SEARCH

SEARCH BY CITATION

Keywords:

  • complex;
  • chloroplast;
  • enzymology;
  • localization;
  • metabolic regulation;
  • mitochondria;
  • phosphorylation

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Compartmentation of the PDC
  5. Plastidial PDC
  6. Expression of plPDC
  7. Mitochondrial PDC
  8. Product inhibition
  9. Reversible phosphorylation
  10. Pyruvate dehydrogenase kinase
  11. Pyruvate dehydrogenase phosphatase
  12. A complex regulatory network
  13. Expression of mtPDC
  14. Future prospects
  15. References

The pyruvate dehydrogenase complex (PDC) is subjected to multiple interacting levels of control in plant cells. The first level is subcellular compartmentation. Plant cells are unique in having two distinct, spatially separated forms of the PDC; mitochondrial (mtPDC) and plastidial (plPDC). The mtPDC is the site of carbon entry into the tricarboxylic acid cycle, while the plPDC provides acetyl-CoA and NADH for de novo fatty acid biosynthesis. The second level of regulation of PDC activity is the control of gene expression. The genes encoding the subunits of the mt- and plPDCs are expressed following developmental programs, and are additionally subject to physiological and environmental cues. Thirdly, both the mt- and plPDCs are sensitive to product inhibition, and, potentially, to metabolite effectors. Finally, the two different forms of the complex are regulated by distinct organelle-specific mechanisms. Activity of the mtPDC is regulated by reversible phosphorylation catalyzed by intrinsic kinase and phosphatase components. An additional level of sensitivity is provided by metabolite control of the kinase activity. The plPDC is not regulated by reversible phosphorylation. Instead, activity is controlled to a large extent by the physical environment that exists in the plastid stroma.


Abbreviations
PDC

pyruvate dehydrogenase complex

mtPDC

mitochondrial pyruvate dehydrogenase complex

plPDC

plastidial pyruvate dehydrogenase complex

E1

pyruvate dehydrogenase

E2

dihydrolipoyl acetyltransferase

E3

dihydrolipoyl dehydrogenase

PDP

phospho-pyruvate dehydrogenase phosphatase

Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Compartmentation of the PDC
  5. Plastidial PDC
  6. Expression of plPDC
  7. Mitochondrial PDC
  8. Product inhibition
  9. Reversible phosphorylation
  10. Pyruvate dehydrogenase kinase
  11. Pyruvate dehydrogenase phosphatase
  12. A complex regulatory network
  13. Expression of mtPDC
  14. Future prospects
  15. References

The pyruvate dehydrogenase complex (PDC) is a multienzyme complex catalyzing the oxidative decarboxylation of pyruvate to yield acetyl-CoA and NADH. The plant PDCs occupy strategic and overlapping positions in plant catabolic and anabolic metabolism (Fig. 1). Similar to other PDCs, the plant complexes contain three primary components: pyruvate dehydrogenase (E1), dihydrolipoyl acetyltransferase (E2) and dihydrolipoyl dehydrogenase (E3). In addition, mitochondrial PDC (mtPDC) has two associated regulatory enzymes: pyruvate dehydrogenase kinase (PDK) and phospho-pyruvate dehydrogenase phosphatase (PDP). Here we briefly describe our current understanding of the regulation of PDC activity in plant cells. Detailed descriptions of the plant complexes are provided by more comprehensive reviews [1–3].

image

Figure 1. Compartmentalization of metabolism in plant cells. PSl, the light reactions of photosynthesis; PSd, the dark reactions of photosynthesis.

Download figure to PowerPoint

Compartmentation of the PDC

  1. Top of page
  2. Abstract
  3. Introduction
  4. Compartmentation of the PDC
  5. Plastidial PDC
  6. Expression of plPDC
  7. Mitochondrial PDC
  8. Product inhibition
  9. Reversible phosphorylation
  10. Pyruvate dehydrogenase kinase
  11. Pyruvate dehydrogenase phosphatase
  12. A complex regulatory network
  13. Expression of mtPDC
  14. Future prospects
  15. References

It is widely believed that eukaryotic cells arose as the result of phagotrophic capture of bacteria and subsequent symbiotic association. The progenitors of mitochondria are thought to be α-proteobacteria [4], possibly related to contemporary Rickettsia [5]. The plastids that are characteristic of plant cells are thought to have been derived from a single common primary symbiotic event with a cyanobacterium [6]. Subsequently, there was extensive gene migration to the nucleus leaving both mitochondria and plastids as semiautonomous organelles. Most mitochondrial and plastidial proteins, including the subunits of the PDC, are encoded within the nuclear genome of land plants, synthesized in the cytoplasm and then post-translationally imported into the organelles [3]. In nonplant eukaryotes the PDC is exclusively localized within the mitochondrial matrix, and serves as an entry point for carbon into the Krebs cycle. The regulatory properties of mtPDC have been specialized to minimize activity in an environment where ATP levels are high. Plant cells contain an mtPDC that is closely related to those of animal cells, but additionally contain a plastidial form of the PDC (plPDC, Fig. 1) that is more closely related to the PDC from cyanobacteria [3,7]. In contrast to mtPDC, the regulatory properties of plPDC are specialized to minimize the effects of an environment with high levels of ATP. The physical environment within the chloroplast stroma changes markedly during the light/dark transition, and specialized regulatory mechanisms have evolved for control of plPDC activity in the dark.

