The Extra-Pathway Interactome of the TCA Cycle: Expected and Unexpected Metabolic Interactions

Abstract

The plant tricarboxylic acid (TCA) cycle provides essential precursors for respiration, amino acid biosynthesis, and general nitrogen metabolism; moreover, it is closely involved in biotic stress responses and cellular redox homeostasis. To further understand the in vivo function of the TCA cycle enzymes, we combined affinity purification with proteomics to generate a comprehensive extra-pathway protein-protein interaction network of the plant TCA cycle. We identified 125 extra-pathway interactions in Arabidopsis () mostly related to the mitochondrial electron transport complex/ATP synthesis and amino acid metabolism but also to proteins associated with redox stress. We chose three high-scoring and two low-scoring interactions for complementary bimolecular fluorescence complementation and yeast two-hybrid assays, which highlighted the reliability of our approach, supported the intimate involvement of TCA cycle enzymes within many biological processes, and reflected metabolic changes reported previously for the corresponding mutant lines. To analyze the function of a subset of these interactions, we selected two mutants of mitochondrial glutaredoxin S15 and Amidase, which have not yet been analyzed with respect to their TCA cycle function, and performed metabolite profiling and flux analysis. Consistent with their interactions identified in this study, TCA cycle metabolites and the relative TCA flux of the two mutants were altered significantly.

On the role of the tricarboxylic acid cycle in plant productivity

“…Second, more recently, both in vitro and reverse genetic studies have revealed an important role for the mitochondrial (and cytosolic) thioredoxin system in the regulation of the TCA cycle, through deactivating both succinate dehydrogenase and fumarase, as well as upregulating the cytosolic enzyme adenosine triphosphate (ATP)‐citrate lyase (Schmidtmann et al ; Daloso et al ; Geigenberger et al ). Third, a range of contemporary molecular and cell biology approaches have revealed that the plant TCA cycle (Zhang et al ; Zhang et al ), like that of microbial (Meyer et al ) and mammalian (Robinson and Srere ) systems, form dynamic enzyme assemblies or metabolons. Indeed, the metabolon concept was established on studying the TCA cycle in rat liver, with the impetus to look for such an enzyme assembly being the fact that the levels of organic acids, within this tissue, were not consistent with the properties of the enzymes when evaluated in isolation (Srere ).…”

Rapid Identification of Protein‐Protein Interactions in Plants

“…Here, we suggest using a GFP tag for the bait protein, which facilitates the detection of the protein localization by confocal microscopy (Dunham, Mullin, & Gingras, ; Keilhauer, Hein, & Mann, ). The efficiency of the trypsin digestion and the recovery of the resulting digested peptides for MS analysis are very important for the success of AP‐MS (Zhang, Swart, et al., ). Instead of in‐gel digestion for performing global proteomics profiling (Huang et al., ; Van Leene et al., ), several studies have used the improved efficiency of in‐solution digestion on the beads, reducing time and steps (León, Schwämmle, Jensen, & Sprenger, ; Zhang, Sun, et al., ).…”

“…The 38 mitochondrial proteins of Arabidopsis thaliana were transformed into a PSB-D Arabidopsis plant cell culture, and a GFP-tagbased modified AP-MS procedure was implemented based on at least three biological replicates (Zhang, Beard, et al, 2017;Zhang, Swart, et al, 2018). Unlike normal AP-MS in which the gel is cut into pieces for several independent trypsin digestions (Morris et al, 2014), we used a proteomics-based in-solution digestion method to directly digest the proteins on the beads following affinity purification (Zhang, Sun, et al, 2017).…”

“…These complexes are thereby “pulled‐down” onto immobilized‐protein agarose beads via affinity purification, prior to their detection and identification via mass spectrometry. Given that AP‐MS experiments have been widely used to generate meaningful interaction networks, it follows that they could also be used to produce information‐rich data concerning extra‐pathway protein‐protein interactions (Bürckstümmer et al., ; Morris et al., ; Puig et al., ; Zhang, Beard, et al., ; Zhang, Sun, Zhang, Brasier, & Zhao, ; Zhang, Swart et al., ). Such interactions could aid in the characterization of the functions of the interacting proteins, provide detailed catalogs of proteins involved in protein complexes and biological processes, or reveal networks of biological processes on both the local and proteome‐wide scale (Morris et al., ).…”

“…Current Protocols in Plant Biology SUBA4 database (Hooper et al, 2016) revealed a total of 257 interactions between mitochondrially localized TCA cycle proteins and 37 of the proteins comprising the mitochondrial interaction network. Of these 257 interactions, 132 interactions between the enzymes of TCA cycle had already been reported (Zhang, Beard, et al, 2017), while there were 125 novel interactions between subunits of enzymes and other pathway enzymes or proteins (Zhang, Swart, et al, 2018).…”