UDP-Glc enters S. pombe ER by both a known antiport and a novel but unknown mechanism.

BUZZI, LI; BREDESTON, LM; PARODI, AJ; D'ALESSIO, C

Buenos Aires

Congreso; Molecular Mechanisms in Cell Signaling and gene expression; 2013

UDP-Glc:glycoprotein glucosyltransferase (UGGT, coded by gpt1 gene) is a glycoprotein folding status sensor in the endoplasmic reticulum (ER) that tags with a glucose moiety incompletely folded glycoproteins, thus favoring their interaction with calnexin and calreticulin. UGGT substrate, UDP-Glc, is synthesized in the cytosol and the mechanism of its import into the ER is still unknown. From six putative nucleotide sugar transporter (NST) homologues in the genome of Schizosaccharomyces pombe, only hut1+ and yea4+ bear an ER retention signal. We first disrupted both genes in a Dalg5 background (Protein-linked Glc1Man9GlcNAc2 formation in Dalg5 or Dalg6 mutants is only due to UGGT activity and indicates entrance of UDP-Glc into the ER). ER vesicles purified from Dalg5Dhut1 but not Dalg5Dyea4 mutants showed a 50% decrease in UDP-Glc transport. However, in vivo labeling of Dalg5Dhut1 mutants resulted in Glc1Man9GlcNAc2 synthesis. We now disrupted all genes coding for NST in S. pombe genome in an Dalg5 background. Only Dalg5Dhut1 mutants showed the same growth lethal phenotype at 37ºC and aberrant morphology at 28ºC as Δalg6Δgpt1 mutants. These results suggest that even though hut1+ appears to be involved in UDP-Glc entrance into the ER, at least another unknown pathway exists in eukaryotic cells.