Abstract
The functional link between glycolipid glycosyltransferases (GT) relies on the ability of these proteins to form organized molecular complexes. The organization, stoichiometry and composition of these complexes may impact their sorting properties, sub-Golgi localization, and may determine relative efficiency of GT in different glycolipid biosynthetic pathways. In this work, by using Förster resonance energy transfer microscopy in live CHO-K1 cells, we investigated homo- and hetero-complex formation by different GT as well as their spatial organization and molecular stoichiometry on Golgi membranes. We find that GalNAcT and GalT2 Ntd are able to form hetero-complexes in a 1:2 molar ratio at the trans-Golgi network and that GalT2 but not GalNAcT forms homo-complexes. Also, GalNAcT/GalT2 complexes exhibit a stable behavior reflected by its clustered lateral organization. These results reveals that particular topological organization of GTs may have functional implications in determining the composition of glycolipids in cellular membranes.
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Acknowledgments
This work was supported in part by Grants from Agencia Nacional de Promoción CientÃfica y Tecnológica (PICT·2006-01239), Universidad Nacional de Córdoba, y MinCyT Córdoba. We thank the technical assistance of G. Schachner and S. Deza with cell cultures and of C. Mas and M. C. Sampedro with confocal microscopy. M.L.F. and G.A.G. were recipients of CONICET Fellowships and H.J.F.M. is Career Investigator of CONICET (Argentina).
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Special Issue: In Honor of Bob Ledeen
This work is dedicated to Bob Ledeen, a pioneer in the study of the biochemistry and neurochemistry of glycolipids, great friend and enthusiastic and clever investigator.
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Ferrari, M.L., Gomez, G.A. & Maccioni, H.J.F. Spatial Organization and Stoichiometry of N-Terminal Domain-Mediated Glycosyltransferase Complexes in Golgi Membranes Determined by Fret Microscopy. Neurochem Res 37, 1325–1334 (2012). https://doi.org/10.1007/s11064-012-0741-1
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DOI: https://doi.org/10.1007/s11064-012-0741-1