Randall1 1 Department of Biochemistry, University of Missouri, Columbia, USA;2Plant Genetics Research Unit, USDA, Agricultural Research Service, Columbia, USA The pyruvate dehydrogenase
Trang 1M I N I R E V I E W
Regulation of pyruvate dehydrogenase complex activity
in plant cells
Alejandro Tovar-Me´ndez1, Jan A Miernyk1,2and Douglas D Randall1
1
Department of Biochemistry, University of Missouri, Columbia, USA;2Plant Genetics Research Unit, USDA,
Agricultural Research Service, Columbia, USA
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
mtPDCis the site of carbon entry into the tricarboxylic acid
cycle, while the plPDCprovides acetyl-CoA and NADH for
de novofatty acid biosynthesis The second level of
regula-tion of PDCactivity 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 mtPDCis regulated by reversible phosphorylation catalyzed
by intrinsic kinase and phosphatase components An addi-tional level of sensitivity is provided by metabolite control of the kinase activity The plPDCis not regulated by reversible phosphorylation Instead, activity is controlled to a large extent by the physical environment that exists in the plastid stroma
Keywords: complex; chloroplast; enzymology; localization; metabolic regulation; mitochondria; phosphorylation
Introduction
The pyruvate dehydrogenase complex (PDC) is a
multien-zyme complex catalyzing the oxidative decarboxylation of
pyruvate to yield acetyl-CoA and NADH The plant PDCs
occupy strategic and overlapping positions in plant
cata-bolic and anacata-bolic metabolism (Fig 1) Similar to other
PDCs, the plant complexes contain three primary
compo-nents: pyruvate dehydrogenase (E1), dihydrolipoyl
acetyl-transferase (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 PDCactivity in plant cells Detailed
descrip-tions of the plant complexes are provided by more
comprehensive reviews [1–3]
Compartmentation of the PDC
It is widely believed that eukaryotic cells arose as the result
of phagotrophic capture of bacteria and subsequent sym-biotic association The progenitors of mitochondria are thought to be a-proteobacteria [4], possibly related to contemporary Rickettsia [5] The plastids that are charac-teristic of plant cells are thought to have been derived from a single common primary symbiotic event with a cyanobac-terium [6] Subsequently, there was extensive gene migration
to the nucleus leaving both mitochondria and plastids as semiautonomous organelles Most mitochondrial and plas-tidial proteins, including the subunits of the PDC, are encoded within the nuclear genome of land plants, synthes-ized in the cytoplasm and then post-translationally imported into the organelles [3] In nonplant eukaryotes the PDCis exclusively localized within the mitochondrial matrix, and serves as an entry point for carbon into the Krebs cycle The regulatory properties of mtPDChave been specialized to minimize activity in an environment where ATP levels are high Plant cells contain an mtPDCthat 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 PDCfrom 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 plPDCactivity in the dark
Mature plastids differentiate from proplastid progenitors
to serve specialized functions in different plant organs Plastid terminology is largely based upon pigmentation,
Correspondence to J A Miernyk, USDA/ARS, Plant Genetics
Research Unit, 108 Curtis Hall, University of Missouri,
Columbia, MO 65211, USA.
Fax: + 1 573 884 7850, Tel.: + 1 573 882 8167,
E-mail: miernykj@missouri.edu
Abbreviations: PDC, pyruvate dehydrogenase complex; mtPDC,
mitochondrial pyruvate dehydrogenase complex; plPDC,
plastidial pyruvate dehydrogenase complex; E1, pyruvate
dehydro-genase; E2, dihydrolipoyl acetyltransferase; and E3, dihydrolipoyl
dehydrogenase; PDP, phospho-pyruvate dehydrogenase phosphatase.
(Received 13 September 2002, accepted 29 November 2002)
Trang 2with 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]
Pos-sibly this type of plastid originated from an endosymbiotic
event involving a red algal cell The fragmentary
informa-tion 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
mtPDCor plasmodial PDCs, but when this becomes
available it should provide us with additional phylogenetic,
evolutionary and regulatory insights
Plastidial PDC
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 plPDCfrom developing castor endosperm has the same kinetic mechan-ism 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 plPDCwas briefly controversial, however all of the subunits have now been cloned [7,16,17] and their plastidial localization verified by
in vitroimport 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 plPDCis not regulated by phosphorylation Early enzymatic studies of plPDCnoted that the pH optimum was significantly more alkaline than that of mtPDC, and that higher Mg2+concentrations were necessary for maxi-mal 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+ concentra-tion [21] Both of these changes would activate plPDC
De novosynthesis of fatty acids in green organs of plant cells
is light-driven and occurs exclusively within the plastids [8] The plPDCprovides acetyl-CoA and NADH for fatty acid biosynthesis [9], so it is essential that PDCactivity parallels that of fatty acid biosynthesis Thus, a unique mechanism for regulating activity of plPDCactivity has evolved based
Fig 1 Compartmentalization of metabolism in plant cells PS l , the light reactions of photosynthesis; PS d , the dark reactions of photosynthesis.
