Synthesis of yeast cell wall glucan and evidence for glucan metabolism in a Saccharomyces cerevisiae whole cell system

Anti-infective Research Division, Abbott Laboratories, Abbott Park, IL 60064-3500, USA ABSTRACT The synthesis and metabolism of yeast cell wall glucan were studied using a Saccharomyces cerevisiae construct in which radiolabelled galactose is metabolized to UDP-glucose and preferentially incorporated into glucan. Greater than 85% of the incorporated radiolabel was found within insoluble cell wall material. Our study also demonstrated that radiolabelled wall glucan is released from cells growing exponentially, and that the released radiolabel is reutilizable low molecular mass material. Size exclusion chromatography and enzymic analysis indicate that laminaribiose comprises approximately 50% of the released fraction. This is consistent with in vitro findings that laminaribiose is a by-product of a newly identified glucosyltransferase (R. P. Hartland, G. W. Emerson & P. A. Sullivan, 1991, Proc R Soc Lond B 246, 155-160) associated with fungal cell walls. Our results also suggest that pre-existing glucan undergoes less metabolic processes than newly synthesized material as evidenced by a decrease in released radiolabel over time. Pulse double labelling of glucan and total cellular protein indicate that glucan metabolism and protein synthesis (ps) are not tightly coupled although they do parallel each other during exponential growth. Inhibitors of glucan synthesis (gs) decrease the glucan to protein ratio. Measurement of ps allows normalization for non-specific decreases in the rate of cell wall synthesis due to general cessation of growth. Cilofungin and papulacandin B, two putative inhibitors of gs, inhibited galactose incorporation into glucan and thus showed a decrease in the glucan to protein ratio, although ps was affected. In contrast, cycloheximide, a known ps inhibitor, displayed an elevated ratio. This whole cell construct affords a simplified system for elucidating the synthesis and metabolic activity of the yeast cell wall and enables the discrimination between specific effectors of gs and ps. Author for correspondence: Robert C. Goldman. Tel: +1 708 937 4477. Fax: +1 708 938 6603. e-mail: goldmanr@randb.abbott.com Keywords: Saccharomyces cerevisiae , glucan metabolism, yeast cell wall, whole cell system