Abstract
Glucosidase I (GI) removes the outermost glucose from protein-linked Glc3Man9GlcNAc2 (G3M9) in the endoplasmic reticulum (ER). Individuals with congenital disorders of glycosylation MOGS-CDG bear mutations in the GI-encoding gene (gls1). Although GI absence has been reported to produce lethality in Schizosaccharomyces pombe yeasts, here we obtained two viable Δgls1 mutants, one with a very sick but not lethal phenotype (Δgls1-S) and the other with a healthier one (Δgls1-H). The sick strain displayed only G3M9 as an ER protein-linked oligosaccharide, whereas the healthier strain had both G3M9 and Man9GlcNAc2 The lipid-linked oligosaccharide patterns of the two strains revealed that the most abundantly formed glycans were G3M9 in Δgls1-S and Glc2Man9GlcNAc2 in Δgls1-H, suggesting reduced Alg10p glucosyltransferase activity in the Δgls1-H strain. A mutation in the alg10+ gene was indeed observed in this strain. Our results indicated that abrogated G3M9 deglucosylation was responsible for the severe defects observed in Δgls1-S cells. Further studies disclosed that the defects could not be ascribed to disruption of glycoprotein entrance into calnexin-folding cycles, inhibition of the oligosaccharyltransferase by transfer reaction products, or reduced proteasomal degradation of misfolded glycoproteins. Lack of triglucosylated glycoprotein deglucosylation neither significantly prevented glycan elongation in the Golgi nor modified the overall cell wall monosaccharide composition. Nevertheless, it resulted in a distorted cell wall and in the absence of underlying ER membranes. Furthermore, Golgi expression of human endomannosidase partially restored normal growth in Δgls1-S cells. We propose that accumulation of G3M9-bearing glycoproteins is toxic and at least partially responsible for defects observed in MOGS-CDG.
Highlights
N-Glycans have multiple functions in the secretory pathway as they influence protein folding in the ER5 and, after being remodeled in the Golgi, mediate interactions among cells and with the environment
According to an analysis of a genome-wide set of gene deletions in the fission yeast S. pombe, deletion of the Glucosidase I (GI)-encoding gene (⌬gls1 mutants) is apparently lethal [11]. This is in contrast to what was reported for S. cerevisiae, in which mutants lacking the GI-encoding gene are viable [12] but in agreement with the report of patients in which mutations in GI-encoding gene result in MOGS-congenital disorders of glycosylation (CDG) with severe and sometimes lethal pathologies [8, 13]
In an attempt to understand the role of GI in MOGS-CDG, we induced meiosis and sporulation in a heterozygous ⌬gls1/ϩ diploid S. pombe strain
Summary
Adds a Glc (residue l, Fig. 1A) exclusively to proteins that have not yet acquired their native conformation [5, 6]. Glycoprotein–lectin interactions enhance folding efficiency and allow participation of a protein-disulfide protein isomerase (ERp57) in the folding process They prevent Golgi exit of folding intermediates and irreparably misfolded glycoproteins (Fig. 1B). Cycles of glucosylation and deglucosylation catalyzed by the opposite activities of UGGT and GII continue until proper folding is achieved (Fig. 1B) [4] Folded glycoproteins pursue their transit through the secretory pathway where their glycans are further modified in the Golgi. Regarding the type II diseases, MOGS-CDG ( known as CDG-IIb) is produced by mutations in GI, the enzyme responsible for the first step in N-glycan processing (Fig. 1). As the protein encoded by that gene is responsible for the addition of the outermost glucose to the Dol-PP derivative, we concluded that accumulation of three Glc moieties in the newly synthesized glycoproteins is the main cause of the growth defect in GI mutants. We propose that at least part of the defects observed in MOGS-CDG patients are caused by the accumulation of triglucosylated glycoproteins
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