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New insights into the organisation and intracellular localisation of the two subunits of glucose-6-phosphatase.
Soty M., Chilloux J., Casteras S., Grichine A., Mithieux G., Gautier-Stein A.
Biochimie 94, 3 (2012) 695-703 - http://www.hal.inserm.fr/inserm-00737760
(21983240)
New insights into the organisation and intracellular localisation of the two subunits of glucose-6-phosphatase.
Maud Soty1, Julien Chilloux1, Sylvie Casteras1, Alexeï Grichine2, Gilles Mithieux1, Amandine Gautier-Stein () 1
1 :  Nutrition et cerveau
INSERM : U855 – Université Claude Bernard - Lyon I
faculte de medecine lyon-rth laennec 7, rue guillaume paradin 69372 LYON CEDEX 08
France
2 :  Institut d'oncologie/développement Albert Bonniot de Grenoble
INSERM : U823 – CHU Grenoble – EFS – Université Joseph Fourier - Grenoble I
Institut Albert Bonniot, BP170, 38042 Grenoble Cedex 9
France
Glucose-6 phosphatase (G6Pase), a key enzyme of glucose homeostasis, catalyses the hydrolysis of glucose-6 phosphate (G6P) to glucose and inorganic phosphate. A deficiency in G6Pase activity causes type 1 glycogen storage disease (GSD-1), mainly characterised by hypoglycaemia. Genetic analyses of the two forms of this rare disease have shown that the G6Pase system consists of two proteins, a catalytic subunit (G6PC) responsible for GSD-1a, and a G6P translocase (G6PT), responsible for GSD-1b. However, since their identification, few investigations concerning their structural relationship have been made. In this study, we investigated the localisation and membrane organisation of the G6Pase complex. To this aim, we developed chimera proteins by adding a fluorescent protein to the C-terminal ends of both subunits. The G6PC and G6PT fluorescent chimeras were both addressed to perinuclear membranes as previously suggested, but also to vesicles throughout the cytoplasm. We demonstrated that both proteins strongly colocalised in perinuclear membranes. Then, we studied G6PT organisation in the membrane. We highlighted FRET between the labelled C and N termini of G6PT. The intramolecular FRET of this G6PT chimera was 27%. The coexpression of unlabelled G6PC did not modify this FRET intensity. Finally, the chimera constructs generated in this work enabled us for the first time to analyze the relationship between GSD-1 mutations and the intracellular localisation of both G6Pase subunits. We showed that GSD1 mutations did neither alter the G6PC or G6PT chimera localisation, nor the interaction between G6PT termini. In conclusion, our results provide novel information on the intracellular distribution and organisation of the G6Pase complex.
Sciences du Vivant/Biochimie, Biologie Moléculaire
Anglais
1638-6183

Articles dans des revues avec comité de lecture
10.1016/j.biochi.2011.09.022
Biochimie (Biochimie)
Publisher Elsevier
ISSN 0300-9084 
non spécifiée
03/2012
29/09/2011
94
3
695-703

Glucose-6-phosphatase – Glucose-6-phosphatase catalytic unit – Glucose-6-phosphate transporter – Fluorescence energy transfer – Glycogen storage disease type 1
Antiporters – Fluorescence Resonance Energy Transfer – Glucose-6-Phosphatase – Glycogen Storage Disease Type I – HeLa Cells – Hep G2 Cells – Humans – Immunoblotting – Immunohistochemistry – Intracellular Membranes – Monosaccharide Transport Proteins – Protein Structure – Secondary – Protein Subunits – Recombinant Proteins
The authors thank INSERM for funding their work, as well as CNRS (G.M.), INRA (A.G.-S.) and the French Ministry of Research (M.S., J.C. and S.C.) for funding their positions.
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