SPECIFIC LABELING OF CELL-WALL PROTEINS BY BIOTINYLATION - IDENTIFICATION OF 4 COVALENTLY-LINKED O-MANNOSYLATED PROTEINS OF SACCHAROMYCES-CEREVISIAE

Intact Saccharomyces cerevisiae cells were biotinylated with the non-p ermeable sulfosuccinimidyl-6-(biotinamido)hexanoate reagent. Twenty sp ecifically labelled cell wall proteins could be extracted and visualiz ed on SDS gels via streptavidin/horseradish peroxidase. Nine cell wall proteins were released by SDS extraction under reducing conditions an d were designated Scw1-9p for (Soluble cell wall proteins); five prote ins were released from SDS-extracted cell walls by laminarinase (Ccw1- 5p for covalently linked cell wall proteins) and six with mild (30 mM- NaOH, 4 degrees C, 14 h) alkali treatment (Ccw6-11p). N-terminal seque nces of the Ccw proteins 6, 7, 8 and 11 showed that these cell wall pr oteins are members of the PIR gene family (predicted proteins with Int ernal repeats), CCW6 being identical to PIR1 and CCW8 to PIR3. Single gene disruptions of all four genes did not yield a phenotype. In the C CW11 disruption the Ccw11p as well as the laminarinase-extracted Ccw5 protein was missing. The new cell wall proteins are O-mannosylated, co ntain a Kex2 processing site, but no C-terminal GPI anchor sequence. ( C) 1997 John Wiley & Sons, Ltd.