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Review
. 2018;17(2):211-227.
doi: 10.1007/s11101-017-9509-1. Epub 2017 Apr 26.

How to prove the existence of metabolons?

Jean-Etienne Bassard  1 , Barbara Ann Halkier  2
Affiliations
Review

How to prove the existence of metabolons?

Jean-Etienne Bassard et al. Phytochem Rev. 2018.

Abstract

Sequential enzymes in biosynthetic pathways are organized in metabolons. It is challenging to provide experimental evidence for the existence of metabolons as biosynthetic pathways are composed of highly dynamic protein-protein interactions. Many different methods are being applied, each with strengths and weaknesses. We will present and evaluate several techniques that have been applied in providing evidence for the orchestration of the biosynthetic pathways of cyanogenic glucosides and glucosinolates in metabolons. These evolutionarily related pathways have ER-localized cytochromes P450 that are proposed to function as anchoring site for assembly of the enzymes into metabolons. Additionally, we have included commonly used techniques, even though they have not been used (yet) on these two pathways. In the review, special attention will be given to less-exploited fluorescence-based methods such as FCS and FLIM. Ultimately, understanding the orchestration of biosynthetic pathways may contribute to successful engineering in heterologous hosts.

Keywords: Fluorescence correlation spectroscopy; Fluorescence lifetime imaging microscopy; Fluorescence-based protein–protein interaction; Yeast-2-hybrid screen.

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Figures

Fig. 1
Fig. 1
Schematic representation of the tandem affinity purification procedure. The two-step affinity purification protocol involves preparation of the cell lysate, followed by the first affinity purification. Subsequently follows cleavage of the first tag, purification using the second tag, and finally elution of the protein complex to be analyzed by mass spectrometry
Fig. 2
Fig. 2
Application of chemically inducible built-in positive control in BiFC technique. Addition of rapamycin induces the interaction between the two proteins FRB and FKBP12. In the glucosinolate pathway, the interaction between the biosynthetic UGT74B1 and different SOT enzymes was investigated in N. benthamiana leaves by co-expressing either YFPn-FRB or YFPn-FRB-UGT74B1 with YFPc-FKBP12, SOT12-FKBP12-YFPc or SOT16-FKBP12-YFPc. Subsequently, the leaves were infiltrated with either water (+DMSO) or 30 μM rapamycin (+Rapa). The rapamycin-induced protein–protein interaction functions as a built-in positive control, to prove that the lack of interaction between the pair SOT12-UGT74B1 is not due to lack of expression and, to show the maximum fluorescence that can be obtained with the pair SOT16-UGT74B1. Scale bars represent 50 μm. (Courtesy of Scientific Reports, Andersen et al. 2016)
Fig. 3
Fig. 3
The principle of FCS and FCCS and application of FCS on the cyanogenic glucoside pathway. a FCS is used to show diffusion or determine local concentration of tagged target protein. All molecules of a specific target protein passing through the confocal volume are recorded to determine the autocorrelation curve. Arrows are indicating trajectories of molecules. b FCCS is used to show that two target proteins tagged with two different fluorophores can interact by following their co-trajectories. All molecules of the two target proteins passing through the confocal volume are recorded to determine the cross-correlation curve. Arrows are indicating trajectories of molecules. c The diffusion of proteins (CYP71E1, CYP79A1, UGT85B1) in dhurrin pathway was investigated by in planta FCS in N. benthamiana leaf epidermal cells transiently expressing GFP-tagged target proteins. GFP and CYP98A1 were used as controls. Letters indicate statistically significant similarities for the recorded values of the t test pairwise comparison with p < 0.05. Error bars indicate ±SD. Red arrows highlight the change of apparent diffusion constant. The apparent average diffusion constant of each partner (71, 79, UGT) was significantly lower when co-expressed with all its partners. These data supported the formation of a dynamic metabolon harboring the enzyme components catalyzing dhurrin synthesis. Interestingly, the P450-supporting reductase (POR) was not affected by co-expression of the dhurrin enzymes. (Courtesy of Science, Laursen et al. 2016)
Fig. 4
Fig. 4
The principle in FRET-based techniques. a The FRET principle for protein–protein interaction between protein “X” and protein “Y” upon excitation of donor fluorophore “D”. If distance and orientation of the FRET donor “D” and acceptor “A” are acceptable, FRET will occur from D to A, when D is excited. b Pseudo-colored image showing lifetime spatial distribution. The image displays CYP73A5-eGFP fusion protein transiently co-expressed with CYP73A5-mRFP1 fusion protein in N. benthamiana leaf epidermal cell. Part of the cortical ER is visible and variation of lifetimes is measured across the ER, indicating subtle local heterogeneity in interaction of both CYP73A5 fusion proteins
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