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. 2013 Apr;161(4):1737-54.
doi: 10.1104/pp.112.210757. Epub 2013 Feb 11.

Time-resolved fluorescence imaging reveals differential interactions of N-glycan processing enzymes across the Golgi stack in planta

Jennifer Schoberer  1 , Eva Liebminger, Stanley W Botchway, Richard Strasser, Chris Hawes
Affiliations

Time-resolved fluorescence imaging reveals differential interactions of N-glycan processing enzymes across the Golgi stack in planta

Jennifer Schoberer et al. Plant Physiol. 2013 Apr.

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.

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Figures

Figure 1.
Figure 1.
Overview of fluorescent protein fusion constructs used for FRET-FLIM. A, Schematic representation of the N-glycan processing pathway in the plant Golgi apparatus and the enzymes involved (for enzyme names, see Table I). Enzymes studied here are highlighted in black. B, Schematic representation of the domain architecture of expressed recombinant proteins, all of them displaying a type II membrane topology (the N terminus on the cytoplasmic side and the C terminus on the luminal side of the Golgi membrane). The CTS regions and the full-length sequences (where available) were C-terminally fused to GFP and/or mRFP, respectively, by insertion into binary plant expression vectors. Details on plasmid construction can be found in “Materials and Methods.” C, Cytoplasmic tail; CD, catalytic domain; S, luminal stem region; T, transmembrane domain.
Figure 2.
Figure 2.
Golgi localization of full-length cis/medial-Golgi protein pairs in tobacco leaves. Confocal images show representative tobacco leaf epidermal cells coexpressing fluorescent protein-tagged full-length protein pairs MNS1-G (A) and MNS1-R (B), GnTI-G (D) and GnTI-R (E), and GnTI-G (G) and MNS1-R (H). C, F, and I show merges of green (GFP fluorescence) and magenta (mRFP fluorescence) channels. White in the merged images indicates areas of colocalization. Bars = 5 µm.
Figure 3.
Figure 3.
co-IP assay and in vivo 2P-FRET-FLIM analysis of full-length cis/medial-Golgi protein pairs. Fluorescent protein-tagged full-length protein pairs MNS1/MNS1, GnTI/GnTI, and GnTI/MNS1 were transiently coexpressed in Nicotiana benthamiana leaf epidermal cells and subjected to co-IP (A) or confocal imaging and 2P-FRET-FLIM (B–J). A, Immunoblot analysis of protein extracts (Input = before incubation with GFP-coupled beads) and eluted samples (Bound = fraction eluted from GFP-coupled beads) with anti-GFP and anti-mRFP antibodies. B to D, Cell showing expression of the donor fusion protein MNS1-G alone as a representative, unquenched negative control with an average fluorescence lifetime of 2.2 ± 0.1 ns following 2P-FRET-FLIM. Blue in the color-coded lifetime image (C) and histogram (D) reflects higher excited-state lifetimes (approximately 3 ns) than green (approximately 2 ns). The distribution curve in the histogram depicts the relative occurrence frequency of the lifetimes within the lifetime image. In C, the asterisk indicates a stoma that gives lower lifetimes than the reference Golgi labeled with an arrow and blue crosshair. E to G, Representative cell that shows quenching of the donor lifetime of MNS1-G in the presence of the acceptor MNS1-R (1.9 ± 0.1 ns), indicating interaction. Quenching is also reflected in the green color in the color-coded lifetime image (F) and histogram (G). H to J, Histograms that show a comparison of the average donor fluorescence lifetimes in the absence and presence of the indicated acceptors following 2P-FRET-FLIM. Each column represents the mean lifetime ± sd. FRET-FLIM data were collected from two independent infiltrations (10–12 cells were analyzed in total) and analyzed statistically using a two-tailed Student’s t test (P ≤ 0.0001). A minimum decrease of the average excited-state fluorescence lifetime of the donor molecule by 0.2 ns or percentage FRET efficiency > 8% in the presence of the acceptor molecule was considered relevant to indicate interaction. For the full data set, see Table II. Bars = 20 µm.
Figure 4.
Figure 4.
