ИСТИНА

Glucantransferase Bgl2p is a major, conservative and constitutive protein of the cell walls (CW) of many yeast species. Bgl2p is one of the key remodeling enzymes that form glucan structural network of CW. It is a non-covalently bound and extraordinary strongly held CW protein. Bgl2p could not be extracted from the CW even during treatment with 1 % SDS at 37ºC during one hour but could be extracted by heating in water. It was demonstrated that S. cerevisiae Bgl2p molecule contains seven amyloidogenic determinants and is able to form amyloid fibrils in vitro. It is unknown whether Bgl2p is able to form amyloid fibrils in the CW or only after isolation from the CW. We assume that Bgl2p in CW is represented in amyloid form and its fibrilization may proceed with involvement of CW lipid components. Here we have demonstrated that Bgl2p can be extracted by chloroform-methanol (2:1) from the CW treated as well as untreated with 1% SDS at 37°C after standard procedure of isolation. After extraction water-methanol and chloroform fractions were analyzed by Western blotting and by MALDI mass-spectrometry. By both methods Bgl2p was detected in the chloroform fraction while in the water-methanol fraction it was absent. Bgl2p can be extracted from the CW obtained from both exponential and logarithmic phases of yeast growth. Importantly to note that Bgl2p extracted by heating in water at 90ºC demonstrated ability to form fibrils, but when analyzed in denaturing conditions was detected as a monomer presumably. At the same time Bgl2p extracted by methanol-chloroform without heating revealed presence of dimers, tetramers and high molecular forms with only negligible amount of monomers when analyzed in denaturing conditions. Our results give the evidence that Bgl2p is associated with S. cerevisiae CW lipid components extracted with methanol-chloroform. Data obtained also confirm our assumption that Bgl2p in the CW could be represented as amyloid fibrils. Our data together with the literature allow us to suggest that lipid component can be involved in the process of Bgl2p fibrillization and strong binding to the CW.