New insights into the organisation and intracellular localisation of the two subunits of glucose-6-phosphatase

Abstract

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.