The Saccharomyces cerevisiae genes KRE6 and SKN1 encode a novel pair of highly homologous proteins involved in cell wall (1$ rightarrow$6)-$ beta$-glucan assembly. Disruption of KRE6 results in a slow growing, killer-toxin resistant mutant possessing reduced levels of structurally wild type (1$ rightarrow$6)-$ beta$-glucan. Although deletion of SKN1 has no effect on killer sensitivity, growth, or (1$ rightarrow$6)-$ beta$-glucan levels, SKN1 appears to overlap in function with KRE6, suppressing kre6 null alleles in a dosage-dependent manner. Strains deleted of both KRE6 and SKN1 possess an exaggerated growth phenotype, enhanced cell wall ultrastructure defects, and more severe (1$ rightarrow$6)-$ beta$-glucan reductions compared with either single disruptant. Moreover, the residual (1$ rightarrow$6)-$ beta$-glucan polymer in kre6 skn1 double mutants is smaller in size and altered in structure. Since single disruptions of either gene lead to structurally wild type (1$ rightarrow$6)-$ beta$-glucan, KRE6 and SKN1 appear to function independently and to act early in the assembly of the polymer, possibly as glucan synthases. Consistent with their direct role in the assembly of this polymer, both Kre6p and Skn1p possess C-terminal domains with significant sequence similarity to two recently identified glucan-binding proteins. / An initial characterization of Kre6p and Skn1p reveal both to be phosphorylated integral-membrane glycoproteins, with Kre6p likely localized to the Golgi apparatus. The topology implied by the post-translational modifications of Kre6p and Skn1p, offers the potential for both proteins to link cytoplasmic regulation with a secretory pathway-based assembly of the (1$ rightarrow$6)-$ beta$-glucan polymer. The observed phosphorylation of both Kre6p and Skn1p prompted an examination for genetic interactions with suspected cell wall regulating kinases. KRE6-dependent suppression of the pkc1 lysis defect, as well as synthetic lethal interactions between several KRE genes and members of the PKC1-mediated MAP kinase pathway, supports a role for the PKC1 pathway in regulating synthesis of cell wall components, including (1$ rightarrow$6)-$ beta$-glucan.

Analyses of genes involved in yeast cell wall $ beta$1,6-glucan assembly have identified CWH41, PTC1/CWH47, EXG1, PBS2 and a family of genes related to the human oxysterol binding protein, OSBP. CWH41 encodes a novel membrane N-glycoprotein located in the ER. Disruption of CWH41 leads to a K1 killer toxin resistant phenotype, and a 50% reduction in the $ beta$1,6-glucan level The $cwh{ it 41 /} Delta$ mutant displayed strong synergistic defects with $kre{ it 1 /} Delta$ or $kre{ it 1 /} Delta$ null mutations: the $cwh{ it 41 /} Delta kre{ it 6 /} Delta$ double mutant showed a slow-growth phenotype and a 75% reduction in $ beta$1,6-glucan level, and cells containing $cwh{ it 41 /} Delta kre{ it 6 /} Delta$ double mutations were nonviable. These results indicate that CWH41 is involved in the assembly of $ beta$1,6-glucan. / PTC1/CWH47 encodes a serine/threonine phosphatase, PBS2 is the structural gene for a MAPK kinase, and EXG1 codes for an exo-$ beta$-glucanase. Overexpression of EXG1 led to a killer resistant phenotype and a reduction in ($ beta$1,6-glucan level; whereas the $exg{ it 1 /} Delta$ mutant displayed modest increases in killer sensitivity and $ beta$1,6-glucan levels. Disruption of PTC1/CWH47 and overexpression of PBS2 resulted in similar $ beta$-glucan related phenotypes, with elevated EXG1 transcription, increased Exg1p activity, reduced $ beta$1,6-glucan levels, and resistance to killer toxin. The killer resistant phenotype caused by PTC1/CWH47 disruption or PBS2 overproduction were partially suppressed by the $exg{ it 1 /} Delta$ null mutation. These results suggest that Ptc1p/Cwh47p and Pbs2p play opposing regulatory roles in $ beta$-glucan assembly, and this is effected in part by modulating Exg1p activity. / Three yeast genes, KES1, HES1 and OSH1, whose products show homology to the human oxysterol binding protein, were also identified. Mutations in these genes resulted in sterol-related phenotypes, which include tryptophan-transport defects and nystatin resistance. In addition, mutant combinations showed small but cumulative reductions in membrane ergosterol levels. The three genes are also functionally related; since overexpression of HES1 or KES1 alleviated the tryptophan-transport defect in $kes{ it 1 /} Delta$ or $osh{ it 1 /} Delta$ mutants, respectively. These observations implicate the KES1-related gene family in ergosterol synthesis and provide comparative evidence of a role for human OSBP in cholesterol synthesis.

Mutations in the yeast gene KRE1 lead to resistance to the K1 killer toxin of S. cerevisiae. The resistant phenotype is associated with a 40% reduction of the cell wall (1 $ to$ 6)-$ beta$-glucan fraction. Yeast cell wall (1 $ to$ 6)-$ beta$-glucan is a highly branched glucose polymer composed predominantly of linear (1 $ to$ 6)-$ beta$- sc D-linked glucopyranosyl residues. This glucan acts as a receptor for the killer toxin, leading to a concentration of active toxin on the yeast cell surface. The KRE1 gene was cloned by complementation of the kre1-1 mutant allele. The KRE1 gene encodes a serine and threonine rich protein, that is directed into the yeast secretory pathway, where it is highly modified, probably through O-linked glycosylation. Haploid strains with a kre1::HIS3 disruption appear to grow somewhat more slowly than wild type, and show an unusual wall structure when examined using electron microscopy. As with strains that carry a mutant kre1-1 allele those with a kre1::HIS3 disruption show a 40% reduced level of cell wall (1 $ to$ 6)-$ beta$-glucan. Structural comparison of the (1 $ to$ 6)-$ beta$-glucan fraction isolated from a wild type strain and a kre1 mutant, showed that the glucan obtained from the mutant had fewer (1 $ to$ 6)-linked residues and displayed a smaller average polymer size. Therefore, the KRE1 gene product appears to be required for the stepwise synthesis of cell wall (1 $ to$ 6)-$ beta$-glucan.

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