2P-FRET-FLIM analysis of the medial-Golgi enzyme GMII coexpressed with the cis/medial-Golgi enzymes MNS1 and GnTI. The GFP-fused full-length enzyme GMII was transiently coexpressed with putative mRFP-tagged interactor(s) in tobacco leaf epidermal cells and subjected to confocal imaging and 2P-FRET-FLIM. A, C, and E, Confocal images of a representative cell showing the colocation of GFP and mRFP full-length enzyme pairs in Golgi stacks of live cells as follows: GMII-G and MNS1-R (A), GMII-G and GnTI-R (C), GMII-G and GMII-R (E). The larger images show merges of the smaller images, which represent GFP fluorescence of the donor in green and mRFP fluorescence of the acceptor in magenta. Colocalization in the merged confocal images appears white. B, D, and F, Histograms showing the donor lifetimes in the absence and presence of the indicated acceptors following 2P-FRET-FLIM. The measurements and analysis were performed as described for Figure 3, H to J. For the full data set, see Table II. Bars = 5 µm.
Figure 5.
Figure 5.
Catalytic domain-deleted cis- and medial-Golgi enzymes form homodimers and heterodimers in vivo. The N-terminal CTS regions of enzymes (lacking their catalytic domain) fused to fluorescent proteins were transiently coexpressed in tobacco leaf epidermal cells and subjected to confocal imaging and 2P-FRET-FLIM. The left panels show confocal images of cells coexpressing GnTI-CTS-G and GnTI-CTS-R (A), GnTI-CTS-G and GMII-CTS-R (C), MNS1-CTS-G and MNS1-CTS-R (E), MNS1-CTS-G and GnTI-CTS-R (G), and MNS1-CTS-G and GMII-CTS-R (I). The larger images show merges of the smaller split images representing the green and magenta channels. The right panels (B, D, F, H, and J) show histograms of the average excited-state lifetimes of each donor in the absence and presence of the indicated acceptor following 2P-FRET-FLIM. The measurements and analysis were performed as described for Figure 3, H to J. For the full data set, see Table III. Bars = 5 µm.
Figure 6.
Figure 6.
Early- and late-Golgi enzymes do not interact. A, Subcellular colocalization (merge) of the trans-Golgi marker ST-CTS-G (green) and the cis/medial-Golgi enzyme GnTI-CTS-R (magenta) in the Golgi. B, Average lifetimes of ST-CTS-G in the absence and presence of GnTI-CTS-R. C, Subcellular colocalization (merge) of GALT1-CTS-G (green) and GnTI-CTS-R (magenta) in the Golgi. D, Average lifetimes of GALT1-CTS-G in the absence and presence of GnTI-CTS-R. The measurements and analysis were performed as described for Figure 3, H to J. For the full data set, see Table IV. Bars = 10 µm.
Figure 7.
Figure 7.
The nonplant trans-Golgi marker ST-CTS does not interact with other plant markers from the trans-Golgi. A and B, Subcellular colocalization (merge) of ST-CTS-G (green) and ST-CTS-R (magenta) in the Golgi (A) and histogram showing the observed average lifetime of the donor in the absence and presence of the acceptor following 2P-FRET-FLIM (B). C and D, Subcellular colocalization (merge) of the trans-Golgi enzyme GALT1-CTS-G (green) and ST-CTS-R (magenta) in the Golgi (C) and the observed average lifetimes (D). E and F, Subcellular colocalization (merge) of the medial-Golgi enzyme XylT-CTS-G (green) and ST-CTS-R (magenta) in the Golgi (E) and the observed average lifetimes (F). The measurements and analysis were performed as described for Figure 3, H to J. For the full data set, see Table V. Bars = 10 µm.
Figure 8.
Figure 8.
The medial-Golgi enzymes XylT-CTS and GMII-CTS form homodimers and heterodimers. A, Subcellular colocalization (merge) of GMII-CTS-G (green) and GMII-CTS-R (magenta) in the Golgi. C and E, Subcellular colocalization (merge) of XylT-CTS-G (green) and XylT-CTS-R (magenta; C) or GMII-CTS-R (magenta; E) in the Golgi. B, D, and F, Histograms showing the average lifetimes of the indicated donors and donor-acceptor combinations. The measurements and analysis were performed as described for Figure 3, H to J. For the full data set, see Table VI. Bars = 10 µm.
Figure 9.
Figure 9.
The trans-Golgi enzyme GALT1 interacts with the medial-Golgi enzyme GMII but not with medial-Golgi XylT or trans-Golgi FUT13. The left panels show the subcellular colocalization (merge) of the donor GALT1-CTS-G (green) with FUT13-R (magenta; A), XylT-CTS-R (magenta; C), or GMII-CTS-R (magenta; E) in the Golgi. The histograms (B, D, and F) at right show the average lifetimes of the indicated donors and donor-acceptor combinations. The measurements and analysis were performed as described for Figure 3, H to J. For the full data set, see Table VII. Bars = 10 µm.
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