engleski

Pir Protein in the S. cerevisiae Cell Wall

S. cerevisiae cell wall is composed of a beta-1, 3-glucan network to which some chitin and more than 20 different mannoproteins are attached. Some of the cell wall proteins are linked to the glucan backbone noncovalently, and can be extracted from the walls by SDS or DTT. Covalently linked cell wall proteins can be divided according to their localisation mechanism in two groups. The first group can be extracted from purified cell walls using different glucanase preparations such as zymolyase, or laminarinase. Identification of nine proteins of this group has shown that they all posses C-terminal signal for the attachment of the GPI-anchor. The second group of covalently linked cell wall proteins was obtained by an extraction of walls with 30 mM NaOH. Four proteins of this group were identified by N-terminal sequencing and they were designated Ccw5p to Ccw8p (Ccw for Covalently linked cell wall). It has been found that Ccw6p, Ccw7p and Ccw8p were products of PIR1, PIR2 and PIR3 genes which contain a characteristic repetitive sequence (PIR for Proteins with internal repeats). The four proteins belong to the same family sharing a high degree of identity and having several common structural features. All four proteins are extensively O-glycosylated and do not posses the signal for the addition of GPI-anchor. So there must be another molecular mechanism for the incorporation of proteins into the cell wall.The fact that, except of the Ccw5p, all other members of the Pir family were not identified in glucanase wall extracts could mean that they were attached to chitin in the wall. Therefore, proteins were isolated from purified cell walls by chitinase. Three distinct electrophoretic protein bands at 47, 67 and 117 kDa were obtained in the extract. The same bands were, however obtained from the walls of the mutant lacking all four proteins of the Pir family, indicating that chitinase-extractable proteins were not Pir-proteins. To find out which part of Pir proteins is responsible for their attachment to the cell wall, Ccw5p was used as a model. The protein was tagged with the haemagglutinin tag at the C-terminal end, and deletions of different parts of the CCW5 gene were analysed. It was found that the protein lacking one repetitive unit was still attached to the cell wall. If both copies of the repetitive sequence were deleted, the protein was secreted into the medium. This strongly suggests that Pir proteins are attached to the carbohydrate network of the wall via their repetitive sequence.

S. cerevisiae; cell wall; CCW; PIR

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