Evidence of altered endomembranes in fission yeasts lacking glucosidase I, a model for human congenital disorder od glycosylation CDG IIb

VALKO, AYELEN; ETCHEGARAY, EMILIANA; ARAMBURU, SOFÍA IVANA; GONZÁLEZ ANA CLARA; GALLO GIOVANNA LUCRECIA; D'ALESSIO CECILIA

Salta

Congreso; LV Reunión anual de la SAIB y XIV Reunión anual de la Panamerican Association for Biochemistry and Molecular Biology; 2019

Sociedad Argentina de Investigación en Bioquímica y Biología Molecular. Panamerican Association for Biochemistry and Molecular Biology

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 human congenital disorders of glycosylation (CDG) IIb, also called MOGS-CDG. Using the fission yeast Schizosaccharomyces pombe lacking GI as a model organism we demonstrated that the main cause of the morphological and growth defects observed in mutant cells was the persistence of G3M9 structures in glycoproteins, as a second mutation in alg10+ gene (which is responsible for the addition of the last Glc during the lipid-linked G3M9 synthesis) substantially suppressed the observed defects. The sick phenotype of Δgls1 mutant cells could not be ascribed to a product inhibition of oligosaccharyltransferase transfer reaction, to the inability of glycoproteins to enter into calnexin-folding cycles, or to a potentially reduced ER-associated degradation. Glycan elongation of glycoproteins in the Golgi and the overall cell wall (CW) monosaccharide composition of Δgls1 mutants were indistinguishable from those observed in cells lacking glucosidase II (Δgls2α), which display a wild type phenotype. However, transmission electron microscopy (TEM) showed that the CW of Δgls1 mutants was thicker than WT and Δgls2α ones, presenting a feathered appearance, and a disorganized arrangement without its characteristic three-layered structure. Endomembrane system was also altered in cells lacking GI as: 1) subcortical ER structures localized below the plasma membrane were apparently absent or mislocalized in mutant cells observed by TEM, 2) CW glycoproteins region was wider in Δgls1 cells than in WT ones as revealed by staining with fluorescent-labeled lectins Griffonia (bandeiraea) simplicifolia (recognizes Galactose terminal residues) and Concanavalin A (recognizes high-Mannose glycans) , and 3) the lack of GI produces cells with highly fragmented vacuoles in hypotonic conditions (revealed by FM4-64 staining) which possibly cannot undergo homotypic fusion. Collectively, these results suggest the occurrence of alterations in the secretory/endocytic pathway in cells lacking GI and shed light on the underlying molecular and cellular mechanisms of CDG IIb disease.