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Plant Physiol. (1999) 119: 635-644
Molecular Cloning and Expression Analysis of the Mitochondrial
Pyruvate Dehydrogenase from Maize1
Jay J. Thelen,
Jan A. Miernyk, and
Douglas D. Randall*
Department of Biological Sciences (J.J.T.), and Department
of Biochemistry (D.D.R., J.A.M.), University of Missouri, Columbia,
Missouri 65211; and University of Missouri, Columbia,
Missouri 65211Mycotoxin Research Unit, United States Department
of Agriculture-Agricultural Research Service, National Center for
Agricultural Utilization Research, Peoria, Illinois 61604 (J.A.M.)
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ABSTRACT |
Four cDNAs, one encoding an
 -subunit and three encoding  -subunits of the mitochondrial
pyruvate dehydrogenase, were isolated from maize (Zea
mays L.) libraries. The deduced amino acid sequences of both
- and -subunits are approximately 80% identical with Arabidopsis
and pea (Pisum sativum L.) homologs. The mature N terminus was determined for the -subunit by microsequencing the protein purified from etiolated maize shoot mitochondria and was resolved by two-dimensional gel electrophoresis. This single
isoelectric species comprised multiple isoforms. Both - and
-subunits are encoded by multigene families in maize, as determined
by Southern-blot analyses. RNA transcripts for both - and
-subunits were more abundant in roots than in young leaves or
etiolated shoots. Pyruvate dehydrogenase activity was also higher in
roots (5-fold) compared with etiolated shoots and leaves. Both subunits
were present at similar levels in all tissues examined, indicating
coordinated gene regulation. The protein levels were highest in
heterotrophic organs and in pollen, which contained about 2-fold more
protein than any other organ examined. The relative abundance of these proteins in nonphotosynthetic tissues may reflect a high cellular content of mitochondria, a high level of respiratory activity, or an
extra plastidial requirement for acetate.
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INTRODUCTION |
The mitochondrial PDC is composed of multiple copies of three
catalytic components, PDH [E1, EC 1.2.4.1], dihydrolipoamide acetyltransferase [E2, EC 2.3.1.12], and dihydrolipoamide
dehydrogenase [E3, EC 1.8.1.4], and catalyzes the overall reaction:
pyruvate + CoA + NAD+  acetyl-CoA + NADH + CO2.
The PDH component is composed of nonidentical - and -subunits,
forming an 22
heterotetramer. In mammals, 20 to 30 of these heterotetramers attach to
the core of the complex, which is formed by 20 homotrimers of
dihydrolipoamide acetyltransferase (for review, see Patel and Roche,
1990 ).
PDH decarboxylates pyruvate and forms a hydroxyethylidene-TPP
intermediate. The C2 group is then transferred
from TPP to the lipoyl moiety of the dihydrolipoamide acetyltransferase
component by reductive acetylation. Dihydrolipoamide acetyltransferase
catalyzes the acetyl transfer to CoA and dihydrolipoamide dehydrogenase reoxidizes the dihydrolipoamide moiety using NAD+
(Reed, 1973).
Most eukaryotic mitochondrial PDCs are regulated by reversible
phosphorylation of the E1-subunit by a specific PDH kinase and
phosphatase (Patel and Roche, 1990, and refs. therein). Phosphorylation of E1 reduces its affinity for TPP and prevents pyruvate binding (Korotchkina et al., 1995), effectively inactivating the complex. Although the phosphorylation of one conserved Ser inactivates PDC, two
other phosphorylation sites are present on mammalian E1 (Yeaman et
al., 1978; Sugden et al., 1979). Plant E1-subunits are also
phosphorylated on Ser residues, but the number and location of the
phosphorylation site(s) have not yet been established (Randall et al.,
1996).
We have purified the maize (Zea mays L.) mitochondrial PDC,
determined its kinetic properties, and established that the E1- and
E1-subunits are 43 and 40 kD, respectively (Thelen et al., 1998a).
The E1-subunit has at least five isoelectric species, whereas the
-subunit has predominantly one. Although most of the
Km and Ki
values for maize PDC were typical of those for other plant PDCs,
differences were noted that could be important for PDC regulation in
C4 plants. For example, the
Km for TPP was approximately 10-fold higher
in maize than in pea (Pisum sativum L.) PDC. Conversely, the
Km for Mg was almost 10-fold lower in maize
compared with pea mitochondrial PDC. To better understand these kinetic
differences and to begin to establish the molecular properties of PDC
in C4 plants, we have undertaken an examination
of the E1- and E1-subunits of the maize complex at the molecular
level.
The accession numbers for the maize PDH isoforms reported in this
article are AF069911 (E1) and AF069908, AF069909, and AF069910 (for
E1 isoforms 1, 2, and 3, respectively).
