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Specific Labelling of Cell Wall Proteins by Biotinylation. Identification of Four Covalently Linked O‐mannosylated Proteins of Saccharomyces cerevisiae
Author(s) -
Mrsă Vladimir,
Seidl Thomas,
Gentzsch Martina,
Tanner Widmar
Publication year - 1997
Publication title -
yeast
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.923
H-Index - 102
eISSN - 1097-0061
pISSN - 0749-503X
DOI - 10.1002/(sici)1097-0061(19970930)13:12<1145::aid-yea163>3.0.co;2-y
Subject(s) - biotinylation , cell wall , biochemistry , biology , saccharomyces cerevisiae , streptavidin , horseradish peroxidase , microbiology and biotechnology , cell , covalent bond , biotin , cell disruption , gene , enzyme , chemistry , organic chemistry
Intact Saccharomyces cerevisiae cells were biotinylated with the non‐permeable sulfosuccinimidyl‐6‐(biotinamido)hexanoate reagent. Twenty specifically labelled cell wall proteins could be extracted and visualized on SDS gels via streptavidin/horseradish 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°C, 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 four genes did not yield a phenotype. In the CCW11 disruption the Ccw11p as well as the laminarinase‐extracted Ccw5 protein was missing. The new cell wall proteins are O‐mannosylated, contain a Kex2 processing site, but no C‐terminal GPI anchor sequence. © 1997 John Wiley & Sons, Ltd.