Trang 3upon 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]
Expression of plPDC
Expression of genes encoding the component enzymes of
plPDCis responsive to developmental and physiological
cues The level of plE1b 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 plPDCin 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 plE1b [25], plE2 [16],
and plE3 [17] in A thaliana flowers However, when the
b-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
Mitochondrial PDC
Product inhibition
As with their mammalian and microbial counterparts, plant PDCs employ a multisite ping-pong kinetic mechanism The forward reaction is irreversible under physiological condi-tions, but activity is sensitive to product inhibition by NADH and acetyl-CoA The Kivalues for NADH (20 lM) and acetyl-CoA (20 lM) 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 mito-chondria suggest that acetyl-CoA/CoA can also have a significant regulatory influence because of the small size of the total CoA pool [29]
Fig 2 Schematic overview of the regulation of pyruvate dehydrogenase complex activity in autotrophic plant cells Distinct regulatory mechanisms control the activity of mtPDCin the light and plPDCin the dark PS, photosynthesis; PR, the photorespiratory pathway; PDC, the pyruvate dehydrogenase complex; P-PDC, the phosphorylated (inactive) form of PDC.
Trang 4Reversible phosphorylation
Plant mtPDCs are regulated in part by reversible multisite
seryl-phosphorylation of the E1a subunit [1,2,30]
Regula-tory phosphorylation is catalyzed by an intrinsic PDK, and
dephosphorylation by an intrinsic PDP The three
phos-phorylation sites of mammalian PDCwere initially mapped
with the native bovine E1a [31] Both the relative positions
of the phosphorylated Ser residues and the flanking
sequences are conserved in mammalian E1a primary
sequences Stoichiometric phosphorylation of any
individ-ual site resulted in total inactivation, but indicated that there
were differences in the relative rates of phosphorylation [32]
Examination of plant mtE1a 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
mam-malian site 3 Recent results obtained from MS analysis of
tryptic peptides from pea seedling mtPDCverified
phos-phorylation of sites 1 and 2 (Ser300, Ser306; N R David,
J A Miernyk & D D Randall, unpublished results)
O2-electrode assays of PDCactivity 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
Although the primary sequences of PDKs closely resemble
those of protein His-kinases, PDKs exclusively
phosphory-late Ser residues [38,39] The Kmvalue of pea seedling PDK
for Mg-ATP is less than 5 lM, and the Vmaxvalues for PDK
are five- to 10-fold higher than those of PDP, implying that
an active PDCrequires tightly regulated PDK activity
[35,40,41] Inhibition of PDK activity by ADP is
competi-tive 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 lMNH4+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 mMcan arise from glycine decarboxylase
complex (GDC) activity during photorespiration
Pyruvate dehydrogenase phosphatase
The PDP is a type 2Cprotein phosphatase, and requires
divalent cations for activity [44] The activity of pea seedling
mtPDP was inhibited 40% by 10 mM Pi, 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 mtPDCis 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 mtPDChas 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 mtPDCactivity 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 photosyn-thesis 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 mtPDCphosphorylation state will most reflect changes in PDK activity
A complex regulatory network The occurrence of multiple sites of regulatory Ser phos-phorylation of PDCE1a, and multiple forms of PDK with distinct specificities, allows tremendous flexibility of meta-bolic 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
There are similarities in expression of mtPDCamong 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 mtPDCactivity [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 phosphory-lation state of mtPDCin pea seedlings [51] As was found with the plPDCsubunits, mtPDCis 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 GDCin pea seedlings [54] The GDCis absent from dark-grown plant organs, but is rapidly synthesized de novo upon transfer of plant organs to the
Trang 5light [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 mtPDCexpression in
monocot plants In maize, the E1 subunits were
coordi-nately 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, E1b reached maximum expression at the end
of cell elongation stage then decreased to relatively low
levels in mature cells By contrast, E1a 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
develop-ment, but is relatively low in autotrophic cells Higher
expression of mtPDCis 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
Despite the advances made in more than 25 years of study,
many aspects of the regulation of PDCactivity 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 PDCgene expression in plants, and prelimi-nary promoter analyses have been conducted only for the E3 genes from A thaliana [19] Thus, the increase in our understanding of regulation of PDCactivity in plant cells over the past 25 years constitutes a classical example of, ÔOne step forward, two steps back.Õ The next 25 years promise to be interesting times!
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