Time-resolved fluorescence imaging reveals differential interactions of N-glycan processing enzymes across the Golgi stack in planta.
N-Glycan processing is one of the most important cellular protein modifications in plants and as such is essential for plant development and defense mechanisms. The accuracy of Golgi-located processing steps is governed by the strict intra-Golgi localization of sequentially acting glycosidases and glycosyltransferases. Their differential distribution goes hand in hand ... with the compartmentalization of the Golgi stack into cis-, medial-, and trans-cisternae, which separate early from late processing steps. The mechanisms that direct differential enzyme concentration are still unknown, but the formation of multienzyme complexes is considered a feasible Golgi protein localization strategy. In this study, we used two-photon excitation-Foerster resonance energy transfer-fluorescence lifetime imaging microscopy to determine the interaction of N-glycan processing enzymes with differential intra-Golgi locations. Following the coexpression of fluorescent protein-tagged amino-terminal Golgi-targeting sequences (cytoplasmic-transmembrane-stem [CTS] region) of enzyme pairs in leaves of tobacco (Nicotiana spp.), we observed that all tested cis- and medial-Golgi enzymes, namely Arabidopsis (Arabidopsis thaliana) Golgi α-mannosidase I, Nicotiana tabacum β1,2-N-acetylglucosaminyltransferase I, Arabidopsis Golgi α-mannosidase II (GMII), and Arabidopsis β1,2-xylosyltransferase, form homodimers and heterodimers, whereas among the late-acting enzymes Arabidopsis β1,3-galactosyltransferase1 (GALT1), Arabidopsis α1,4-fucosyltransferase, and Rattus norvegicus α2,6-sialyltransferase (a nonplant Golgi marker), only GALT1 and medial-Golgi GMII were found to form a heterodimer. Furthermore, the efficiency of energy transfer indicating the formation of interactions decreased considerably in a cis-to-trans fashion. The comparative fluorescence lifetime imaging of several full-length cis- and medial-Golgi enzymes and their respective catalytic domain-deleted CTS clones further suggested that the formation of protein-protein interactions can occur through their amino-terminal CTS region.
Mesh Terms:
Animals, Arabidopsis, Biological Markers, Catalytic Domain, Fluorescence Resonance Energy Transfer, Glycosyltransferases, Golgi Apparatus, Immunoprecipitation, Mannosidases, Microscopy, Fluorescence, Molecular Sequence Data, Photons, Plant Cells, Plant Leaves, Polysaccharides, Protein Binding, Protein Multimerization, Protein Structure, Tertiary, Rats, Recombinant Fusion Proteins, Subcellular Fractions, Time Factors, Tobacco
Animals, Arabidopsis, Biological Markers, Catalytic Domain, Fluorescence Resonance Energy Transfer, Glycosyltransferases, Golgi Apparatus, Immunoprecipitation, Mannosidases, Microscopy, Fluorescence, Molecular Sequence Data, Photons, Plant Cells, Plant Leaves, Polysaccharides, Protein Binding, Protein Multimerization, Protein Structure, Tertiary, Rats, Recombinant Fusion Proteins, Subcellular Fractions, Time Factors, Tobacco
Plant Physiol.
Date: Apr. 01, 2013
PubMed ID: 23400704
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