Stoichiometry and dynamics of Golgi glycolipid glycosyltransferase complexes

FERRARI, M. L.; GOMEZ, G. A.; MACCIONI, H. J. F.

Puerto Madryn

Congreso; XLVI Reunión Anual de la Sociedad Argentina de Bioquímica y Biología Molecular; 2010

Sociedad Argentina de Bioquímica y Biología Molecular

Glycolipid glycosyltransferases (GT) are Golgi resident, type II transmembrane proteins, with a N-terminal domain (Ntd), constituted by a short cytoplasmic tail, a transmembrane, and a lumenal stem region, linked to a large C-terminal catalytic domain facing the lumen. They concentrate selectively in different sub-Golgi compartments, in an overlapped manner, acting in succession in the transfer of a sugar moiety from an activated donor onto ceramide-linked oligosaccharide acceptors. GT are known to exist as monomers, or to form a variety of enzyme complexes, comprising homodimers, heterodimers or oligomers, through their different domains. Up to now, the stoichiometry of reported complexes is unknown. To approach this issue, we performed Förster resonance energy transfer (FRET) microscopy experiments in living CHO-K1 cells to elucidate the spatio-temporal relationships between GalT2 Ntds and between GalT2 and GalNAcT. We found that GalT2 NTds are able to form homo-complexes in the Golgi membranes. Also, we determined that the stoichiometry of association in the hetero-complex GalNAcT/GalT2 in the Golgi apparatus is 1:2. This molar ratio of two enzymes acting in succession in the pathway of synthesis of glycolipids may have functional implications in determining the composition of glycolipids in cellular membranes.