title: Functional organization of Golgi N- and O-glycosylation pathways involves pH-dependent complex formation that is impaired in cancer cells.
abstract: Glycosylation is one of the most common modifications of proteins and lipids and also a major source of biological diversity in eukaryotes. It is critical for many basic cellular functions and recognition events that range from protein folding to cell signaling, immunological defense, and the development of multicellular organisms. Glycosylation takes place mainly in the endoplasmic reticulum and Golgi apparatus and involves dozens of functionally distinct glycosidases and glycosyltransferases. How the functions of these enzymes, which act sequentially and often competitively, are coordinated to faithfully synthesize a vast array of different glycan structures is currently unclear. Here, we investigate the supramolecular organization of the Golgi N- and O-glycosylation pathways in live cells using a FRET flow cytometric quantification approach. We show that the enzymes form enzymatically active homo- and/or heteromeric complexes within each pathway. However, no complexes composed of enzymes that operate in different pathways, were detected, which suggests that the pathways are physically distinct. In addition, we show that complex formation is mediated almost exclusively by the catalytic domains of the interacting enzymes. Our data also suggest that the heteromeric complexes are functionally more important than enzyme homomers. Heteromeric complex formation was found to be dependent on Golgi acidity, markedly impaired in acidification-defective cancer cells, and required for the efficient synthesis of cell surface glycans. Collectively, the results emphasize that the Golgi glycosylation pathways are functionally organized into complexes that are important for glycan synthesis.
authors.affiliation: Department of Biochemistry and the Finnish Glycoscience Graduate School, University of Oulu, FIN-90014 Oulu, Finland.
authors.forename: Antti
authors.initials: A
authors.lastname: Hassinen
authors.affiliation: Department of Biochemistry and the Finnish Glycoscience Graduate School, University of Oulu, FIN-90014 Oulu, Finland.
authors.forename: Francois M
authors.initials: FM
authors.lastname: Pujol
authors.affiliation: Department of Biochemistry and the Finnish Glycoscience Graduate School, University of Oulu, FIN-90014 Oulu, Finland.
authors.forename: Nina
authors.initials: N
authors.lastname: Kokkonen
authors.affiliation: Department of Biochemistry and the Finnish Glycoscience Graduate School, University of Oulu, FIN-90014 Oulu, Finland.
authors.forename: Caroline
authors.initials: C
authors.lastname: Pieters
authors.affiliation: Department of Biochemistry and the Finnish Glycoscience Graduate School, University of Oulu, FIN-90014 Oulu, Finland.
authors.forename: Minna
authors.initials: M
authors.lastname: Kihlström
authors.affiliation: Department of Biochemistry and the Finnish Glycoscience Graduate School, University of Oulu, FIN-90014 Oulu, Finland.
authors.forename: Kati
authors.initials: K
authors.lastname: Korhonen
authors.affiliation: Department of Biochemistry and the Finnish Glycoscience Graduate School, University of Oulu, FIN-90014 Oulu, Finland. Electronic address: sakari.kellokumpu@oulu.fi.
authors.forename: Sakari
authors.initials: S
authors.lastname: Kellokumpu
mesh_terms: D000818:Animals; D019556:COS Cells; D020134:Catalytic Domain; D045744:Cell Line, Tumor; D002522:Chlorocebus aethiops; D002845:Chromatography / Q000379:methods; D005434:Flow Cytometry / Q000379:methods; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods; D006031:Glycosylation; D006056:Golgi Apparatus / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D006863:Hydrogen-Ion Concentration; D019158:N-Acetylneuraminic Acid / Q000737:chemistry; D009369:Neoplasms / Q000378:metabolism* / Q000473:pathology
chemical_list: D019158:N-Acetylneuraminic Acid