Mature plastids differentiate from proplastid progenitors to serve specialized functions in different plant organs. Plastid terminology is largely based upon pigmentation, with leucoplasts, etioplasts, chloroplasts and chromoplasts being, respectively, unpigmented, pale yellow, green and red/orange. The chlorophyll-containing green plastids (chloroplasts) are the site of photosynthesis in autotrophic plant cells. Plastids, regardless of pigmentation or degree of differentiation, are the sole site of de novo fatty acid biosynthesis in plant cells [8]. All forms of plastids contain the plPDC, which provides the acetyl-CoA and NADH necessary for fatty acid biosynthesis [9].

Recently it has been discovered that certain animal cell parasites, such as Plasmodium spp., contain a type of nonphotosynthetic plastid termed the apicoplast [10]. Possibly this type of plastid originated from an endosymbiotic event involving a red algal cell. The fragmentary information available indicates that red algal plPDCs are more closely related to other plPDCs than to any mtPDC [3,7]. There is as yet no sequence information concerning red algal mtPDC or plasmodial PDCs, but when this becomes available it should provide us with additional phylogenetic, evolutionary and regulatory insights.

Plastidial PDC

  1. Top of page
  2. Abstract
  3. Introduction
  4. Compartmentation of the PDC
  5. Plastidial PDC
  6. Expression of plPDC
  7. Mitochondrial PDC
  8. Product inhibition
  9. Reversible phosphorylation
  10. Pyruvate dehydrogenase kinase
  11. Pyruvate dehydrogenase phosphatase
  12. A complex regulatory network
  13. Expression of mtPDC
  14. Future prospects
  15. References

Based upon the results of cell-fractionation, it was proposed that developing oilseeds contain a plastidial glycolytic pathway in addition to the classical cytoplasmic glycolysis [11]. It was additionally reported that these same plastids contain a unique form of the PDC [12–14]. The plPDC from developing castor endosperm has the same kinetic mechanism as mtPDC, but has distinct catalytic and enzymatic properties. It was later reported that green leaves from pea seedlings also contain both mitochondrial and plastidial forms of the PDC [15]. The occurrence of plPDC was briefly controversial, however all of the subunits have now been cloned [7,16,17] and their plastidial localization verified by in vitro import studies [16,18] and confocal microscopy of GFP-fusion proteins [19].

Similar to bacterial and mtPDC, the activity of plPDC is sensitive to product inhibition by NADH and acetyl-CoA [9,20]. Another property that is shared with bacterial PDCs is that plPDC is not regulated by phosphorylation. Early enzymatic studies of plPDC noted that the pH optimum was significantly more alkaline than that of mtPDC, and that higher Mg2+ concentrations were necessary for maximal activity [9,12]. When plant leaves are shifted from dark to light there is a rapid alkalinization of the chloroplast stroma along with an increase in the free Mg2+ concentration [21]. Both of these changes would activate plPDC. De novo synthesis of fatty acids in green organs of plant cells is light-driven and occurs exclusively within the plastids [8]. The plPDC provides acetyl-CoA and NADH for fatty acid biosynthesis [9], so it is essential that PDC activity parallels that of fatty acid biosynthesis. Thus, a unique mechanism for regulating activity of plPDC activity has evolved based upon the physical conditions present in the chloroplast stroma (Fig. 2). It is additionally possible that the activity of plPDC [22] might be sensitive to light:dark changes in the redox state of the chloroplast stroma [23] as are several chloroplast regulatory enzymes [24].

image

Figure 2. Schematic overview of the regulation of pyruvate dehydrogenase complex activity in autotrophic plant cells. Distinct regulatory mechanisms control the activity of mtPDC in the light and plPDC in the dark. PS, photosynthesis; PR, the photorespiratory pathway; PDC, the pyruvate dehydrogenase complex; P-PDC, the phosphorylated (inactive) form of PDC.

Download figure to PowerPoint

Expression of plPDC

  1. Top of page
  2. Abstract
  3. Introduction
  4. Compartmentation of the PDC
  5. Plastidial PDC
  6. Expression of plPDC
  7. Mitochondrial PDC
  8. Product inhibition
  9. Reversible phosphorylation
  10. Pyruvate dehydrogenase kinase
  11. Pyruvate dehydrogenase phosphatase
  12. A complex regulatory network
  13. Expression of mtPDC
  14. Future prospects
  15. References

Expression of genes encoding the component enzymes of plPDC is responsive to developmental and physiological cues. The level of plE1β mRNA expressed in A. thaliana siliques increased to a peak six to seven days after flowering, then decreased with seed maturity [25]. This pattern of developmental expression is parallel to that of plastidial acetyl-CoA carboxylase, consistent with a role for both enzymes in seed oil synthesis and accumulation [25]. The importance of plPDC in seed oil synthesis has been further supported by results from both digital Northern [26] and microarray [27] analyses of developing A. thaliana seeds.

