IDENTIFICATION OF ER UDP-Glc TRANSPORTERS IN YEAST: THE SEARCH CONTINUES

CECILIA D'ALESSIO; LUIS BREDESTON; ARMANDO J. PARODI

Rosario, Santa Fe, Argentina

Congreso; XLII Reunión Anual de la Sociedad Argentina de Investigación en Bioquímica y Biología Molecular; 2006

Sociedad Argentina de Investigación en Bioquímica y Biología Molecular

The UDP-Glc:glycoprotein glucosyltransferase (GT) labels incompletely folded glycoproteins in the ER lumen with a Glc tag. Its donor substrate, UDP-Glc, is synthesized in the cytosol. Several nucleotide sugar transporters (NST) have been identified, but only AtUTR1 from Arabidopsis thaliana has been proposed as an ER UDP-Glc transporter. To identify an ER UDP-Glc transporter gene in yeasts we looked for NST homologues bearing the canonical ER retention signals in the genomes of S. cerevisiae and S. pombe. Two of such genes occur in each yeast: hut1+ (an orthologue of AtUTR1) and yea4+. We disrupted both genes in S. pombe and S. cerevisiae alg5 or alg6 mutant backgrounds, and transformed the last yeast with S. pombe GT cDNA (gpt1+). The use of alg mutants allows assignation of protein-linked Glc1Man9GlcNAc2 formation to GT activity in the ER, and thus entrance of UDP-Glc into the lumen. Both S.p. alg6 gpt1 and alg6 hut1 double mutants, showed similar aberrant morphology and thermal sensitivity, thus suggesting glycoprotein misfolding as the common defect. Nevertheless, in vivo labeling of S.c. alg5 hut1, S.c. alg5 yea4,  S.p. alg6 hut1, S.p. alg6 yea4 and S.p. alg6 hut1 yea4 resulted in Glc1Man9GlcNAc2 synthesis, thus suggesting UDP-Glc entrance into the ER lumen through a NST not bearing the classic ER retention signal or, alternatively, by a totally novel mechanism.