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

Abstract

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...

Golgi and Endoplasmic Reticulum Functions Take Place in Different Subcellular Compartments of Entamoeba histolytica

“…Previously we had described that GA and ER vesicles from higher and lower eukaryotes contain luminal nucleoside di-and triphosphatases whose role is to give rise to nucleoside monophosphates, which are necessary for mediating the nucleotide sugar transport/antiport cycle (14,31). To answer the above question, a cell suspension of Eh trophozoites was subjected to Dounce homogenization as previously described (22).…”

“…To answer the above question, a cell suspension of Eh trophozoites was subjected to Dounce homogenization as previously described (22). Following centrifugation at 130,000 ϫ g a membrane pellet was obtained, resuspended, and assayed for latency of different nucleoside di-and triphosphatases, which had been shown to be luminal enzymatic activities of the GA and ER (31). As shown in Fig.…”

APY-1, a NovelCaenorhabditis elegansApyrase Involved in Unfolded Protein Response Signalling and Stress Responses

“…We have previously identified and characterized the uridine diphosphatase (UDPase)/guanine diphosphatase (GDPase) UDA-1, involved in ER "quality control"; UDA-1 showed high similarity of sequence and of substrate specificity with S. cerevisiae Golgi Gda1p and with a human ER NDPase (Abeijon et al, 1993;Trombetta and Helenius, 1999;Uccelletti et al, 2004).…”

“…Loss of MIG-23 function results in altered gonad morphogenesis, demonstrating the importance of this enzyme (Nishiwaki et al, 2004). Transcription of uda-1 but not ntp-1 and mig-23 was up-regulated by conditions causing ER stress and the accumulation of unfolded proteins such as tunicamycin, or high temperature; however, no relevant phenotypes were associated with loss-of-function mutation in uda-1 obtained by RNA interference (RNAi; Uccelletti et al, 2004).We report here the characterization of the C. elegans open reading frame (ORF) F08C6.6 coding for an NDPase similar to the apyrases of blood-sucking insects. The gene transcription was up-regulated by ER stress conditions under ire-1 and atf-6 control.…”

“…C. elegans is an attractive model to study the relevance of intracellular E-NTPDases in alleviating ER stress and regulating protein and lipid glycosylation. Its genome encodes at least three proteins belonging to the former family of ENTPDases with sequence similarity to the Saccharomyces cerevisiae Golgi apparatus (GA) Gda1p: UDA-1 related, in substrate specificity, to the yeast Gda1p, NTP-1 an apyrase related to the yeast Ynd1p (Abeijon et al, 1993;Xiao-Dong et al, 1999;Zhong and Guidotti, 1999;Uccelletti et al, 2004) and a nucleotide diphosphatase functionally homologous to the yeast Ynd1p, encoded by the mig-23 gene. Loss of MIG-23 function results in altered gonad morphogenesis, demonstrating the importance of this enzyme (Nishiwaki et al, 2004).…”