Naslov
Localisation of NaOH-extractable proteins (PIR-protein family) in the Saccharomyces cerevisiae cell wall

Autori
Mrša, Vladimir ; Kokanj, Dejana ; Ecker, Margit ; Tanner, Widmar

Vrsta, podvrsta i kategorija rada
Sažeci sa skupova, sažetak, znanstveni

Izvornik
Molecular Mechanisms of Fungal Cell Wall Biogenesis : abstracts / Aebi, Markus ; Tanner, Widmar - Monte Verità : Ascona, 2001

Skup
Molecular Mechanisms of Fungal Cell Wall Biogenesis

Mjesto i datum
Ascona, Švicarska, 26.08.2001. - 31.08.2001

Vrsta sudjelovanja
Poster

Vrsta recenzije
Međunarodna recenzija

Ključne riječi
yeast cell wall; NaOH-extractable proteins

Sažetak
S. cerevisiae cell walls contain more than 20 different mannoproteins. Some of them are linked to the glucan backbone noncovalently, and can be extracted by SDS or DTT. Two kinds of covalently linked cell wall proteins can be distinguished. The first group can be extracted from purified cell walls using different glucnase preparations. They all posses C-terminal signal for the attachment of the GPI-anchor. The second group was obtained by NaOH extraction. Four proteins of this group were identified and designated Ccw5p to Ccw8p ( Ccw for Covalently linked cell wall). Ccw6p, Ccw7p and Ccw8p were products of PIR1, PIR2, and PIR3 genes , which contain a characteristic repetiitve sequence (PIR for Proteins with internal repeats). They belong to the same family sharing a high degree of identity, all are extensively O-glycosylated and do not posses the GPI-anchoring signal. Therefore, they must be incorporated in the cell wall in a different way. To test whether Pir-proteins are targeted to the wall chitin, proteins were isolated from purified cell walls by chitinase. Three protein bands at 47, and 117kDa were obtained in the extract. The same bands were, however obtained from the mutant lacking all four Pir-proteins, indicating that chitnase-extractable proteins were not Pir-proteins. To find out which part of Pir-proteins is responsible for their attachment to glucan, Ccw5p was tagged with the haemagglutinin tag at the C-terminus, and deletions of different parts of the CCW5 were analysed. Protein lacking one repetitive unit was still attached to the cell wall, but if both copies were deleted, Ccw5p was secreted into the medium. This strongly suggests that Pir-proteins are attached to the carbohydrate network via their repetitive sequence. To check whether this sequence is a sufficient requirement for the localisation, a hybrid protein with the Ccw5p repetitive sequence added to the N-terminal end of the lacZ galactosidase was constructed. Results showed that the hybrid was indeed directed to the cell wall. Its overproduction however, affeceted the incorporation of Pir-proteins into wall. To investigate the nature of linker between Pir-proteins and glucan, the sizes of Ccw5p isolated by glucanase, NaOH and the overproduced protein secreted into the medium, were compared. The NaOH-extracted protein was about 500 Dalton smaller than the other two protein species indicating the linker is a small molecule added to the protein before its attachment to glucan.

Izvorni jezik
Engleski

Znanstvena područja
Prehrambena tehnologija

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