In addition to developing seeds, it has been reported that there were high levels of expression of plE1β[25], plE2 [16], and plE3 [17] in A. thaliana flowers. However, when the β-glucuronidase (GUS) reporter gene was fused to the A. thaliana plE3 promoter, and this chimera expressed in tobacco plants, high levels of expression were seen in developing seeds and mature pollen grains while low levels were present in young leaves and flowers [19]. This result suggests the previously reported elevated levels of plPDC subunit expression in flowers might instead reflect mRNAs present in the pollen.

Product inhibition

  1. Top of page
  2. Abstract
  3. Introduction
  4. Compartmentation of the PDC
  5. Plastidial PDC
  6. Expression of plPDC
  7. Mitochondrial PDC
  8. Product inhibition
  9. Reversible phosphorylation
  10. Pyruvate dehydrogenase kinase
  11. Pyruvate dehydrogenase phosphatase
  12. A complex regulatory network
  13. Expression of mtPDC
  14. Future prospects
  15. References

As with their mammalian and microbial counterparts, plant PDCs employ a multisite ping-pong kinetic mechanism. The forward reaction is irreversible under physiological conditions, but activity is sensitive to product inhibition by NADH and acetyl-CoA. The Ki values for NADH (20 μm) and acetyl-CoA (20 μm) are within the physiological concentration range [28]. While the results from in vitro studies suggest that NAD+/NADH is the more important regulator, results from analyses using isolated intact mitochondria suggest that acetyl-CoA/CoA can also have a significant regulatory influence because of the small size of the total CoA pool [29].

Reversible phosphorylation

  1. Top of page
  2. Abstract
  3. Introduction
  4. Compartmentation of the PDC
  5. Plastidial PDC
  6. Expression of plPDC
  7. Mitochondrial PDC
  8. Product inhibition
  9. Reversible phosphorylation
  10. Pyruvate dehydrogenase kinase
  11. Pyruvate dehydrogenase phosphatase
  12. A complex regulatory network
  13. Expression of mtPDC
  14. Future prospects
  15. References

Plant mtPDCs are regulated in part by reversible multisite seryl-phosphorylation of the E1α subunit [1,2,30]. Regulatory phosphorylation is catalyzed by an intrinsic PDK, and dephosphorylation by an intrinsic PDP. The three phosphorylation sites of mammalian PDC were initially mapped with the native bovine E1α[31]. Both the relative positions of the phosphorylated Ser residues and the flanking sequences are conserved in mammalian E1α primary sequences. Stoichiometric phosphorylation of any individual site resulted in total inactivation, but indicated that there were differences in the relative rates of phosphorylation [32]. Examination of plant mtE1α sequences reveals that the Ser residue corresponding to mammalian site 1 is present, and there is a Ser one residue upstream of mammalian site 2 [33,34]. There is, however, no Ser corresponding to mammalian site 3. Recent results obtained from MS analysis of tryptic peptides from pea seedling mtPDC verified phosphorylation of sites 1 and 2 (Ser300, Ser306; N. R. David, J. A. Miernyk & D. D. Randall, unpublished results).

O2-electrode assays of PDC activity in isolated pea seedling mitochondria verified that PDK and PDP are simultaneously active [35], and that steady-state PDC activity reflects this antagonism. In contrast to the response of mammalian PDC, changes in ATP/ADP over a 20-fold range had no effect on phosphorylation state/activity [36]. Furthermore, Ca2+ had no affect on steady-state PDC activity [37].

Pyruvate dehydrogenase kinase

  1. Top of page
  2. Abstract
  3. Introduction
  4. Compartmentation of the PDC
  5. Plastidial PDC
  6. Expression of plPDC
  7. Mitochondrial PDC
  8. Product inhibition
  9. Reversible phosphorylation
  10. Pyruvate dehydrogenase kinase
  11. Pyruvate dehydrogenase phosphatase
  12. A complex regulatory network
  13. Expression of mtPDC
  14. Future prospects
  15. References

Although the primary sequences of PDKs closely resemble those of protein His-kinases, PDKs exclusively phosphorylate Ser residues [38,39]. The Km value of pea seedling PDK for Mg-ATP is less than 5 μm, and the Vmax values for PDK are five- to 10-fold higher than those of PDP, implying that an active PDC requires tightly regulated PDK activity [35,40,41]. Inhibition of PDK activity by ADP is competitive with respect to ATP but, unlike mammalian PDK, K+ does not effect ADP inhibition of the plant enzyme [40]. Pyruvate inhibition of PDK activity is also competitive with respect to ATP [37,40,42]. Pyruvate and ADP are synergistic inhibitors of PDK [42], which might allow the Krebs cycle to operate despite high matrix ATP concentrations. Pea seedling PDK activity is stimulated by 5–40 μm NH4+ and 10–80 mm K+, but inhibited by 10–100 mm Na+. The NH4+ decreases the Km for Mg-ATP by about sixfold [41,43]. Stimulation of PDK activity by NH4+ is additive with stimulation by K+. Mitochondrial concentrations of NH4+ as high as 3 mm can arise from glycine decarboxylase complex (GDC) activity during photorespiration.