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MATERIALS AND METHODS |
Plant Materials
Maize (Zea mays L. cv B73; Illinois Seed Foundation,
Urbana, IL) seedlings were grown in a growth chamber (10-h photoperiod) for about 14 d and etiolated maize seedlings were grown in a
darkened growth chamber (30°C) for 5 d. Pea (Pisum
sativum L. cv Little Marvel), Arabidopsis (cv Columbia), and
Kalanchöe daigremontianum seedlings (provided by J. Ringbauer, University of Missouri, Columbia) were grown in a growth
chamber (10-h photoperiod, 18°C) for about 14 d (pea), 40 d
(Arabidopsis), and 25 d (K. daigremontianum). Mitochondria were isolated from etiolated maize shoots according to the
method of Hayes et al. (1991) and from the other tissues using the
procedure of Fang et al. (1987).
Radiochemicals
[1-14C]Pyruvate was purchased from New
England Nuclear in the solid crystalline form and was dissolved in 6 mL
of 20 mM sodium pyruvate containing 3 mM HCl;
aliquots (50 µCi; 1 Ci = 37 GBq) were stored at  20°C.
Isolation of cDNAs and Sequencing Strategies
A maize cDNA clone obtained from the Maize EST Stock Center
(Columbia, MO) encoded a polypeptide with homology to the C-terminal half of the Arabidopsis PDH E1-subunit (accession no. U80186). The
partial maize cDNA was used as a probe to screen a maize 2-week-old seedling  ZAP (Stratagene) expression library (generously provided by
Professor Alice Barkan, University of Oregon, Eugene). A screen of more
than 3 million transformants yielded approximately 50 positive plaques.
DNA was excised and prepared according to the manufacturer's
instructions. Nested Exo III deletions were prepared for the longest
cDNA according to the "Erase-A-Base" protocol (Promega). The
deletion constructs were sequenced using the dye-deoxynucleotide chain
termination method using AmpliTaq polymerase (Perkin-Elmer Cetus).
Reaction products were analyzed on an automated sequencer (model 373, ABI, Foster City, CA) at the University of Missouri DNA Core Facility.
Two additional unique cDNAs encoding E1 polypeptides were obtained
from the Pioneer Hi-Bred International (Johnston, IA) maize EST
facility. These cDNAs were prepared and sequenced as described
previously. Sequence alignment and phylogenetic analysis were performed
with GeneWorks software (IntelliGenetics, Mountain View, CA). The
Pioneer Hi-Bred International maize EST database was also examined for
cDNAs encoding the E1-subunit. One cDNA long enough to encode the
entire E1-subunit was found, along with several partial E1
clones. The full-length E1 cDNA was sequenced as described
previously.
Protein Microsequencing
Approximately 200 mg of mitochondrial protein from etiolated maize
shoots was used to obtain 0.4 mg of highly enriched PDC (as described
by Thelen et al., 1998a). For N-terminal protein microsequencing, 100 µg of enriched PDC was resolved by two-dimensional gel
electrophoresis according to standard procedures with the following
modifications. Sodium thioglycolate (0.25 mM) and
glutathione (0.1 mM) were added to the first- and
second-dimension gels. The polyacrylamide gel for the second dimension
was prerun for 30 min to remove any nonpolymerized acrylamide and
persulfate. The protein was blotted to a PVDF membrane using transfer
buffer (10 mM 3-[cyclohexylamino]-1-propanesulfonic acid
[APS]-NaOH, pH 11.0, and 10% [v/v] methanol). After transfer the
protein was stained with amido black (0.1% [w/v] amido black, 40%
[v/v] methanol, and 1% [v/v] acetic acid), washed with 50% (v/v)
methanol, and then dried. The immobilized proteins were excised and
submitted to the University of Nebraska Protein Core Facility (Lincoln, NE) for sequencing by Edman degradation.
Antibodies
Monoclonal antibodies to the -subunit of the mitochondrial PDH
(E1), the -subunit to the ATPase, and the heat-shock chaperone (HSP70) were raised in mice immunized with total maize mitochondrial proteins (Luethy et al., 1993, 1995b; Lund et al., 1998). Polyclonal antibodies to the -subunit of the mitochondrial PDH (E1) were raised in rabbits immunized with purified recombinant Arabidopsis E1-maltose-binding fusion protein (M.H. Luethy, unpublished data).
