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-Forster 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 alpha-mannosidase I, Nicotiana tabacum beta 1,2-N-acetylglucosaminyltransferase I, Arabidopsis Golgi alpha-mannosidase II (GMII), and Arabidopsis beta 1,2-xylosyltransferase, form homodimers and heterodimers, whereas among the late-acting enzymes Arabidopsis beta 1,3-galactosyltransferase1 (GALT1), Arabidopsis alpha 1,4-fucosyltransferase, and Rattus norvegicus alpha 2,6-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.