The Post-Translational Modifications, Localization, and Mode of Attachment of Non-Covalently Bound Glucanosyltransglycosylases of Yeast Cell Wall as a Key to Understanding their Functioning.

Glucan linked to proteins is a natural mega-glycoconjugate (mGC) playing the central role as a structural component of a yeast cell wall (CW). Regulation of functioning of non-covalently bound glucanosyltransglycosylases (ncGTGs) that have to remodel mGC to provide CW extension is poorly understood. We demonstrate that the main ncGTGs Bgl2 and Scw4 have phosphorylated and glutathionylated residues and are represented in CW as different pools of molecules having various firmness of attachment. Identified pools contain Bgl2 molecules with unmodified peptides, but differ from each other in the presence and combination of modified ones, as well as in the presence or absence of other CW proteins. Correlation of Bgl2 distribution among pools and its N-glycosylation was not found. Glutathione affects Bgl2 conformation, probably resulting in the mode of its attachment and enzymatic activity. Bgl2 from the pool of unmodified and monophosphorylated molecules demonstrates the ability to fibrillate after isolation from CW. Revealing of Bgl2 microcompartments and their mosaic arrangement summarized with the results obtained give the evidence that the functioning of ncGTGs in CW can be controlled by reversible post-translational modifications and facilitated due to their compact localization. The hypothetical scheme of distribution of Bgl2 inside CW is represented.

Dissection of differential vanadate sensitivity in two Ogataea species links protein glycosylation and phosphate transport regulation.

The closely related yeasts Ogataea polymorpha and O. parapolymorpha differ drastically from each other by sensitivity to the toxic phosphate analog vanadate. Search for genes underlying this difference revealed two genes, one designated as ABV1 (Alcian Blue staining, Vanadate resistance), which encodes a homologue of Saccharomyces cerevisiae Mnn4 responsible for attachment of mannosylphosphate to glycoside chains of secretory proteins, and the other designated as its S. cerevisiae homologue PHO87, encoding the plasma membrane low affinity phosphate sensor/transporter. The effect of Pho87 on vanadate resistance was bidirectional, since it decreased the resistance on phosphate-depleted medium, but was required for pronounced protection against vanadate by external phosphate. This highlights the dual function of this protein as a low affinity phosphate transporter and an external phosphate sensor. Involvement of Pho87 in phosphate sensing was confirmed by its effects on regulation of the promoter of the PHO84 gene, encoding a high affinity phosphate transporter. The effect of Abv1 was also complex, since it influenced Pho87 level and enhanced repression of the PHO84 promoter via a Pho87-independent pathway. Role of the identified genes in the difference in vanadate resistance between O. polymorpha and O. parapolymorpha is discussed.