KEG1/YFR042w of Saccharomyces cerevisiae is an essential gene that encodes a 200-amino acid polypeptide with four predicted transmembrane domains. The green fluorescent proteinor Myc 6 -tagged Keg1 protein showed the typical characteristics of an integral membrane protein and was found in the endoplasmic reticulum by fluorescence imaging. Immunoprecipitation from the Triton X-100-solubilized cell lysate revealed that Keg1 binds to Kre6, which has been known to participate in ␤-1,6-glucan synthesis. To analyze the essential function of Keg1 in more detail, we constructed temperature-sensitive mutant alleles by error-prone polymerase chain reaction. The keg1-1 mutant cells showed a common phenotype with ⌬kre6 mutant including hypersensitivity to Calcofluor white, reduced sensitivity to the K1 killer toxin, and reduced content of ␤-1,6-glucan in the cell wall. These results suggest that Keg1 and Kre6 have a cooperative role in ␤-1,6-glucan synthesis in S. cerevisiae.The fungal cell wall is an essential extracellular network composed of polysaccharides and proteins that contributes to protect the cell from internal turgor pressure and external harmful agents. It also contributes in the selective uptake and excretion of various materials and works in communication with the environment. The yeast Saccharomyces cerevisiae cell wall is composed of polysaccharides (ϳ85%) and proteins (ϳ15%) and represents ϳ30% of the cell dry weight (1). Polysaccharides form a basal fibril network with branches and covalent linkage among the components. The proteins are heavily glycosylated with N-and O-linked saccharides mostly composed of mannose. Many of these mannoproteins are covalently linked to polysaccharide networks through glycosylphosphatidylinositol-anchor remnant or alkali-labile linkage (1, 2). Some proteins are physically trapped in the mesh of networks or are chemically linked with other proteins by a disulfide bond. Polysaccharides are long fibrillar and tensile ␤-1,3-glucans of ϳ1500 glucose units/chain, amorphous and flexible ␤-1,6-glucans of ϳ350 glucose units/chain, and linear microfibril chitins of ␤-1,4-linked N-acetylglucosamine (1-2%). ␤-1,3-Glucan and chitin are synthesized by specific polymerases embedded in the plasma membrane using cytoplasmic UDP-sugars as the substrates. The localization and activity of polymerases are controlled by the actin cytoskeleton and secretory system according to the environmental stimuli and cell cycle regulatory system. Conversely, ␤-1,6-glucan synthesis is more complex, and the gene products whose mutations have defects in the ␤-1,6-glucan content are localized throughout the secretory pathway and at the cell surface (3). KRE5 and CNE1, which encode UDP-glucose:glycoprotein glucosyltransferase-and calnexin-related proteins, CWH41 and ROT2, which encode glucosidases I and II, respectively, fungus-specific genes BIG1, ROT1, KRE9 and its homologue KNH1, KRE6, and SKN1, which encode type II membrane protein with sequence and structural similarity to family 16 glycoside hydrolases, a...