engleski

Specific labelling of cell wall proteins by biotinylation. Identification of four covalently linked O-mannosylated proteins of Saccharomyces cerevisiae.

Intact S. cerevisiae cells were biotinylated with the non-permeable Sulfosuccinimidyl-6-(biotinamido)hexanoate reagent. 20 specifically labelled cell wall proteins could be extracted and visualized on SDS gels via Streptavidin/horse radish peroxidase. Nine cell wall proteins were released by SDS extraction under reducing conditions and were designated Scw1-9p (for soluble cell wall proteins) ; five proteins 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^oC, 14 h) alkali treatment (Ccw6-11p). N-terminal sequences of the Ccw proteins 6, 7, 8 and 11 showed that these cell wall proteins are members of the PIR gene family (predicted proteins with internal repeats), CCW6 being identical to PIR1 and CCW8 to PIR3. Single gene disruptions of all 4 genes did not yield a phenotype. In the CCW11 disruption the Ccw11p as well as the laminarinase extracted Ccw5 protein was missing. The four new cell wall proteins are highly O-mannosylated, contain a Kex2 processing site, but no C-terminal GPI anchor sequence.

cell wall; mannosylation; KEX2

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