Regulation of the mitochondrial tricarboxylic acid cycle

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Recent years have seen considerable advances in our understanding of the particular physiological roles of the constituent enzymes of the tricarboxylic acid (TCA) cycle. Despite acquiring a fairly comprehensive overview of the functional importance of these proteins relatively little is known concerning how this important pathway is regulated. In this review we concentrate on the mitochondrial reactions since this organelle is the only one in which a full cycle can, at least theoretically, operate. We summarize what is known about the regulation of the enzymes of the pathway both from historical kinetic studies as well as discussing more recent transcriptional and proteomic studies and our enhanced understanding of subcellular compartmentation within the context of metabolic regulation.

Highlights

► The functional roles of the constituent enzymes of the plant TCA cycle are largely clarified. ► By contrast, the regulation of the flux through this vital pathway is currently poorly understood. ► Detailed enzyme kinetic studies alongside gene expression studies hint for a complex regulatory hierarchy. ► Proteomics approaches hint towards at least some conservation of regulatory principals with those characterized in non-plant systems.

Introduction

There is now considerable cumulative evidence that mitochondrial respiratory function is associated with proper maintenance of cellular metabolism as a whole. Reverse molecular genetic approaches have proven particularly instrumental in establishing the physiological and metabolic basis for mitochondrial function in living plant cells [1]. This was particularly true for increasing our knowledge of mitochondrial metabolism as of fundamental importance in many other important cellular processes such as photosynthesis, photorespiration, nitrogen metabolism, redox regulation and signalling [2, 3, 4]. That said, and quite remarkably since it was demonstrated over 50 years ago that exactly the same reactions occur in plant cells that were first described by Hans Krebs in pigeon muscle [5], relatively little is known concerning the regulation and control of this pathway [1, 6•, 7•].

The tricarboxylic acid (TCA) cycle is composed by a set of eight enzymes primarily linking the product of the oxidation of pyruvate and malate (generated in the cytosol) to CO2 with the generation of NADH for the oxidation by the mitochondrial respiratory chain [8]. The genomic organization and the subcellular localization of the enzymes involved in the Arabidopsis TCA cycle are summarized in Supplementary Table 1; however those are not described in detail since firstly it has been expertly reviewed elsewhere [9] and secondly we intend to concentrate here on the mitochondrial reactions since this organelle is the only one in which a full cycle can, at least theoretically, operate [10]. Whilst the presence of organic acids, particularly TCA cycle intermediates, in all plants is known to support numerous and diverse functions within and beyond cellular metabolism, the level of accumulation of the various organic acids is extremely variable between species, developmental stages and tissue types [11] providing further support that the enzymes involved in the interconversion of these metabolic intermediates are subject to tight regulatory control. The sequestration of organic acids into the vacuole and secretion into the rhizosphere additionally represent important mechanisms of regulating these intermediates; however, these have been recently reviewed elsewhere [11, 12].

Hints to the regulation of the TCA cycle have been provided by a recent metabolic control analysis which show that much of the control through this pathway is resident in fumarase, malate dehydrogenase (MDH) and 2-oxoglutarate dehydrogenase [1], suggesting that these would be sensitive targets for flux regulation. A lack of subcellular information concerning the levels of intermediates of the cycle [13] currently precludes us from being able to assess the potential of the constituent enzymes to play regulatory roles on the basis of disequilibrium ratios. However, there is a growing body of information concerning allosteric and post-translational regulation of specific enzymes from which mechanisms of regulation can be inferred (Figure 1). We shall discuss these studies in two separate sections. In the first we will describe focused in vitro and in vivo studies investigating the role of enzyme effectors. The second section will focus on broad-based studies in which changes in transcripts, protein abundance, post-translational modification of proteins or metabolite levels were assessed in response to environmental or experimental perturbation and from which potential elements of regulation were identified. Finally we discuss regulatory principles which have already been described in non-plant systems and conclude with a perspective for future efforts aimed at unraveling the regulation of this vital pathway.

Section snippets

TCA cycle effectors

In the following paragraphs we will briefly detail the known metabolite regulators of each enzyme which are additionally summarized as an overview in Table 1. For the purpose of the discussion, we have also included the pyruvate dehydrogenase complex (PDC) reaction which is not strictly part of the TCA cycle but is, nevertheless, intimately associated with it. The PDC is a complex consisting of three components: pyruvate dehydrogenase (E1), dihydrolipoyl acetyltransferase (E2) and dihydrolipoyl

Mechanisms of regulation revealed by profiling studies

By contrast to the directed studies described above, we will now approach insights achieved from the application of broad profiling techniques in a more general manner describing studies based at the transcript, protein and finally metabolite level. At the transcriptional level it has been frequently observed that genes associated with the TCA cycle are down regulated during the night in an essentially co-ordinated manner. However, a more recent analysis [48] reveals that genes associated to

Inferences from the regulation of the TCA cycle in non-plant systems and future perspectives

As alluded to above, a further mode of regulation is that played by compartmentation whilst studies in plants have provided a fairly comprehensive view as to how this is controlled at the organellar level as yet little is known there in plants at finer sub-compartment resolution. This is in sharp contrast to mammalian and microbial systems. Indeed the coining of the term ‘metabolon’ to define supermolecular complexes composed of sequential metabolic enzymes was first used in the context of the

References and recommended reading

Papers of particular interest, published within the period of review, have been highlighted as:

  • • of special interest

  • •• of outstanding interest

Acknowledgements

Financial support from the Max-Planck-Society (to WLA, ANN and ARF), the Deutsche Forschungsgemeinschaft (grant no. DFG-SFB429 to ARF), and the National Council for Scientific and Technological Development CNPq-Brazil (grant numbers 478261/2010-1 to ANN and 472787/2011-0 to WLA) are gratefully acknowledged.

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