Protein-protein interactions and metabolite channelling in the plant tricarboxylic acid cycle

Youjun Zhang,Katherine F M Beard,Alisdair R Fernie,Susan Bergmann,Ina Krahnert,R George Ratcliffe,Toshihiro Obata,Lee J Sweetlove,Corné Swart,Alexander Graf,Zoran Nikoloski

doi:10.1038/ncomms15212

Abstract

Protein complexes of sequential metabolic enzymes, often termed metabolons, may permit direct channelling of metabolites between the enzymes, providing increased control over metabolic pathway fluxes. Experimental evidence supporting their existence in vivo remains fragmentary. In the present study, we test binary interactions of the proteins constituting the plant tricarboxylic acid (TCA) cycle. We integrate (semi-)quantitative results from affinity purification-mass spectrometry, split-luciferase and yeast-two-hybrid assays to generate a single reliability score for assessing protein–protein interactions. By this approach, we identify 158 interactions including those between catalytic subunits of sequential enzymes and between subunits of enzymes mediating non-adjacent reactions. We reveal channelling of citrate and fumarate in isolated potato mitochondria by isotope dilution experiments. These results provide evidence for a functional TCA cycle metabolon in plants, which we discuss in the context of contemporary understanding of this pathway in other kingdoms.

Highlights

Protein complexes of sequential metabolic enzymes, often termed metabolons, may permit direct channelling of metabolites between the enzymes, providing increased control over metabolic pathway fluxes
High-throughput split luciferase assays in Arabidopsis mesophyll protoplasts[40] and semiquantitative yeast-two-hybrid (Y2H) assays were employed for binary protein interaction analysis
We identified a total of 312 cliques, with the largest clique composed of five proteins, namely, ACO2, ACO3, citrate synthase (CSY) 4, IDH1 and IDH2

Summary

Introduction

Protein complexes of sequential metabolic enzymes, often termed metabolons, may permit direct channelling of metabolites between the enzymes, providing increased control over metabolic pathway fluxes Experimental evidence supporting their existence in vivo remains fragmentary. We integrate (semi-)quantitative results from affinity purification-mass spectrometry, split-luciferase and yeast-two-hybrid assays to generate a single reliability score for assessing protein–protein interactions By this approach, we identify 158 interactions including those between catalytic subunits of sequential enzymes and between subunits of enzymes mediating non-adjacent reactions. We reveal channelling of citrate and fumarate in isolated potato mitochondria by isotope dilution experiments These results provide evidence for a functional TCA cycle metabolon in plants, which we discuss in the context of contemporary understanding of this pathway in other kingdoms. Additional isotope-based dilution experiments performed with isolated mitochondria provided evidence for substrate channelling that is mediated by only a subset of the enzymes

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