings on the linkage between mannoproteins and ␤(136)-glucan, it is concluded that the latter polysaccharide has a central role in the organization of the yeast cell wall. The possible mechanism of synthesis and physiological significance of the cross-links is discussed.Cell walls are essential for the survival of fungal cells. Digestion of cell walls in the absence of an osmotic protector leads to cell lysis due to the high internal turgor pressure. Thus, substances that interfere with cell wall synthesis may be considered as potential antifungal agents (1). Because of its rigidity, the cell wall determines the shape of fungal cells. For that reason, cell wall formation has been used as a model for morphogenesis (1).The major components of fungal cell walls are polysaccharides and glycoproteins (2). In the yeast, Saccharomyces cerevisiae, the cell wall contains ␤(133)-D-glucan, ␤(136)-D-glucan, chitin, and mannoprotein(s) (3). The polysaccharides appear to have a structural function, whereas the mannoprotein(s) may act as "filler" and are important for the permeability of the cell wall (4, 5). How can one explain the strength and resilience of the fungal cell wall? Recent results with S. cerevisiae suggest that the answer may be found in the existence of covalent linkages between the different components of the wall that would give rise to a continuous and consequently stronger fabric. Thus, previous studies in our laboratories showed the presence of linkages between chitin and ␤(133)-glucan (6) as well as among glycoproteins, ␤(136)-glucan, and ␤(133)-glucan (7).The strategy for the investigation of interconnections between chitin and ␤(133)-glucan consisted in the digestion of cell walls with ␤(133)-glucanase, followed by labeling of the exposed reducing ends with borotritide and enzymati...

In the vegetative (mitotic) cycle and during sexual conjugation, yeast cells display polarized growth, giving rise to a bud or to a mating projection, respectively. In both cases one can distinguish three steps in these processes: choice of a growth site, organization of the growth site, and actual growth and morphogenesis. In all three steps, small GTP-binding proteins (G proteins) and their regulators play essential signaling functions. For the choice of a bud site, Bud1, a small G protein, Bud2, a negative regulator of Bud1, and Bud5, an activator, are all required. If any of them is defective, the cell loses its ability to select a proper bud position and buds randomly. In the organization of the bud site or of the site in which a mating projection appears, Cdc42, its activator Cdc24, and its negative regulators play a fundamental role. In the absence of Cdc42 or Cdc24, the actin cytoskeleton does not become organized and budding does not take place. Finally, another small G protein, Rho1, is required for activity of β(1 → 3)glucan synthase, the enzyme that catalyzes the synthesis of the major structural component of the yeast cell wall. In all of the above processes, G proteins can work as molecular switches because of their ability to shift between an active GTP-bound state and an inactive GDP-bound state.

Brain-derived neurotrophic factor (BDNF) affects the development of brain neurotransmitter systems, including dopamine and serotonin systems that are important for cocaine's rewarding and locomotor stimulatory properties. Human genomic markers within or near the BDNF locus have been linked to or associated with substance abuse. Post-mortem human brain specimens reveal individual differences in the levels of BDNF mRNA and in mRNA splicing patterns. To assess the effects of lifelong alterations in the levels of BDNF expression on a measure of psychostimulant reward, we have compared locomotor stimulant and rewarding effects of cocaine in heterozygous BDNF knockout mice with effects in their wild-type littermates. Heterozygous BDNF knockout mice displayed less locomotion during habituation and less locomotion after cocaine injections. Cocaine-conditioned place preferences were reduced in the BDNF heterozygotes. These mice displayed no significant difference from saline control values at a dose of 10 mg/kg s.c. cocaine, although they exhibited cocaine-induced preference at a 20 mg/kg dose. These data confirm important roles for BDNF in psychostimulant actions, presumably via neurotrophic effects on dopamine and serotonin systems. Furthermore, these data support suggestions that differences in human BDNF expression may underlie associations between markers near the human BDNF gene locus and drug addiction.