Nucleic Acid Analyses
Total genomic DNA was isolated from 10-d-old green maize leaves
according to the method of Sambrook et al. (1989). Digested DNA (20 µg) was separated by electrophoresis on a 0.8% (w/v) agarose gel and
then transferred to a Nytran membrane (Schleicher & Schuell). Following
transfer, the membrane was UV cross-linked and rinsed in 2× SSC, 0.1%
(w/v) SDS prior to prehybridization. Prehybridization was performed at
65°C for 6 h in 2.5× SSPE, 1% (w/v) SDS, 1% nonfat dry milk
(Schnuck's, St. Louis, MO), and 0.025% (w/v) denatured salmon-sperm
DNA. Hybridization was performed in the same solution and temperature
with the entire E1 (EcoRI-XbaI) or E1
isoform 2 (XbaI) cDNA fragment labeled by random hexamer
extension (specific activity = 2500 mCi/mg). Subsequent to
hybridization, the membrane was washed twice with 2× SSC, 0.1% (w/v)
SDS for 1 h, twice with 0.2× SSC, 0.1% (w/v) SDS for 2 h,
and was then dried for autoradiographic exposure.
Total RNA was isolated from fresh maize organs using guanidinium
extraction (Sambrook et al., 1989). The RNA (40 µg) samples were
separated by electrophoresis on a 1.0% (w/v) agarose gel containing
2.2 M formaldehyde and subsequently transferred to a Nytran
membrane. Following transfer the membrane was UV cross-linked and
stained with 0.03% (w/v) methylene blue and 0.3 M sodium
acetate, pH 6.0, to visualize RNA markers and rRNA. Hybridization and
washing were carried out as described for the Southern analysis.
RT-PCR of Maize RNA
Approximately 60 µg of maize total RNA isolated from
various organs was treated with 5 units of RNase-free DNase (Boehringer Mannheim) for 2 h at 37°C in 10 mM
MgCl2, 1 mM DTT, and 50 units of
RNase inhibitor (RNasin, Promega). The RNA was then extracted with
phenol, precipitated with ethanol, and resuspended in nuclease-free water. The RNA was quantitated by A260 and
diluted to 10 ng/µL for use in RT-PCR.
Each RT-PCR reaction contained the following RNase-free reagents: 1.5 mM magnesium sulfate, 0.2 mM
deoxyribonucleotide triphosphates, 1.5 pmol/µL oligonucleotides, 0.1 unit/µL avian myeloblastosis virus RT, 0.1 unit/µL Tfl
DNA polymerase (Promega), 1× avian myeloblastosis virus RT buffer, 2.5 ng/µL DNase-free RNA. Reverse transcription proceeded for 45 min at
48°C. The PCR cycling was as follows: 2 min at 94°C (one cycle);
30 s at 94°C, 1 min at 60°C, 2 min at 68°C (40 cycles); 7 min at 68°C (one cycle). The oligonucleotides used for RT-PCR span
the putative intron of the E1 cDNA (Fig. 2) and are denoted: DDR206,
5 -ccgcgacatgtccctcatgc-3 (sense oligonucleotide); DDR212,
5-ctctcaacatttcggccctc-3 (sense oligonucleotide); and DDR 229, 5-gcccttcttataaacatttgt-3 (antisense oligonucleotide).
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| Figure 2.
Nucleotide and deduced amino acid sequence for
maize E1 cDNA. Amino acids are denoted by the single-letter
abbreviations. The stop codon is indicated by a period. Underlined
region indicates the putative TPP-binding domain. The targeting
peptide-processing site, indicated by the triangle, is putative and
based on characteristics of cleavage sites (von Heijne et al., 1989).
The putative intron splice site is shaded and the polyadenylation
signal is underlined. Oligonucleotides used as primers for RT-PCR
in Figure 6B are overlined. Numbers to the right indicate base
pairs or amino acid number. Asterisks and  denote residues referred
to in the text.
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Isolation of Proteins and Assay for PDC Activity
Total proteins from various maize organs were prepared by
homogenization with a mortar and pestle in liquid nitrogen. Homogenized powder was immediately suspended in SDS-PAGE sample buffer (8 M urea, 4% [v/v] 2-mercaptoethanol and 4% [w/v] SDS)
containing 0.01% (w/v) bromphenol blue, mixed thoroughly by vortexing,
and incubated at 70°C for 30 min.
In vivo steady-state PDC activity was determined by the rapid-sampling
technique of Budde and Randall (1990). Minus-enzyme and
acid-precipitated protein controls were performed for each data point
to determine background rates. Activity was linear and stable for up to
15 min. Each data point represents the mean of five determinations.
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RESULTS |
Three E1 cDNA clones were obtained from immature ear (AP9
inbred), green seedling (B73 inbred), and callus culture (B73 inbred) maize libraries and were designated isoforms 1, 2, and 3, respectively. These three cDNAs shared 65% (isoforms 1 and 2), 70% (1 and 3), and
89% (2 and 3) identity at the nucleotide level, whereas 90% identity
was observed at the amino acid level (Fig.
1). The three E1 cDNAs were 1511, 1679, and 1655 bp in length, with ORFs starting at bases 82, 258, and
233 and in-frame stop codons at bases 1200, 1379, and 1354 for isoforms
1, 2, and 3, respectively. They encoded polypeptides 373, 374, and 374 amino acids in length with calculated Mrs
of 39,767, 39,982, and 39,917. The calculated pI values were 5.3 and 4.8 for the precursor and mature proteins, respectively.