Pyruvate dehydrogenase phosphatase

  1. Top of page
  2. Abstract
  3. Introduction
  4. Compartmentation of the PDC
  5. Plastidial PDC
  6. Expression of plPDC
  7. Mitochondrial PDC
  8. Product inhibition
  9. Reversible phosphorylation
  10. Pyruvate dehydrogenase kinase
  11. Pyruvate dehydrogenase phosphatase
  12. A complex regulatory network
  13. Expression of mtPDC
  14. Future prospects
  15. References

The PDP is a type 2C protein phosphatase, and requires divalent cations for activity [44]. The activity of pea seedling mtPDP was inhibited 40% by 10 mmPi, but was not affected by any of an extensive array of mitochondrial metabolites tested. In contrast to mammalian PDP, plant PDP is not stimulated by polyamines or Ca2+, either in vitro[44] or in isolated intact mitochondria [37]. Ca2+ actually antagonizes the Mg2+ activation of PDP from pea seedling mitochondria. There are two forms of mammalian PDP; the activity of PDP1 is enhanced by Ca2+, while that of PDP2 is not, which resembles the plant enzyme [45].

Results from both in vitro and in vivo studies have established that leaf mtPDC is rapidly phosphorylated in the light, and dephosphorylated in the dark [46,47]. Any conditions that inhibit photorespiration or glycine oxidation decrease the light-dependent phosphorylation. However, the in vivo light-dependent phosphorylation of mtPDC has been observed in leaves of C3 species as well as maize, a C4 plant that does not typically exhibit photorespiration or glycine oxidation. Our model for light-induced inhibition of mtPDC activity includes elevated matrix ATP levels, NH4+ (which activates PDK) produced by photorespiratory glycine metabolism, and NADH, which inhibits PDC (Fig. 2). In the dark, photorespiration is curtailed and NH4+ levels drop, and the phosphatase reactivates mtPDC. This mechanism regulates or limits unnecessary carbon oxidation by the Krebs cycle in the leaf during photosynthesis. Because pyruvate is an inhibitor of PDK, mtPDC can be in a more active status under light conditions if metabolic conditions cause high pyruvate levels to be present. Evidence to date indicates that only PDK is under regulation. Thus, changes in mtPDC phosphorylation state will most reflect changes in PDK activity.

A complex regulatory network

  1. Top of page
  2. Abstract
  3. Introduction
  4. Compartmentation of the PDC
  5. Plastidial PDC
  6. Expression of plPDC
  7. Mitochondrial PDC
  8. Product inhibition
  9. Reversible phosphorylation
  10. Pyruvate dehydrogenase kinase
  11. Pyruvate dehydrogenase phosphatase
  12. A complex regulatory network
  13. Expression of mtPDC
  14. Future prospects
  15. References

The occurrence of multiple sites of regulatory Ser phosphorylation of PDC E1α, and multiple forms of PDK with distinct specificities, allows tremendous flexibility of metabolic control in mammalian cells [45,48]. Such flexibility is necessary to adjust to changes in nutrition, developmental and physiological states, and health. While there is now a considerable body of knowledge concerning the regulatory complex in animal cells, there is very little comparable information about plant cells. It remains a challenge to understand how metabolism can be regulated in a complex eukaryote that has only one [49] or two [50] forms of PDK.

Expression of mtPDC

  1. Top of page
  2. Abstract
  3. Introduction
  4. Compartmentation of the PDC
  5. Plastidial PDC
  6. Expression of plPDC
  7. Mitochondrial PDC
  8. Product inhibition
  9. Reversible phosphorylation
  10. Pyruvate dehydrogenase kinase
  11. Pyruvate dehydrogenase phosphatase
  12. A complex regulatory network
  13. Expression of mtPDC
  14. Future prospects
  15. References

There are similarities in expression of mtPDC among dicot plants. In a detailed study of pea (Pisum sativum) seedling development, it was observed that changes in the levels of E1 and E2 proteins and mRNAs were coordinated with changes in mtPDC activity [34]. The highest activities were found in cells or organs that were rapidly expanding or differentiating; etiolated seedlings or the youngest leaves of light-grown plants. Activities decreased in mature leaves and were virtually nonexistent in senescing leaves. A similar pattern was observed in an earlier study of the phosphorylation state of mtPDC in pea seedlings [51]. As was found with the plPDC subunits, mtPDC is highly expressed in pollen [52,53].

Changes in E3 mRNA, protein and activity did not follow the same pattern [34]. This is not unexpected. In addition to being a component of the PDC, E3 is also associated with the GDC in pea seedlings [54]. The GDC is absent from dark-grown plant organs, but is rapidly synthesized de novo upon transfer of plant organs to the light [55,56]. In contrast to pea, A. thaliana has two mtE3 genes that are expressed at similar levels in stems, flowers and siliques [57]. Expression of the E3 gene, designated mtLPD1, was higher in leaves, and was strongly induced by light, whereas expression of mtLPD2 is higher in roots and is only moderately induced by light. The expression pattern of mtLPD2 resembles that of mtE1[33,58]. It appears that in A. thaliana mitochondria, mtLPD1 is preferentially associated with the GDC, although it is capable of also associating with all of the a-keto acid dehydrogenase complexes [57].

