EVIDENCE FOR MORE THAN ONE PATHWAY TO IMPORT UDP-GLC INTO THE ENDOPLASMIC RETICULUM OF S. POMBE

BREDESTON, L; PRUCCA, CG; LUJAN, HD; PARODI, AJ; D'ALESSIO, C

Buenos Aires, Argentina

Congreso; XII Jornadas Anuales de la Sociedad Argentina de Biología; 2010

Sociedad Argentina de Biología

The UDP-Glc:glycoprotein glucosyltransferase (GT) serves as a glycoprotein folding sensor, labeling incompletely folded glycoproteins in the endoplasmic reticulum (ER) lumen with a glucose (Glc) tag. Its donor substrate, UDP-Glc, is synthesized in the cytosol. From six putative nucleotide sugar transporter (NST) homologues identified in the genome of the fission yeast Schizosaccharomyces pombe, only hut1+ and yea4+ bear the ER retention signal. We disrupted both genes in S. pombe Dalg5 background. This allows assignation of protein-linked Glc1Man9GlcNAc2 formation to GT activity and thus entrance of UDP-Glc into the ER lumen. ER vesicles purified from Dalg5Dhut1 mutants showed a 50% decrease in UDP-Glc transport compared with that of Dalg5, suggesting that hut1+ is involved in UDP-Glc transport into the ER. However, in vivo labeling of S.p Dalg5Dhut1 (and also of Dalg5Dyea4 and Dalg5Dhut1Dyea4 mutants) resulted in Glc1Man9GlcNAc2 synthesis. This suggests that there is at least another pathway for importing UDP-Glc into the ER: either through a NST not bearing the classic ER retention signal or by an novel mechanism. The parasite Giardia lamblia provides a new system for testing heterologous UDP-Glc transporters as its membranes only transport UDP-GlcNAc. We expressed S.p hut1+ and S.p. yea4+ in the parasite and preliminary results confirmed that hut1+ is a UDP-Glc transporter. We are currently expressing the rest of the NSTs in Giardia to determine their substrate transport specificity.