The UDP-Glc:Glycoprotein Glucosyltransferase Is Essential for <i>Schizosaccharomyces pombe</i> Viability under Conditions of Extreme Endoplasmic Reticulum Stress

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Nucleoside diphosphates generated by glycosyltransferases in the fungal, plant, and mammalian cell secretory pathways are converted into monophosphates to relieve inhibition of the transferring enzymes and provide substrates for antiport transport systems by which the entrance of nucleotide sugars from the cytosol into the secretory pathway lumen is coupled to the exit of nucleoside monophosphates. Analysis of the yeast Schizosaccharomyces pombe genome revealed that it encodes two enzymes with potential nucleoside diphosphatase activity, Spgda1p and Spynd1p. Characterization of the overexpressed enzymes showed that Spgda1p is a GDPase/UDPase, whereas Spynd1p is an apyrase because it hydrolyzed both nucleoside tri and diphosphates. Subcellular fractionation showed that both activities localize to the Golgi. Individual disruption of their encoding genes did not affect cell viability, but disruption of both genes was synthetically lethal. Disruption of Spgda1 ؉ did not affect Golgi N-or O-glycosylation, whereas disruption of Spynd1 ؉ affected Golgi N-mannosylation but not O-mannosylation. Although no nucleoside diphosphatase activity was detected in the endoplasmic reticulum (ER), N-glycosylation mediated by the UDP-Glc:glycoprotein glucosyltransferase (GT) was not severely impaired in mutants because first, no ER accumulation of misfolded glycoproteins occurred as revealed by the absence of induction of BiP mRNA, and second, in vivo GT-dependent glucosylation monitored by incorporation of labeled Glc into folding glycoproteins showed a partial (35-50%) decrease in Spgda1 but was not affected in Spynd1 mutants. Results show that, contrary to what has been assumed to date for eukaryotic cells, in S. pombe nucleoside diphosphatase and glycosyltransferase activities can localize to different subcellular compartments. It is tentatively suggested that ER-Golgi vesicle transport might be involved in nucleoside diphosphate hydrolysis.Almost all nucleotide sugar-dependent glycosyltransferases in the secretory pathway generate nucleoside diphosphates that are converted into monophosphates to relieve inhibition of the transferring enzymes and provide substrates for antiport transport systems by which the entrance of nucleotide sugars from the cytosol into the lumen of the secretory pathway is coupled to the exit of nucleoside monophosphates (1). Several secretory pathway nucleoside diphosphatases have been described already. There are two enzymes displaying such enzymatic activity in Saccharomyces cerevisiae, a GDPase/UDPase and an apyrase (denominated Gda1p and Ynd1p, respectively) (2-4). Apyrases differ from nucleoside diphosphatases, as the former are able to degrade not only nucleoside diphosphates but also triphosphates. Both enzymatic activities are localized to the Golgi apparatus. Irrespective of their cellular origin, all enzymes able to hydrolyze nucleoside diphosphates (nucleoside diphosphatases proper and apyrases) share four highly similar sequences that have been called apyrase conserved regions. Analysis of th...

Section: Methodsmentioning

confidence: 99%

“…The construction was characterized by Southern blotting analysis as described previously (20 :ura4 ϩ were conjugated in malt extract medium, and diploids were selected in minimal medium solely supplemented with leucine. Diploids spontaneously sporulate after 7 days in this medium.…”

Section: Methodsmentioning

confidence: 99%

Nucleoside Diphosphatase and Glycosyltransferase Activities Can Localize to Different Subcellular Compartments in Schizosaccharomyces pombe

D’Alessio

Trombetta

Parodi

2003

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“…The proper folding and assembly of proteins in the endoplasmic reticulum (ER) 6 is maintained by quality control mechanisms that avoid the release of misfolded proteins by retaining them within the organelle.…”

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confidence: 99%

AtUTr1, a UDP-glucose/UDP-galactose Transporter from Arabidopsis thaliana, Is Located in the Endoplasmic Reticulum and Up-regulated by the Unfolded Protein Response