There have been fewer analyses of mtPDC expression in monocot plants. In maize, the E1 subunits were coordinately expressed with the highest mRNA levels found in pollen and roots [53]. There was not a substantial difference in maize E1 expression between dark and light-grown organs. A somewhat different developmental pattern was seen with barley leaves. While there was coordinate expression of the E1 proteins during the early stages of leaf development, E1β reached maximum expression at the end of cell elongation stage then decreased to relatively low levels in mature cells. By contrast, E1α reached the maximum expression level later and remained high in mature leaf cells [59].

In contrast to the single PDK gene present in A. thaliana[49], there are two maize PDKs [50]. A parallel pattern of PDK expression, with ZmPDK1 more highly expressed than ZmPDK2, was seen in most maize organs and tissues. The exception was green maize leaves, where the mRNA levels for both PDKs were similar. The seemingly higher expression of total PDK might be related to the increased concentration of leaf ATP during photosynthesis [50].

In summary, there is evidence for organ- and tissue-specific developmental control of the expression of mtPDC, and expression is responsive to light. The complex is highly expressed in heterotrophic stages of dicot plant development, but is relatively low in autotrophic cells. Higher expression of mtPDC is correlated with the metabolic and structural changes that accompany membrane expansion and remodeling. In most instances, there is coordinate expression of the E1 and E2 components. The transient accumulation of mRNA preceding changes in protein levels and catalytic activity is consistent with transcriptional regulation.

Future prospects

  1. Top of page
  2. Abstract
  3. Introduction
  4. Compartmentation of the PDC
  5. Plastidial PDC
  6. Expression of plPDC
  7. Mitochondrial PDC
  8. Product inhibition
  9. Reversible phosphorylation
  10. Pyruvate dehydrogenase kinase
  11. Pyruvate dehydrogenase phosphatase
  12. A complex regulatory network
  13. Expression of mtPDC
  14. Future prospects
  15. References

Despite the advances made in more than 25 years of study, many aspects of the regulation of PDC activity in plant cells remain enigmatic. Some components of both plastidial and mtPDCs are encoded by small multigene families [3,16,17,33,50,53,57,58,60]. Are there distinct functions for these subunits, as has been found in Ascaris suum[61,62]? As yet there is no evidence for the occurrence of an E3BP in either plastidial or mtPDCs. Is this because this subunit does not exist in plants, or is it simply that it has it not yet been discovered? This is an intriguing question, considering the importance of E3BP under conditions of elevated NADH/NAD+[63]. In contrast to the dilipoyl forms of E2 ubiquitous in mammalian PDCs, there are additionally monolipoyl forms of mtE2 in plants [60]. This raises obvious questions concerning the nature of the association of PDK and PDP with mtPDC. Although experiments are currently in progress, there is virtually no knowledge about plant PDPs. In the current mammalian regulatory paradigm, specific forms of PDK have specific roles in overall regulation [45,48]. How is it that plants can be adequately responsive with only one or two forms of PDK [49,50]? To date very little is known about the post-transcriptional regulation of PDC gene expression in plants, and preliminary promoter analyses have been conducted only for the E3 genes from A. thaliana[19]. Thus, the increase in our understanding of regulation of PDC activity in plant cells over the past 25 years constitutes a classical example of, ‘One step forward, two steps back.’ The next 25 years promise to be interesting times!

