Guanosine diphosphatase is required for protein and sphingolipid glycosylation in the Golgi lumen of Saccharomyces cerevisiae

Abeijón, Claudia; Yanagisawa, Ken; Mandon, Elisabet C.; Häusler, Alex; Moremen, Kelley W.; Hirschberg, Carlos B.; Robbins, Phillips W.

doi:10.1083/jcb.122.2.307

Cited by 172 publications

(179 citation statements)

References 75 publications

(109 reference statements)

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“…The GDP-Man transporter in yeast is believed to operate via a coupled antiporter reaction (29). A guanosine diphosphatase has been shown to be required for protein and sphingolipid mannosylation in the Golgi lumen of Saccharomyces cerevisiae, implicating the significance of GDP-Man/GMP exchange (30). In the Leishmania system, coupling of entry of GDP-Man into the Golgi compartment to exit of GMP is not known nor has a guanosine diphosphatase been demonstrated.…”

Section: Subcellular Localization Of the Protein Encoded By Lpg2mentioning

confidence: 99%

Golgi GDP-mannose Uptake Requires Leishmania LPG2

Russell

Beverley

et al. 1997

Journal of Biological Chemistry

142

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The synthesis of glycoconjugates within the secretory pathway of eukaryotes requires the provision of lumenal nucleotide-sugar substrates. This is particularly important for eukaryotic microbes such as Leishmania because they must synthesize considerable amounts of extracellular and cell surface glycoconjugates that play significant roles in the infectious cycle. Here we used properly oriented sealed microsomes to characterize lumenal uptake of GDP-Man in Leishmania donovani. In this system, GDP-Man uptake was saturable with an apparent K m for GDP-Man of 0.3 M and facilitated its use as a donor substrate for lipophosphoglycan (LPG) synthesis. A lpg2 ؊ deletion mutant showed loss of GDPMan but not UDP-Gal uptake, which was restored by introduction of the gene LPG2. Immunoelectron microscopy localized an active, epitope-tagged LPG2 protein to the Golgi apparatus. Thus, LPG2 is required for nucleotide-sugar transport activity and probably encodes this Golgi transporter. LPG2 belongs to a large family of eukaryotic genes that potentially encode transporters with different substrate specificities and/or cellular locations. In the future, the amenability of the Leishmania system to biochemical and genetic manipulation will assist in functional characterization of nucleotide-sugar transports from this and other eukaryotes. Furthermore, since LPG2 plays an important role in the Leishmania infectious cycle and mammalian cells lack a Golgi GDP-Man transporter, this activity may offer a new target for chemotherapy.

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Section: Subcellular Localization Of the Protein Encoded By Lpg2mentioning

confidence: 99%

Golgi GDP-mannose Uptake Requires Leishmania LPG2

Russell

Beverley

et al. 1997

Journal of Biological Chemistry

142

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“…In contrast, analysis of the amino acid sequence of SmATPDase2 shows that it more closely resembles intracellular and soluble enzymes of the NTPDase 5 and 6 families [18]. Proteins in these families have been reported to support glycosylation reactions in the Golgi apparatus or the endoplasmic reticulum [30][31][32]. It is possible that SmATPDase2 likewise participates in Golgi and/ or ER glycosylation reactions inside schistosomes.…”

Section: Discussionmentioning

confidence: 97%

Schistosome apyrase SmATPDase1, but not SmATPDase2, hydrolyses exogenous ATP and ADP

Da’dara¹,

Bhardwaj²,

Skelly³

2014

Purinergic Signalling

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Schistosomes are parasitic worms that can live in the bloodstream of their vertebrate hosts for many years. It has been proposed that the worms impinge on host purinergic signalling by degrading proinflammatory molecules like ATP as well as prothrombotic mediators like ADP. This capability may help explain the apparent refractoriness of the worms to both immune elimination and thrombus formation. Three distinct ectoenzymes, expressed at the host-exposed surface of the worm's tegument, are proposed to be involved in the catabolism of ATP and ADP. These are alkaline phosphatase (SmAP), phosphodiesterase (SmNPP-5), and ATP diphosphohydrolase (SmATPDase1). It has recently been shown that only one of these enzymes-SmATPDase1-actually degrades exogenous ATP and ADP. However, a second ATP diphosphohydrolase homolog (SmATPDase2) is located in the tegument and has been reported to be released by the worms. It is possible that this enzyme too participates in the cleavage of exogenous nucleotide tri-and di-phosphates. To test this hypothesis, we employed RNA interference (RNAi) to suppress the expression of the schistosome SmATPDase1 and SmATPDase2 genes. We find that only SmATPDase1-suppressed parasites are significantly impaired in their ability to degrade exogenously added ATP or ADP. Suppression of SmATPDase2 does not appreciably affect the worms' ability to catabolize ATP or ADP. Furthermore, we detect no evidence for the secretion or release of an ATP-hydrolyzing activity by cultured parasites. The results confirm the role of tegumental SmATPDase1, but not SmADTPDase2, in the degradation of the exogenous proinflammatory and prothrombotic nucleotides ATP and ADP by live intravascular stages of the parasite.

