Biochemistry

Abstract

CS-11-3 - Protein-protein interactions around the plant tricarboxylic acid cycle

Monday, July 16
1:43 PM - 2:03 PM

Plant tricarboxylic acid (TCA) cycle is operated in an exceptionally flexible manner but the molecular mechanism regulating this cycle is largely unclear. The enzymes of mammalian TCA cycle form multi-enzyme complexes. Some of those are composed of the enzymes catalyzing sequential reactions and channel intermediate metabolites between the catalytic cores of them. Multi-enzyme complexes and metabolite channeling potentially offer various advantages in metabolic regulation and likely involved in it. In the present study, the protein-protein interactions including subunits of the plant TCA cycle enzymes were examined to identify multi-enzyme complexes potentially involved in the regulation of the pathway.


The protein-protein interactions were evaluated using a compromised score generated from (semi-)quantitative scores obtained by three independent methods, namely affinity purification-mass spectrometry (AP-MS), split luciferase and yeast two hybrid (Y2H) assays. All possible binary interactions between 38 proteins composing mitochondrial TCA cycle were tested and 158 interactions were detected. These include interactions of catalytic subunits mediating sequential reactions of the pathway. The composition of the TCA cycle multi-enzyme complexes is conserved in mammals, bacteria and plants. Isotope dilution experiments demonstrated that citrate and fumarate were channeled in isolated mitochondria. These results indicate the multi-enzyme complex formation and metabolite channeling between citrate synthase and aconitase and probably in some other enzyme pairs as well.


The analysis of AP-MS data further identified 125 possible interactions between the TCA cycle enzymes and other mitochondrial proteins. Additionally, the interaction between citrate synthase (CSY4) and a subunit of mitochondrial electron transport chain complex I (B13) was detected by co-purification with CSY4 in illuminated Arabidopsis leaves. Although the B13 knock-down Arabidopsis lines showed reduction of mature complex I, their growth phenotypes were distinct from those of previously characterized complex I deficient mutants. The functions of these extra-TCA cycle interactions will also be discussed.


 

Co-Authors

Youjun Zhang – Max-Planck-Institute of Molecular Plant Physiology; Katherine Beard – University of Oxford; Corne Swart – Max-Planck-Institute of Molecular Plant Physiology; Etienne Meyer – Max-Planck-Institute of Molecular Plant Physiology; Zoran Nikoloski – Max-Planck-Institute of Molecular Plant Physiology; Alexandar Graf – Max-Planck-Institute of Molecular Plant Physiology; George Ratcliffe – University of Oxford; Lee Sweetlove – University of Oxford; Alisdair Fernie – Max-Planck-Institute of Molecular Plant Physiology

Toshihiro Obata

Assistant Professor
University of Nebraska Lincoln

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