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Compartmentation of the PDC
  5. Plastidial PDC
  6. Expression of plPDC
  7. Mitochondrial PDC
  8. Product inhibition
  9. Reversible phosphorylation
  10. Pyruvate dehydrogenase kinase
  11. Pyruvate dehydrogenase phosphatase
  12. A complex regulatory network
  13. Expression of mtPDC
  14. Future prospects
  15. References
  • 1
    Luethy, M.H., Miernyk, J.A., David, N.R. & Randall, D.D. (1996) Plant pyruvate dehydrogenase complexes. In α-Keto Acid Dehydrogenase Complexes (Patel, M.S., Roche, T.E. & Harris, R.A., eds.), pp. 7192. Birkhauser-Verlag, Basel, Switzerland.
  • 2
    Randall, D.D., Miernyk, J.A., David, N.R., Gemel, J. & Luethy, M.H. (1996) Regulation of leaf mitochondrial pyruvate dehydrogenase complex activity by reversible phosphorylation. In Protein Phosphorylation in Plants (Shewry, P.R., Halford, N.G. & Hooley, R., eds.), pp. 87103. Oxford Press, Clarendon, UK.
  • 3
    Mooney, B.P., Miernyk, J.A. & Randall, D.D. (2002) The complex fate of α-ketoacids. Annu. Rev. Plant Biol. 53, 357375.
  • 4
    Martin, W., Hoffmeister, M., Rotte, C. & Henze, K. (2001) An overview of endosymbiotic models for the origins of eukaryotes, their ATP-producing organelles (mitochondria and hydrogenosomes), and their heterotrophic lifestyle. Biol. Chem. 382, 15211539.
  • 5
    Emelyanov, V.V. (2001) Rickettsiaceae, rickettsia-like endosymbionts, and the origin of mitochondria. Biosci. Report 21,117.
  • 6
    Moreira, D. & Philippe, H. (2001) Sure facts and open questions about the origin and evolution of photosynthetic plastids. Res. Microbiol. 152, 771780.
  • 7
    Johnston, M.L., Luethy, M.H., Miernyk, J.A. & Randall, D.D. (1997) Cloning and molecular analyses of the Arabidopsis thaliana plastid pyruvate dehydrogenase subunits. Biochim. Biophys. Acta 1321, 200206.
  • 8
    Rawsthorne, S. (2002) Carbon flux and fatty acid synthesis in plants. Prog. Lipid Res. 41, 182196.
  • 9
    Camp, P.J. & Randall, D.D. (1985) Purification and characterization of the pea chloroplast pyruvate dehydrogenase complex: a source of acetyl-CoA and NADH for fatty acid biosynthesis. Plant Physiol. 77, 571577.
  • 10
    Wilson, R.J. (2002) Progress with parasite plastids. J. Mol. Biol. 319, 257274.
  • 11
    Dennis, D.T. & Miernyk, J.A. (1982) Compartmentation of non-photosynthetic carbohydrate metabolism. Annu. Rev. Plant Physiol. 33, 2750.
  • 12
    Reid, E.E., Thompson, P., Lyttle, C.R. & Dennis, D.T. (1977) Pyruvate dehydrogenase complex from higher plants mitochondria and proplastids. Plant Physiol. 59, 842848.
  • 13
    Thompson, P., Reid, E.E., Lyttle, C.R. & Dennis, D.T. (1977) Pyruvate dehydrogenase complex from higher plants mitochondria and proplastids. Kinetics. Plant Physiol. 59, 849853.
  • 14
    Lyttle, C.R., Thompson, P., Reed, E.E. & Dennis, D.T. (1977) Pyruvate dehydrogenase complex from higher plants mitochondria and proplastids. Regulation. Plant Physiol. 59, 854858.
  • 15
    Williams, M. & Randall, D.D. (1979) Pyruvate dehydrogenase complex from chloroplasts of Pisum sativum L. Plant Physiol. 64, 10991103.
  • 16
    Mooney, B.P., Miernyk, J.A. & Randall, D.D. (1999) Cloning and characterization of the dihydrolipoamide S-acetyltransferase subunit of the plastid pyruvate dehydrogenase complex (E2) from Arabidopsis. Plant Physiol. 120, 443452.
  • 17
    Lutziger, I. & Oliver, D.J. (2000) Molecular evidence of a unique lipoamide dehydrogenase in plastids: analysis of plastidic lipoamide dehydrogenase from Arabidopsis thaliana. FEBS Lett. 484, 1216.
  • 18
    Johnston, M.L., Miernyk, J.A. & Randall, D.D. (2000) Import, processing, and assembly of the α- and β-subunits of chloroplast pyruvate dehydrogenase. Planta 211, 7276.
  • 19
    Drea, S.C., Mould, R.M., Hibberd, J.M., Gray, J.C. & Kavanagh, T.A. (2001) Tissue-specific and developmental-specific expression of an Arabidopsis thaliana gene encoding the lipoamide dehydrogenase component of the plastid pyruvate dehydrogenase complex. Plant Mol. Biol. 46, 705715.
  • 20
    Camp, P.J., Miernyk, J.A. & Randall, D.D. (1988) Some kinetic and regulatory properties of the pea chloroplast pyruvate dehydrogenase complex. Biochim. Biophys. Acta 933, 269275.
  • 21
    Igamberdiev, A.U. & Kleczkowski, L.A. (2001) Implications of adenylate kinase-governed equilibrium of adenylates on contents of free magnesium in plant cells and compartments. Biochem. J. 360, 225231.
  • 22
