EVIDENCE OF ALTERED ENDOMEMBRANES IN FISSION YEASTS LACKING GLUCOSIDASE I, A MODEL FOR HUMAN CONGENITAL DISORDER OF GLYCOSYLATION CDG IIb

VALKO, AYELÉN; GONZALEZ, ANA C.; ETCHEGARAY, EMILIANA; GALLO, GIOVANNA L.; ARAMBURU, SOFÍA I.; D'ALESSIO, CECILIA

Salta

Congreso; Joint LV Annual SAIB Meeting and XIV PABMB conference en Salta; 2019

SAIB

Glucosidase I (GI) is an endoplasmic reticulum(ER) membrane protein that removes the outermost glucose from the glycan Glc3Man9GlcNAc2(G3M9) immediately after protein N-glycosylation. Mutations in GI-encoding gene (gls1+) result in humancongenital disorders of glycosylation (CDG) IIb, also called MOGS-CDG. Usingthe fission yeast Schizosaccharomyces pombe lacking GI as a modelorganism we demonstrated that the main cause of the morphological and growthdefects observed in mutant cells was the persistence of G3M9 structures inglycoproteins, as a second mutation in alg10+ gene (which is responsible for the addition of the last Glcduring the lipid-linked G3M9 synthesis) substantially suppressed the observed defects. The sickphenotype of Δgls1 mutant cells could not be ascribedto a product inhibition of oligosaccharyltransferase transfer reaction, to theinability of glycoproteins to enter into calnexin-folding cycles, or to apotentially reduced ER-associated degradation. Glycan elongation of glycoproteins in the Golgiand the overall cell wall (CW) monosaccharide composition of Δgls1 mutants were indistinguishablefrom those observed in cells lacking glucosidase II (Δgls2α), which display a wild typephenotype.However, transmission electron microscopy (TEM) showed that the CW of Δgls1 mutants was thicker than WTand Δgls2α ones, presenting a feathered appearance, and adisorganized arrangement without its characteristic three-layered structure. Endomembranesystem was also altered in cells lacking GI as: 1) subcortical ER structureslocalized below the plasma membrane were apparently absent or mislocalized inmutant cells observed by TEM, 2) CW glycoproteins region was wider in Δgls1 cells than in WT ones as revealed by staining with fluorescent-labeledlectins Griffonia(bandeiraea) simplicifolia (recognizes Galactoseterminal residues) and Concanavalin A (recognizes high-Mannose glycans) , and 3) thelack of GI producescells with highly fragmented vacuoles in hypotonic conditions (revealed by FM4-64 staining) whichpossibly cannot undergo homotypic fusion. Collectively, these results suggest theoccurrence of alterations in the secretory/endocytic pathway in cells lackingGI and shed light on the underlying molecular and cellular mechanisms of CDGIIb disease.