Transmembrane domain of glucosidase I is involved in its function in the fission yeast endoplasmic reticulum

IDROVO-HIDALGO, TOMMY; MIQUET, LEANDRO; ALONSO, GUILLERMO D; DALESSIO, CECILIA

Mendoza

Congreso; LVIII Annual Meeting of the Argentine Society for Biochemistry and Molecular Biology Research; 2022

Sociedad Argentina de Investigaciones en Bioquímica y Biología Molecular

Protein N-glycosylation is a highly conserved process and one of the most relevant post-translational modifications in eukaryotes. It begins with oligosaccharyltransferase (OST), a complex present in the membrane of the endoplasmic reticulum (ER), transferring the pre-assembled oligosaccharide Glc3Man9GlcNAc2 (G3M9) to asparagine residues of proteins that are being translocated into the ER. ERglucosidase I (GI), trims immediately the outermost glucose of G3M9 converting it to G2M9 and allowing glucosidase II (GII), to convert it to G1M9, which is in turn recognized by the Endoplasmic Reticulum Quality Control (ERQC) mechanism that facilitates folding and of secretory and membrane proteins and marks terminally misfolded polypeptides for degradation. Defects associated with GI, produce type IIb “Congenital disorders of glycosylation” (CDG-IIb). This phenotypically manifests with multisystemic failures that appear at different ages in human patients. CDG-IIb has been associated with an unactive GI that would be responsible for not trimming G3M9 into G2M9. Our previous findings demonstrated that the accumulation of G3M9 due to the lack of GI in Schizosaccharomyces pombe is extremely toxic for the cell, resulting in a sick phenotype of GI yeast mutants. This adverse phenotype could be partially -but not fully- rescued by knocking out an alpha-1,2-glucosyltransferase (alg10p) responsible for adding theoutermost residue of glucose during glycan biosynthesis, the same that is a substrate for GI. Interestingly, mutations found in patients show that most are not within the proposed catalytic pair residues of GI. Both results could imply that not only the catalytic function of the enzyme is critical for the correct development of the cell, but that other domain/s of GI could also be important. While GII is a soluble resident protein of the ER, GI is a membrane bound protein that contains three domains: a globular catalytic domain inside the ER, a transmembrane domain, and a short cytosolic tail. In this work, we evaluated the phenotypic effect of the genetic complementation of a catalytic-domain-only GI (cGI) of ΔGI fission yeast mutants. GI variant’s proper expression and localization was confirmed by fluorescence microscopy and western blot. We assessed viability and growth rate by spot assay in solid and liquid media, respectively. Also, phenotypic cell differences were observed by transmission microscopy, and cells lengths were measured and compared by a one-way ANOVA. Our findings show that adding back cGI to ΔGI mutants does not rescue the sick phenotype of fission yeasts lacking full length GI, supporting the idea that GI has an additional unknown role yet to be discovered and that membrane localization might be involved in it.