    Johnston, M.L., Miernyk, J.A. & Randall, D.D. (2000) Use of sulfhydryl-directed inhibitors in vitro to distinguish activities of the mitochondrial and plastidic forms of pyruvate dehydrogenase. Arch. Biochem. Biophys. 378, 192193.
  • 23
    Savchenko, G., Wiese, C., Neimanis, S., Hedrich, R. & Heber, U. (2000) pH regulation in apoplastic and cytoplasmic cell compartments of leaves. Planta 211, 246255.
  • 24
    Zhang, N. & Portis, A.R. Jr (1999) Mechanism of light regulation of Rubisco: a specific role for the larger Rubisco activase isoform involving reductive activation by thioredoxin-f. Proc. Natl Acad. Sci. USA 96, 94389443.
  • 25
    Ke, J., Behal, R.H., Back, S.L., Nikolau, B.J., Wurtele, E.S. & Oliver, D.J. (2000) The role of pyruvate dehydrogenase and acetyl-Coenzyme A synthetase in fatty acid synthesis in developing Arabidopsis seeds. Plant Physiol. 123, 497508.
  • 26
    White, J.A., Todd, J., Newman, T., Focks, N., Girke, T., de Ilarduya, O.M., Jaworski, J.G., Ohlrogge, J.B. & Benning, C. (2000) A new set of Arabidopsis expressed sequence tags from developing seeds. The metabolic pathway from carbohydrates to seed oil. Plant Physiol. 124, 15821594.
  • 27
    Ruuska, S.A., Girke, T., Benning, C. & Ohlrogge, J.B. (2002) Contrapuntal networks of gene expression during Arabidopsis seed filling. Plant Cell 14, 11911206.
  • 28
    Randall, D.D. & Miernyk, J.A. (1990) The mitochondrial pyruvate dehydrogenase complex. In Methods in Plant Biochemistry, Vol. 3, Enzymes of Primary Metabolism, (Lea, P.J., ed.), pp. 175192. Academic Press, London, UK.
  • 29
    Budde, R.J., Fang, T.K., Randall, D.D. & Miernyk, J.A. (1991) Acetyl-Coenzyme A can regulate activity of the mitochondrial pyruvate dehydrogenase complex in situ. Plant Physiol. 95, 131136.
  • 30
    Miernyk, J.A., Thelen, J.J. & Randall, D.D. (1998) Reversible phosphorylation as a mechanism for regulating activity of the mitochondrial PDC. In: Plant Mitochondria: from Gene to Function (Gardeström, P., Møller, I.M., Glimelius, K. & Glaser, E., eds.), pp. 321327. Backhuys Publishers, Leiden, the Netherlands.
  • 31
    Yeaman, S.J., Hutcheson, E.T., Roche, T.E., Pettit, F.H., Brown, J.R., Reed, L.J., Watson, D.C. & Dixon, G.H. (1978) Sites of phosphorylation on pyruvate dehydrogenase from bovine kidney and heart. Biochemistry 17, 23642370.
  • 32
    Korotchkina, L.G. & Patel, M.S. (2001) Probing the mechanism of inactivation of human pyruvate dehydrogenase by phosphorylation of three sites. J. Biol. Chem. 276, 57315738.
  • 33
    Luethy, M.H., Miernyk, J.A. & Randall, D.D. (1995) The mitochondrial pyruvate dehydrogenase complex: nucleotide and deduced amino-acid sequence of a cDNA encoding the Arabidopsis thaliana E1α-subunit. Gene 164, 251254.
  • 34
    Luethy, M.H., Gemel, J., Johnston, M.L., Mooney, B.P., Miernyk, J.A. & Randall, D.D. (2001) Developmental expression of the mitochondrial pyruvate dehydrogenase complex in pea (Pisum sativum) seedlings. Physiologia Plantarum 112, 559566.
  • 35
    Budde, R.J.A. & Randall, D.D. (1988) Regulation of the steady state pyruvate dehydrogenase complex activity in plant mitochondria. Reactivation constants. Plant Physiol. 88, 10261030.
  • 36
    Budde, R.J.A. & Randall, D.D. (1987) Regulation of pea mitochondrial pyruvate dehydrogenase complex activity. Inhibition of ATP-dependent inactivation. Arch. Biochem. Biophys. 258, 600606.
  • 37
    Budde, R.J.A., Fang, T.K. & Randall, D.D. (1988) Regulation of the phosphorylation of mitochondrial pyruvate dehydrogenase complex in situ. Effects of respiratory substrates and calcium. Plant Physiol. 88, 10311036.
  • 38
    Thelen, J.J., Miernyk, J.A. & Randall, D.D. (2000) Pyruvate dehydrogenase kinase from Arabidopsis thaliana: a protein histidine kinase that phosphorylates serine residues. Biochem. J. 349, 195201.
  • 39
    Tovar-Méndez, A., Miernyk, J.A. & Randall, D.D. (2002) Histidine mutagenesis of Arabidopsis thaliana pyruvate dehydrogenase kinase. Eur. J. Biochem. 269, 26012606.
  • 40
    Miernyk, J.A. & Randall, D.D. (1987) Some properties of plant mitochondrial pyruvate dehydrogenase kinase. In Plant Mitochondria (Moore, A.L. & Beechy, R.B., eds.), pp. 223226. Plenum Press, N.Y., USA.
  • 41
    Shuller, K.A. & Randall, D.D. (1989) Regulation of pea mitochondrial pyruvate dehydrogenase complex: Does photorespiratory ammonium influence mitochondrial carbon metabolism? Plant Physiol. 89, 12071212.
  • 42
    Shuller, K.A. & Randall, D.D. (1990) Mechanism of pyruvate inhibition of plant pyruvate dehydrogenase complex and synergism with ADP. Arch. Biochem. Biophys. 278, 211216.
  • 43
