A new stress protein: synthesis of Schizosaccharomyces pombe UDP-Glc:glycoprotein glucosyltransferase mRNA is induced by stress conditions but the enzyme is not essential for cell viability.

Lumenal ecto-nucleoside tri-and di-phosphohydrolases (ENTPDases) of the secretory pathway of eukaryotes hydrolyze nucleoside diphosphates resulting from glycosyltransferase-mediated reactions, yielding nucleoside monophosphates. The latter are weaker inhibitors of glycosyltransferases than the former and are also antiporters for the transport of nucleotide sugars from the cytosol to the endoplasmic reticulum (ER) and Golgi apparatus (GA) lumen. Here we describe the presence of two cation-dependent nucleotide phosphohydrolase activities in membranes of Caenorhabditis elegans: one, UDA-1, is a UDP/GDPase encoded by the gene uda-1, whereas the other is an apyrase encoded by the gene ntp-1. UDA-1 shares significant amino acid sequence similarity to yeast GA Gda1p and mammalian UDP/GDPases and has a lumenal active site in vesicles displaying an intermediate density between those of the ER and GA when expressed in S. cerevisiae. NTP-1 expressed in COS-7 cells appeared to localize to the GA. The transcript of uda-1 but not those of two other C. elegans ENTPDase mRNAs (ntp-1 and mig-23) was induced up to 3.5-fold by high temperature, tunicamycin, and ethanol. The same effectors triggered the unfolded protein response as shown by the induction of expression of green fluorescent protein under the control of the BiP chaperone promoter and the UDP-glucose:glycoprotein glucosyltransferase. Up-regulation of uda-1 did not occur in ire-1-deficient mutants, demonstrating the role of this ER stress sensor in this event. We hypothesize that up-regulation of uda-1 favors hydrolysis of the glucosyltransferase inhibitory product UDP to UMP, and that the latter product then exits the lumen of the ER or pre-GA compartment in a coupled exchange with the entry of UDP-glucose, thereby further relieving ER stress by favoring protein re-glycosylation.The occurrence of the eukaryotic endomembrane system has brought forth a need for a mechanism to coordinate the metabolic flux of nucleotides, nucleotide sugars, and nucleotide sulfate between the cytosol and the lumen of secretory organelles. E-type ATPases play an important role in this function. They have been conserved through evolution with their catalytic site in the ecto-position, facing the outer surface of the plasma membrane or its topological equivalent, the lumen of intracellular organelles such as the endoplasmic reticulum (ER), 1 Golgi apparatus (GA), and lysosomes/vacuoles. These enzymes, members of the ecto-nucleoside triphosphate diphosphohydrolase (ENTPDase) family, hydrolyze nucleoside tri-and/or diphosphates in the presence of cations and share, at the amino acid sequence level, five conserved motifs called apyrase-conserved regions (ACRs) (1).Extracellular nucleotides such as ATP and ADP are intercellular signaling molecules in virtually every tissue where, modulated by ecto-apyrases, they participate in a broad range of biological processes such as the regulation of immune responses (2) and modulation of signaling by neuronal cells (3). Specifically, mammalian ENTPDase1/CD...

Section: Discussionsupporting

confidence: 78%

ire-1-dependent Transcriptional Up-regulation of a Lumenal Uridine Diphosphatase from Caenorhabditis elegans

Uccelletti

O'Callaghan

Berninsone

et al. 2004

“…This reaction is not essential for cell viability under normal growth conditions (31). Moreover, complete or severe reduction in monoglucosylated glycan formation caused by disruption of glucosidase II subunit ␣-or ␤-or GT-encoding genes did not affect cell growth or morphology.…”

Section: Analysis Of S Pombementioning

confidence: 88%

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

D’Alessio

Trombetta

Parodi

2003

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...

“…Since catalysis of glucose transfer by UGTR is a Ca 2ϩ -dependent process (49) it has been suggested that hydrolysis of UDP-glucose, which is coupled to glucose transfer, may provide the energy necessary for dissociating bound glycoproteins from UGTR (8). We have been unable to demonstrate transfer of glucose to oligosaccharides of null(Hong Kong) during Ca 2ϩ -dependent dissociation of UGTR, but this may reflect poor detection limits associated with our analysis.…”

Section: Discussionmentioning

confidence: 82%

“…Reglucosylation of high mannose-type glycans has been detected in microsomal preparations from mammals, plants, fungi, yeast, and protozoa (7,8) and is catalyzed by the ER resident protein UDP-glucose:glycoprotein glucosyltransferase (UGTR) (8 -10). Importantly, only high mannose-type oligosaccharides attached to unfolded proteins function as acceptors in the glucose transfer reaction (11)(12)(13).…”

mentioning

confidence: 99%

Intracellular Association between UDP-glucose:Glycoprotein Glucosyltransferase and an Incompletely Folded Variant of α1-Antitrypsin

Choudhury

Liu

Bick³

et al. 1997

In eukaryotes, proteins destined for secretion are translocated as nascent polypeptides into the lumen of the endoplasmic reticulum (ER) 1 (for a review, see Ref. 1). Folding into the native conformation, a structure dictated by the primary amino acid sequence (2), is facilitated through transient interaction with one or more molecular chaperones (3). Conformational fidelity of folded structures is monitored by a poorly understood quality control system (4) which prevents transport of incompletely folded and unassembled proteins beyond the ER (5).Cotranslational addition of Glc 3 Man 9 GlcNAc 2 to specific asparagine residues and hydrolysis of attached glucose units can accompany translocation of the nascent polypeptide (6). Reglucosylation of high mannose-type glycans has been detected in microsomal preparations from mammals, plants, fungi, yeast, and protozoa (7,8) and is catalyzed by the ER resident protein UDP-glucose:glycoprotein glucosyltransferase (UGTR) (8 -10). Importantly, only high mannose-type oligosaccharides attached to unfolded proteins function as acceptors in the glucose transfer reaction (11-13). Results from a cell-free system indicate that the unfolded polypeptide and asparagine-linked GlcNAc are responsible for eliciting glucose transfer (13).Several nascent proteins (14 -18) form transient associations with calnexin (also designated p88 or IP90), a calcium-binding protein of the ER membrane (19). Since calnexin functions as a molecular chaperone for glycoproteins (17,20) and interacts with monoglucosylated oligosaccharides (21), Hammond et al. (15) proposed that reglucosylation by UGTR may function to initiate assembly between unfolded glycoproteins and the molecular chaperone. In support of this idea, Labriola et al. (22) have reported that delivery of a nascent acid hydrolase to lysosomes of Trypanosoma cruzi is delayed by inhibition of ER ␣-glucosidase activity and is a predictable response if attached monoglucosylated oligosaccharides are interacting with calnexin.Recent evidence suggests that protein folding and quality control machinery may participate in the molecular pathogenesis of several human diseases caused by defective intracellular transport of an aberrantly folded protein through the secretory pathway (23-25). Human ␣ 1 -antitrypsin (AAT) is a 394-amino acid protein (26, 27) glycosylated at three specific asparagine residues (28). It is folded into a highly ordered tertiary structure containing three ␤-sheets, nine ␣ helices, and three internal salt bridges (29). The human AAT structural gene is highly polymorphic (30), and several alleles exhibit a distinct mutation predicted to preclude conformational maturation of the encoded polypeptide following biosynthesis (31). Secretion of AAT from hepatocytes (32, 33) is impaired in response to incomplete folding of the polypeptide (34,35).AAT is a member of the serine proteinase inhibitor superfamily (36). Since elastase released by activated neutrophils is rendered inactive by the inhibitor (37), diminished circulating levels...