Development of a molecular tool to measure in vivo protein hypoglycosylation in fission yeasts

GALLO GIOVANNA LUCRECIA; HERRERA AGUILAR NATALIA; ORSI RAMIRO; MATTERA VANESA SOLEDAD; PARODI ARMANDO; D'ALESSIO CECILIA

Rosario

Congreso; L Reunión Anual de la Sociedad de Investigación en Bioquímica y Biología Molecular; 2014

Sociedad Argentina de Investigación en Bioquímica y Biología Molecular

During N-glycosylation the endoplasmic reticulum (ER) membrane oligosaccharytransferase complex (OST) transfers Glc3Man9GlcNAc2 from a dolichol-PP donor to the sequence Asn-X-Ser/Thr of proteins that are entering into the ER. The addition of a bulky hydrophilic glycan enhances protein folding efficiency as it reduces aggregation of folding intermediates. Defects in the glycan transfer reaction due either to OST mutations or to truncated glycan structures may result in protein hypoglycosylation (the N-glycosylation sites normally occupied with a glycan are empty) thus causing ubiquitous defects and in human diseases known as congenital disorders of glycosylation type I. It has been recently shown that a GFP variant in which an N-glycosylation site was introduced (Gly-GFP) loses fluorescence when such site is occupied by a glycan. We expressed GFP and Gly-GFP variants fused to an N-terminal S. pombe signal peptide and a C-terminal ER retention signal (ER-GFP and ER-GlyGFP), both in a wild type and in an delta alg6 mutant in which hypoglycosylation occurs. Our results showed that while expressed ER-GFP fluoresces in the ER of both strains, ER-GlyGFP only fluoresces in the ER of delta alg6 mutant, indicating that the fluorescence intensity of this construction may be used to test the glycoprotein hypoglycosylation in vivo in S. pombe.