The conundrum of UDP-Glc entrance into the yeast ER lumen.

UDP-Glc entrance into the endoplasmic reticulum (ER) of eukaryotic cells is a key step in the quality control of glycoprotein folding, a mechanism requiring transfer of a Glc residue from the nucleotide sugar (NS) to glycoprotein folding intermediates by the UDP-Glc:glycoprotein glucosyltransferase (UGGT). According to a bioinformatics search there are only eight genes in the Schizosaccharomyces pombe genome belonging to the three Pfam families to which all known nucleotide-sugar transporters (NSTs) of the secretory pathway belong. The protein products of two of them (hut1+ and yea4+) localize to the ER, those of genes gms1+, vrg4+, pet1+, pet2+ and pet3+ to the Golgi, whereas that of gms2+ has an unknown location. Here we demonstrate that (1) Δhut1 and Δgpt1 (UGGT null) mutants share several phenotypic features; (2) Δhut1 mutants show a 50% reduction in UDP-Glc transport into ER-derived membranes; (3) in vivo UDP-Glc ER entrance occurred in Δhut1Δyea4Δgms2 mutants and in cells in which Δhut1 disruption was combined with that of each of four of the genes encoding Golgi-located proteins. Therefore, disruption of all genes whose products localize to the ER or have an unknown location did not obliterate UDP-Glc ER entrance. We conclude that the hut1+ gene product is involved in UDP-Glc entrance into the ER, but that at least another as yet unknown NST displaying an unconventional sequence operates in the yeast secretory pathway. This conclusion agrees with our previous results showing that UDP-Glc entrance into the yeast ER does not follow the classical NST antiport mechanism.

Origin and Evolution of Two Independently Duplicated Genes Encoding UDP- Glucose: Glycoprotein Glucosyltransferases in Caenorhabditis and Vertebrates.

UDP- glucose: glycoprotein glucosyltransferase (UGGT) is a protein that operates as the gatekeeper for the endoplasmic reticulum (ER) quality control mechanism of glycoprotein folding. It is known that vertebrates and Caenorhabditis genomes harbor two uggt gene copies that exhibit differences in their properties.Bayesian phylogenetic inference based on 195 UGGT and UGGT-like protein sequences of an ample spectrum of eukaryotic species showed that uggt genes went through independent duplications in Caenorhabditis and vertebrates. In both lineages, the catalytic domain of the duplicated genes was subjected to a strong purifying selective pressure, while the recognition domain was subjected to episodic positive diversifying selection. Selective relaxation in the recognition domain was more pronounced in Caenorhabditis uggt-b than in vertebrates uggt-2 Structural bioinformatics analysis revealed that Caenorhabditis UGGT-b protein lacks essential sequences proposed to be involved in the recognition of unfolded proteins. When we assayed glucosyltrasferase activity of a chimeric protein composed by Caenorhabditis uggt-b recognition domain fused to S. pombe catalytic domain expressed in yeast, no activity was detected.The present results support the conservation of the UGGT activity in the catalytic domain and a putative divergent function of the recognition domain for the UGGT2 protein in vertebrates, which would have gone through a specialization process. In Caenorhabditis, uggt-b evolved under different constraints compared to uggt-a which, by means of a putative neofunctionalization process, resulted in a non-redundant paralog. The non-canonical function of uggt-b in the worm lineage highlights the need to take precautions before generalizing gene functions in model organisms.

The two Caenorhabditis elegans UDP-glucose:glycoprotein glucosyltransferase homologues have distinct biological functions.

The UDP-Glc:glycoprotein glucosyltransferase (UGGT) is the sensor of glycoprotein conformations in the glycoprotein folding quality control as it exclusively glucosylates glycoproteins not displaying their native conformations. Monoglucosylated glycoproteins thus formed may interact with the lectin-chaperones calnexin (CNX) and calreticulin (CRT). This interaction prevents premature exit of folding intermediates to the Golgi and enhances folding efficiency. Bioinformatic analysis showed that in C. elegans there are two open reading frames (F48E3.3 and F26H9.8 to be referred as uggt-1 and uggt-2, respectively) coding for UGGT homologues. Expression of both genes in Schizosaccharomyces pombe mutants devoid of UGGT activity showed that uggt-1 codes for an active UGGT protein (CeUGGT-1). On the other hand, uggt-2 coded for a protein (CeUGGT-2) apparently not displaying a canonical UGGT activity. This protein was essential for viability, although cnx/crt null worms were viable. We constructed transgenic worms carrying the uggt-1 promoter linked to the green fluorescent protein (GFP) coding sequence and found that CeUGGT-1 is expressed in cells of the nervous system. uggt-1 is upregulated under ER stress through the ire-1 arm of the unfolded protein response (UPR). Real-time PCR analysis showed that both uggt-1 and uggt-2 genes are expressed during the entire C. elegans life cycle. RNAi-mediated depletion of CeUGGT-1 but not of CeUGGT-2 resulted in a reduced lifespan and that of CeUGGT-1 and CeUGGT-2 in a developmental delay. We found that both CeUGGT1 and CeUGGT2 play a protective role under ER stress conditions, since 10 µg/ml tunicamycin arrested development at the L2/L3 stage of both uggt-1(RNAi) and uggt-2(RNAi) but not of control worms. Furthermore, we found that the role of CeUGGT-2 but not CeUGGT-1 is significant in relieving low ER stress levels in the absence of the ire-1 unfolding protein response signaling pathway. Our results indicate that both C. elegans UGGT homologues have distinct biological functions.