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“…Fractions derived from RNAi individuals showed a drastic reduction of UDPase activity with respect to control ones; GDP was hydrolyzed to a smaller extent but the activity inhibition was still evident. No significant differences were observed in the very low catalytic activity with ADP as substrate, and no activity was detected with NMPs ( Figure 1A); these results are consistent with the hypothesis that APY-1 has NDPase activity.To further confirm these data a heterologous approach was followed by expressing apy-1 in a S. cerevisiae gda1⌬ mutant strain, which has a low background in nucleotide phosphatase activities (Abeijon et al, 1993). UDPase and GDPase activities were significantly increased in membranes from gda1⌬ cells transformed with the vector containing apy-1 cDNA compared with those transformed with the empty vector (Supplementary Figure S1).…”

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confidence: 97%

“…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).…”

Section: Introductionmentioning

confidence: 99%

“…The following C. elegans strains were used in this study: Bristol strain N2 as standard wild-type strain, SJ30 [ire-1(zcI4) II; :GFP] V], SU93 (jcIs1) IV, and CB1370 [daf-2 (e1370) III], kindly provided by the C. elegans Genetics Center (CGC). The S. cerevisiae yeast strain Scgda1⌬ gda1::LEU2) was used for heterologous expression and was described previously (Abeijon et al, 1993). Yeast cells were grown at 30°C in yeast extract/peptone/dextrose or SD medium supplemented with amino acids as needed.…”

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confidence: 99%

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

Uccelletti

Pascoli

Farina

et al. 2008

MBoC

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Protein glycosylation modulates a wide variety of intracellular events and dysfunction of the glycosylation pathway has been reported in a variety of human pathologies. Endo-apyrases have been suggested to have critical roles in protein glycosylation and sugar metabolism. However, deciphering the physiological relevance of Endo-apyrases activity has actually proved difficult, owing to their complexity and the functional redundancy within the family. We report here that a UDP/GDPase, homologous to the human apyrase Scan-1, is present in the membranes of Caenorhabditis elegans, encoded by the ORF F08C6.6 and hereinafter-named APY-1. We showed that ER stress induced by tunicamycin or high temperature resulted in increased transcription of apy-1. This increase was not observed in C. elegans mutants defective in ire-1 or atf-6, demonstrating the requirement of both ER stress sensors for up-regulation of apy-1. Depletion of APY-1 resulted in constitutively activated unfolded protein response. Defects in the pharynx and impaired organization of thin fibers in muscle cells were observed in adult worms depleted of APY-1. Some of the apy-1(RNAi) phenotypes are suggestive of premature aging, because these animals also showed accumulation of lipofuscin and reduced lifespan that was not dependent on the functioning of DAF-2, the receptor of the insulin/IGF-1 signaling pathway. INTRODUCTIONFrom observations on human diseases and mutant mice, it has become clear that glycosylation plays a major role in metazoan development (Schachter, 2004). Given the numerous physiological roles of glycoproteins, which encompass many aspects of normal cellular function and survival, the structures of glycan moieties vary markedly providing the chemical diversity required to fulfill the vast range of their biological tasks. The diversity of natural sugar structures is ultimately derived from the activities of the enzymes responsible for sugar attachment, namely glycosyltransferases, which determine the feasibility of natural product enzymatic glycodiversification. Lumenal ecto-nucleoside triand diphosphohydrolases (E-NTPDases) of the secretory pathway of eukaryotes hydrolyzes the nucleoside diphosphates (NDPs) generated by glycosyltransferases. The resulting nucleoside monophosphates (NMPs) are weaker inhibitors of glycosyltransferases than NDPs. NMPs also serve as antiporters in the transport of nucleotide-sugars from the cytosol to the lumen of the endoplasmic reticulum (ER) and Golgi apparatus. Glycosylation plays an important role in the biogenesis of secreted and transmembrane proteins and an elaborate mechanism ensure that only properly folded and assembled proteins exit the ER, a process termed "quality control."However, The ER's capacity to process proteins is relatively limited and the stress caused by accumulation of unfolded and misfolded proteins (ER stress) contributes to a number of important human diseases (Zhang and Kaufman, 2006). Cells respond to ER stress by activating the unfolded protein response (UPR), which limits new pr...

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Guanosine diphosphatase is required for protein and sphingolipid glycosylation in the Golgi lumen of Saccharomyces cerevisiae

Cited by 172 publications

References 75 publications

Golgi GDP-mannose Uptake Requires Leishmania LPG2

Golgi GDP-mannose Uptake Requires Leishmania LPG2

Schistosome apyrase SmATPDase1, but not SmATPDase2, hydrolyses exogenous ATP and ADP

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

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