engleski

Localization of the PIR Protein Family in the S. cerevisiae Cell Wall

S. cerevisiae cell wall is composed of a beta-1, 3-gucan network to which more then 20 different mannoproteins are attached (1). Besides, small amounts of chitin and beta-1, 6-glucan are also found. Some of the cell wall proteins are linked to the glucan backbone noncovalently or solely via S-S links, and can be extracted from the walls by SDS or DTT (2). Six proteins of this group have been indentified so far and it has been found that they are all either hydrolytic enzymes, or at least are homologous to known yeast glucanases (2). Covalently linked cell wall proteins can be divided according to their attachment in two groups. The first group represents proteins, which can be extracted from purified cell walls depleted of SDS-extractable proteins, using different glucanase preparations such as zymolyase, or laminarinase. Identification of nine proteins of this group has shown that they all posses a C-terminal signal for the attachment of the GPI-anchor (3, 4, 5, 6). It has, therefore, been postulated that these proteins get plasma membrane anchored through GPI, and are then translocated, by a so far unknown mechanism, to beta-1, 6-glucan, which serves as a link to the beta-1, 3-glucan network of the cell wall (7). The second group of covalently linked cell wall proteins was obtained from walls after SDS extraction with 30 mM NaOH (8, 9). 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 for which it has previously been reported to contain a characteristic repetitive sequence (PIR for Proteins with internal repeats, 10). The four proteins belong to the same family sharing a high degree of identity and having several common structural features. All are proteolytically processed by the Kex2p protease so that the repetitive sequence occupies the N-terminal part of mature proteins. The number of repetitive units ranges from 2 in Ccw5p, to 9 in Ccw7p. Besides, all four proteins are extensively O-glycosylated and do not posses the signal for the addition of a GPI-anchor. The latter indicates that there must be another molecular mechanism for the incorporation of proteins into the cell wall, different from the one suggested for proteins extracted by glucanases. Therefore, the aim of this investigation was to get an insight into the way of attachment of this group of proteins to the cell wall. Since members of the Pir family are generally not found in glucanase wall extracts they may be attached to chitin in the wall, rather than to glucan. 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 obteined from the walls of the mutant lacking all four proteins of the Pir family, indicating that chitinase-extractable proteins were not Pir-proteins. The same bands were also obtained from cell walls of the mutant lacking five major glucanase-extractable proteins i.e. Ccw12p, Ccw13p, Cwp1p, Icwp1p and Tip1p, suggesting that the three proteins were so far unidentified. 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-trminal end, and deletions of different parts of the CCW5 gene were analysed. It was found taht the protein lacking one repetitive unit was attached to the cell wall as was the wild type. 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, most probably to beta-1, 3-glucan, via their repetitive sequence and that the single copy of this sequence is sufficient for the effective binding.The study of the linker which connects Pir proteins to wall carbohydrates is a subject of current investigation and the results obtained so far indicate that it most probably does not contain phosphate.

yeast; cell wall; ccw proteins; pir proteins

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