Reyes

Marchant

Norambuena

et al. 2006

The folding of glycoproteins in the endoplasmic reticulum (ER) depends on a quality control mechanism mediated by the calnexin/ calreticulin cycle. During this process, continuous glucose trimming and UDP-glucose-dependent re-glucosylation of unfolded glycoproteins takes place. To ensure proper folding, increases in misfolded proteins lead to up-regulation of the components involved in quality control through a process known as the unfolded protein response (UPR). Reglucosylation is catalyzed by the ER lumenal located enzyme UDP-glucose glycoprotein glucosyltransferase, but as UDP-glucose is synthesized in the cytosol, a UDPglucose transporter is required in the calnexin/calreticulin cycle. Even though such a transporter has been hypothesized, no protein playing this role in the ER yet has been identified. Here we provide evidence that AtUTr1, a UDP-galactose/glucose transporter from Arabidopsis thaliana, responds to stimuli that trigger the UPR increasing its expression around 9-fold. The accumulation of AtUTr1 transcript is accompanied by an increase in the level of the AtUTr1 protein. Moreover, subcellular localization studies indicate that AtUTr1 is localized in the ER of plant cells. We reasoned that an impairment in AtUTr1 expression should perturb the calnexin/calreticulin cycle leading to an increase in misfolded protein and triggering the UPR. Toward that end, we analyzed an AtUTr1 insertional mutant and found an up-regulation of the ER chaperones BiP and calnexin, suggesting that these plants may be constitutively activating the UPR. Thus, we propose that in A. thaliana, AtUTr1 is the UDP-glucose transporter involved in quality control in the ER.The proper folding and assembly of proteins in the endoplasmic reticulum (ER) 6 is maintained by quality control mechanisms that avoid the release of misfolded proteins by retaining them within the organelle.Most of the proteins synthesized in the ER are glycoproteins that contain the oligosaccharide Glc 3 Man 9 GlcNAc 2 . This structure is co-translationally transferred to the protein and immediately trimmed to GlcMan 9 GlcNAc 2 (1-3). The chaperones calnexin and calreticulin bind this monoglucosylated form retaining the glycoprotein in the ER, facilitating its folding (4). To release the protein from the chaperone, the glucose residue is removed by glucosidase II, and if the protein is correctly folded, it can exit the ER (2, 5). However, if the protein is misfolded, it is recognized by UDP-glucose glycoprotein glucosyltransferase (UGGT) and reglucosylated, thereby allowing its re-association with calnexin and calreticulin (6 -9). Therefore, UGGT plays an essential role in quality control by allowing the re-entry of the glycoprotein into the folding cycle.UGGT is located in the lumen of the ER and utilizes UDP-glucose as substrate. However, UDP-glucose is synthesized in the cytosol and needs to be translocated into the lumen. The protein involved in this process likely belongs to the family of nucleotide sugar transporters (NSTs). Although several NSTs have...

“…In addition to the suggested role in the recognition of misfolded conformers, the results presented here indicate that the Nterminal domains are required for proper folding of the catalytic C-terminal portions, as the latter were enzymatically and functionally inactive and rapidly degraded in vivo when expressed in the absence of the former. ϩ 33A and expression vector pREP3Xgpt1 ϩ , which encode S. pombe GT, were generated in our laboratory (13). Plasmid pOT2gpt1 ϩ (D. melanogaster GT) was a generous gift from Dr. S. E. Trombetta (Department of Cell Biology, Yale University Medical School).…”

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confidence: 99%

The UDP-glucose:Glycoprotein Glucosyltransferase Is Organized in at Least Two Tightly Bound Domains from Yeast to Mammals

Guerin

Parodi

2003

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The endoplasmic reticulum UDP-Glc:glycoprotein glucosyltransferase (GT) exclusively glucosylates nonnative glycoprotein conformers. GT sequence analysis suggests that it is composed of at least two domains: the N-terminal domain, which composes 80% of the molecule, has no significant similarity to other known proteins and was proposed to be involved in the recognition of non-native conformers and the C-terminal or catalytic domain, which displays a similar size and significant similarity to members of glycosyltransferase family 8. Here, we show that N-and C-terminal domains from Rattus norvegicus and Schizosaccharomyces pombe GTs remained tightly but not covalently bound upon a mild proteolytic treatment and could not be separated without loss of enzymatic activity. The notion of a two-domain protein was reinforced by the synthesis of an active enzyme upon transfection of S. pombe GT null mutants with two expression vectors, each of them encoding one of both domains. Transfection with the Cterminal domain-encoding vector alone yielded an inactive, rapidly degraded protein, thus indicating that the N-terminal domain is required for proper folding of the C-terminal catalytic portion. If, indeed, the N-terminal domain is, as proposed, also involved in glycoprotein conformation recognition, the tight association between N-and C-terminal domains may explain why only Nglycans in close proximity to protein structural perturbations are glucosylated by the enzyme. Although S. pombe and Drosophila melanogaster GT N-terminal domains display an extremely poor similarity (16.3%), chimeras containing either yeast N-terminal and fly Cterminal domains or the inverse construction were enzymatically and functionally active in vivo, thus indicating that the N-terminal domains of both GTs shared three-dimensional features.