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

IDROVO, TOMMY; MIQUET, LEANDRO; ALONSO, GUILLERMO D.; D'ALESSIO, CECILIA

Congreso; Congreso de la sociedad Argentina de Bioquímica y Biología Molecular; 2022

Protein N-glycosylationis a highly conserved process and one of the most relevant post-translationalmodifications in eukaryotes. It begins with oligosaccharyltransferase (OST), acomplex present in the membrane of the endoplasmic reticulum (ER), transferringthe pre-assembled oligosaccharide Glc3Man9GlcNAc2(G3M9) to asparagine residues of proteins that are being translocated into the ER.ER-aglucosidase I (GI), trims immediately the outermost glucose of G3M9 convertingit to G2M9 and allowing glucosidase II (GII), to convert it to G1M9, which is inturn recognized by the Endoplasmic Reticulum Quality Control (ERQC) mechanismthat facilitates folding and of secretory and membrane proteins and marksterminally misfolded polypeptides for degradation. Defects associated with GI,produce type IIb “Congenital disorders of glycosylation” (CDG-IIb). Thisphenotypically manifests with multisystemic failures that appear at differentages in human patients. CDG-IIb has been associated with an unactive GI thatwould be responsible for not trimming G3M9 into G2M9. Our previous findingsdemonstrated that the accumulation of G3M9 due to the lack of GI in Schizosaccharomyces pombe is extremely toxicfor the cell, resulting in a sick phenotype of DGI yeast mutants. This adverse phenotype could bepartially -but not fully- rescued by knocking out an alpha-1,2-glucosyltransferase(alg10p) responsible for adding the outermost residue of glucose during glycanbiosynthesis, the same that is a substrate for GI. Interestingly, mutationsfound in patients show that most are not within the proposed catalytic pairresidues of GI. Both results could imply that not only the catalytic functionof the enzyme is critical for the correct development of the cell, but that otherdomain/s of GI could also be important. While GII is a soluble resident proteinof the ER, GI is a membrane bound protein that contains three domains: aglobular catalytic domain inside the ER, a transmembrane domain, and a shortcytosolic tail. In this work, we evaluated the phenotypic effect of the geneticcomplementation of a catalytic-domain-only GI (cGI) of ΔGI fission yeast mutants.GI variant’s proper expression and localization was confirmed by fluorescencemicroscopy and western blot. We assessed viability and growth rate by spotassay in solid and liquid media, respectively. Also, phenotypic celldifferences were observed by transmission microscopy, and cells lengths were measuredand compared by a one-way ANOVA. Our findings show that adding back cGI to ΔGI mutantsdoes 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 discoveredand that membrane localization might be involved in it.