The Two Caenorhabditis elegans UDP- Glucose:Glycoprotein Glucosyltransferase Homologues Have Distinct Biological Functions Lucila I. Buzzi 1 , Sergio H. Simonetta 2 , Armando J. Parodi 1,3 , Olga A. Castro 1,3,4 * 1 Laboratory of Glycobiology, Fundacio ´ n Instituto Leloir, Buenos Aires, Argentina, 2 Laboratory of Genetics and Molecular Physiology, Fundacio ´ n Instituto Leloir, Buenos Aires, Argentina, 3 Instituto de Investigaciones Bioquı ´micas de Buenos Aires, Consejo Nacional de Investigaciones Cientı ´ficas y Te ´ cnicas, Buenos Aires, Argentina, 4 School of Sciences, University of Buenos Aires, Buenos Aires, Argentina Abstract 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 mg/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. Citation: Buzzi LI, Simonetta SH, Parodi AJ, Castro OA (2011) The Two Caenorhabditis elegans UDP-Glucose:Glycoprotein Glucosyltransferase Homologues Have Distinct Biological Functions. PLoS ONE 6(11): e27025. doi:10.1371/journal.pone.0027025 Editor: Diane Bassham, Iowa State University, United States of America Received May 6, 2011; Accepted October 9, 2011; Published November 2, 2011 Copyright: ß 2011 Buzzi et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: Grants: PICT 2008-700 www.agencia.gov.ar and PIP 20080100567 www.conicet.gov.ar (both to Olga Castro). Howard Hughes Medical Institute, and National Institutes of Health grant GM-44500 to Armando Parodi. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: ocastro@leloir.org.ar Introduction The endoplasmic reticulum (ER) is the subcellular compartment where glycoproteins acquire their tertiary and quaternary structures. The quality control of glycoprotein folding allows cells to discriminate between native and non native protein conforma- tions, selectively transporting properly folded proteins to their final destinations through the secretory pathway, or alternatively, retrotranslocating proteins recognized by cells as irreparably misfolded or incompletely formed glycoprotein complexes to the cytosol to be degraded by proteasomes. The N-glycosylation of proteins starts with the ‘‘en bloc’’ addition of a glycan of composition Glc 3 Man 9 GlcNAc 2 to polypeptide chains in the ER lumen by the oligosaccharyltransferase complex. The glycan is first processed by two ER-resident enzymes, glucosidase I which removes the outermost glucose residue and glucosidase II (GII) that removes the middle and innermost glucose units. An ER mannosidase (s) may also excise several mannose units. Mono- glucosylated glycoproteins bearing glycans of compositions Glc 1 Man 7–9 GlcNAc 2 may interact with two ER-resident lectin chaperones, membrane bound calnexin (CNX) or its soluble homologue calreticulin (CRT). Monoglucosylated N-glycans may also be formed by reglucosylation of deglucosylated glycans of structure Man 7–9 GlcNAc 2 by the UDP-Glc:glycoproteinglucosyl- transferase (UGGT). This enzyme is the key component of the folding quality control mechanism. It behaves as a sensor of glycoprotein conformations as it exclusively glucosylates glycopro- teins not displaying their native conformations. Lectin–glycopro- tein binding and unbinding as a result of the opposing activities of UGGT and GII continues until glycoproteins either acquire their native structures or, alternatively are recognized by cells as irreparably misfolded species or as complexes unable to acquire their full subunit complement. The interaction of folding intermediates, incomplete complexes and irreparably misfolded glycoproteins with the lectin-chaperones not only prevents Golgi exit of the former but also decreases the folding rate and increases PLoS ONE | www.plosone.org 1 November 2011 | Volume 6 | Issue 11 | e27025