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 formation of multi-enzyme complexes is considered a feasible Golgi protein localization strategy. In this study we used two-photon (2P)-excitation Förster resonance energy transfer (FRET)-fluorescence lifetime imaging microscopy (FLIM) to determine the interaction of N-glycan processing enzymes with differential intra-Golgi locations. Following the coexpression of fluorescent protein-tagged N-terminal Golgi targeting sequences (cytoplasmic-transmembrane-stem region, designated CTS) of enzyme pairs in leaves of tobacco (Nicotiana tabacum or Nicotiana benthamiana), we observed that all tested cis- and medial-Golgi enzymes, namely MNS1, GnTI, GMII and XylT, form homo- and heterodimers, whereas among the late-acting enzymes GALT1, FUT13 and ST (a non-plant Golgi marker) only GALT1 and 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 2P-FRET-FLIM analysis 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 N-terminal CTS region
