Time-Resolved Fluorescence Imaging Reveals Differential Interactions ofN-Glycan Processing Enzymes across the Golgi Stack in Planta

Jennifer Schoberer,Stanley W. Botchway,Richard Strasser,Chris Hawes,Eva Liebminger

doi:10.1104/pp.112.210757

Abstract

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-Förster 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.

Highlights

Using the noninvasive 2P-Förster resonance energy transfer (FRET)-fluorescence lifetime imaging (FLIM) biophysical method, we show that several cis- and medial-Golgi enzymes, namely MNS1, GnTI, Golgi a-mannosidase II (GMII), and XylT, are able to assemble into homodimers and heterodimers with the participation of their N-terminal CTS Golgitargeting domains
Among the late-acting enzymes GALT1, FUT13, and ST, only the CTS region of GALT1 was found to interact with GMII-CTS
We confirm that the determination of FRET by measuring the decrease in donor fluorescence lifetime by means of 2P-FLIM has the potential to become a gold standard for screening protein-protein interactions in planta, as it enables direct access to interactions in their natural environment inside live cells and by doing so overcomes the limitations of other methods, many of them performed by invasive cell disruption assays

Summary

Introduction

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. We used two-photon excitation-Förster resonance energy transfer-fluorescence lifetime imaging microscopy to determine the interaction of N-glycan processing enzymes with differential intra-Golgi locations. Golgi-mediated N-glycan processing steps are catalyzed by numerous glycosidases and glycosyltransferases that follow a nonuniform subcompartmentspecific distribution pattern along the cis-to-trans axis of the Golgi stack in the order in which they function in the processing pathway (Fig. 1A; Schoberer and Strasser, 2011).

Methods

Results

Discussion

Conclusion