    Shuller, K.A., Gemel, J. & Randall, D.D. (1993) Monovalent cation activation of plant pyruvate dehydrogenase kinase. Plant Physiol. 102, 139143.
  • 44
    Miernyk, J.A. & Randall, D.D. (1987) Some properties of pea mitochondrial phospho-pyruvate dehydrogenase-phosphatase. Plant Physiol. 83, 306310.
  • 45
    Roche, T.E., Baker, J.C., Yan, X., Hiromasa, Y., Gong, X., Peng, T., Dong, J., Turkan, A. & Kasten, A. (2001) Distinct regulatory properties of pyruvate dehydrogenase kinase and phosphatase isoforms. Prog. Nucleic Acid Res. Mol. Biol. 70, 3375.
  • 46
    Budde, R.J.A. & Randall, D.D. (1990) Pea leaf mitochondrial pyruvate dehydrogenase complex is inactivated in vivo in a light-dependent manner. Proc. Natl Acad. Sci. USA 87, 673676.
  • 47
    Gemel, J. & Randall, D.D. (1992) Light regulation of leaf mitochondrial pyruvate dehydrogenase complex. Plant Physiol. 100, 908914.
  • 48
    Patel, M.S. & Korotchkina, L.G. (2001) Regulation of mammalian pyruvate dehydrogenase complex by phosphorylation: complexity of multiple phosphorylation sites and kinases. Exp. Mol. Med. 33, 191197.
  • 49
    Thelen, J.J., Miernyk, J.A. & Randall, D.D. (1998) Nucleotide and deduced amino acid sequences of the pyruvate dehydrogenase kinase from Arabidopsis thaliana (accession, AF039406) (PGR 98–192). Plant Physiol. 118, 1533.
  • 50
    Thelen, J.J., Muszynski, M.G., Miernyk, J.A. & Randall, D.D. (1998) Molecular analysis of two pyruvate dehydrogenase kinases from maize. J. Biol. Chem. 273, 2661826623.
  • 51
    Miernyk, J.A., Camp, P.J. & Randall, D.D. (1985) Regulation of plant pyruvate dehydrogenase complexes. Curr. Top. Plant Biochem. Physiol. 4, 175190.
  • 52
    Grof, C.P.L., Winning, B.M., Scaysbrook, T.P., Hill, S.A. & Leaver, C.J. (1995) Mitochondrial pyruvate dehydrogenase. Molecular cloning of the E1α subunit and expression analysis. Plant Physiol. 108, 16231629.
  • 53
    Thelen, J.J., Miernyk, J.A. & Randall, D.D. (1999) Molecular cloning and expression analysis of the mitochondrial pyruvate dehydrogenase from maize. Plant Physiol. 119, 635643.
  • 54
    Bourguignon, J., Merand, V., Rawsthorne, S., Forest, E. & Douce, R. (1996) Glycine decarboxylase and pyruvate dehydrogenase complexes share the same dihydrolipoamide dehydrogenase in pea leaf mitochondria: Evidence from mass spectrometry and primary-structure analysis. Biochem. J. 313, 229234.
  • 55
    Walker, J.L. & Oliver, D.J. (1986) Light-induced increases in the glycine decarboxylase multienzyme complex from pea leaf mitochondria. Arch. Biochem. Biophys. 248, 626638.
  • 56
    Bourguignon, J., Macherel, D., Neuburger, M. & Douce, R. (1992) Isolation, characterization, and sequence analysis of a cDNA clone encoding L-protein, the dihydrolipoamide dehydrogenase component of the glycine cleavage system from pea-leaf mitochondria. Eur. J. Biochem. 204, 865873.
  • 57
    Lutziger, I. & Oliver, D.J. (2001) Characterization of two cDNAs encoding mitochondrial lipoamide dehydrogenase from Arabidopsis. Plant Physiol. 127, 615623.
  • 58
    Luethy, M.H., Miernyk, J.A. & Randall, D.D. (1994) The nucleotide and deduced amino acid sequences of the E1β subunit of pyruvate dehydrogenase from Arabidopsis thaliana. Biochim. Biophys. Acta 1187, 9598.
  • 59
    Thompson, P., Bowsher, C.G. & Tobin, A.K. (1998) Heterogeneity of mitochondrial protein biogenesis during primary leaf development in barley. Plant Physiol. 118, 10891099.
  • 60
    Thelen, J.J., Muszynski, M.G., David, N.R., Luethy, M.H., Elthon, T.E., Miernyk, J.A. & Randall, D.D. (1999) The dihydrolipoamide S-acetyltransferase subunit of the mitochondrial pyruvate dehydrogenase complex from maize contains a single lipoyl domain. J. Biol. Chem. 274, 2176921775.
  • 61
    Huang, Y.J. & Komuniecki, R. (1997) Cloning and characterization of a putative testis-specific pyruvate dehydrogenase beta subunit from the parasitic nematode, Ascaris suum. Mol. Biochem. Parasitol. 90, 391394.
  • 62
    Huang, Y.J., Walker, D., Chen, W., Klingbeil, M. & Komuniecki, R. (1998) Expression of pyruvate dehydrogenase isoforms during the aerobic/anaerobic transition in the development of the parasitic nematode Ascaris suum: Altered stoichiometry of phosphorylation/inactivation. Arch. Biochem. Biophys. 352, 263270.
  • 63
    Klingbeil, M.M., Walker, D.J., Arnette, R., Sidawy, E., Hayton, K., Komuniecki, P.R. & Komuniecki, R. (1996) Identificationof a novel dihydrolipoyl dehydrogenase-binding protein in the pyruvate dehydrogenase complex of the anaerobic parasitic nematode, Ascaris suum. J. Biol. Chem. 271, 54515457.