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| Figure 1.
Amino acid sequence comparison for maize E1
isoforms. Consensus sequence is noted at the top. Dots indicate
identity. Gaps, indicated by dashes, were inserted to maximize
homology. Overlines indicate the four conserved domains as first
pointed out by Wexler et al. (1991). Asterisks denote residues referred
to in text. The targeting peptide-processing site is indicated by the
inverted triangle. GeneWorks (IntelliGenetics) software was used to
perform the alignment algorithm.
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The E1 cDNA was obtained from an immature ear (B73 inbred) maize
library and was 1747 bp in length with a 930-bp ORF (Fig. 2). However, amino acid identity with the
Arabidopsis E1 stopped at base 980 with Ile-282. A second ORF was
found 285 bp downstream of Ile-282. This ORF started with Val-283 at
base 1267 and had an in-frame stop codon at base 1597. This second ORF
encoded a polypeptide with strong amino acid homology to the C-terminal 109 amino acids of Arabidopsis E1. When joined, the two
ORFs encoded a full-length E1 polypeptide 392 amino acids in length with a mass of 42,867 D. The calculated pI values were 8.26 and 6.89 for the precursor and mature protein, respectively. The 285-bp region
connecting the two ORFs was likely an unspliced intron. One
polyadenylation signal was observed in the E1 cDNA, 52 bases downstream of the stop codon (Fig. 2).
Among all eukaryotes, three distinct classes of E1 polypeptides were
apparent in dendrogram analyses (Fig.
3A). The maize E1 polypeptides shared
77% amino acid identity with other plant mitochondrial copies. The
E1-subunits from plants were more closely related to the yeast (48%
amino acid identity) than to the animal (43%-47%) polypeptides and
were more distantly related to the plastidial (36%), cyanobacterial
(30%), and Bacillus subtilis (23%) homologs. The maize
mitochondrial E1 was also closely related to other plant
mitochondrial E1-subunits (80% amino acid identity). As is the case
for E1, the plant mitochondrial E1 was more similar to yeast
(58% amino acid identity) than to the animal (55%), plastidial (36%), cyanobacterial (36%), or B. subtilis (32%)
homologs (Fig. 3B).
By comparison with other TPP-binding enzymes (Hawkins et al., 1989;
Robinson and Chun, 1993), a potential TPP-binding domain for E1 can
be predicted (Fig. 2, underlined). This potential TPP-binding domain
was highly conserved and contained one of only two Trp residues present
in plant mitochondrial E1-subunits (L-216-WKLP-220). The three Ser
residues phosphorylated in mammalian E1-subunits are indicated by
asterisks in Figure 2. Phosphorylation site 1 (Ser-292), conserved in
plastidial and mitochondrial E1 polypeptides, was the site
responsible for inactivation (Sugden et al., 1979). Ser-300 at site 2 was conserved only in animal sequences, although the plant
mitochondrial proteins contained a Ser one residue upstream of this
site. Phosphorylation site 3 (corresponding to Ser-232 of human
sequence) is an Ala in all plant mitochondrial E1-subunits described
to date, but is conserved in the mammalian and plastidial subunits.
A comparison of the primary sequence of E1 polypeptides from various
organisms (Fig. 1, overlined) revealed the four regions of homology
first noted by Wexler et al. (1991). Although conserved, the functional
significance of these four regions is uncertain. Regions 2 and 3 are
proposed to be involved in oxidative decarboxylation, and chemical
covalent modification studies of mammalian PDH have shown that Trp and
Arg residues are essential for catalytic activity (Ali et al., 1995;
Eswaran et al., 1995). Surprisingly, Trp-173 (Fig. 1, asterisk) was
conserved in the mitochondrial but not in the plastidial or bacterial
E1-subunits, whereas Arg-277 was conserved in all organisms except
cyanobacteria.
Genomic Southern analysis revealed that the maize genome contains
multiple copies of both E1 and genes (Fig.
4). The multigenic nature of maize E1
was confirmed by the cloning of three unique cDNAs. Northern analysis
indicated that the E1 and transcripts are approximately 1.5 and
1.6 kb in length, respectively (Fig. 5A).
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| Figure 4.
Genomic Southern analysis of maize E1 (left)
and E1 (right). Genomic DNA isolated from maize leaves was digested
with the indicated restriction enzymes. Approximately 30 µg of DNA
was fractionated by electrophoresis on an agarose gel, transferred to a
Nytran membrane, and probed with random-prime-labeled DNA. Marker sizes
are indicated to the right in kilobases.
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| Figure 5.
RNA, RT-PCR, and activity analysis of PDH from
maize tissues. A, Approximately 30 µg of total RNA isolated from
dark-grown seedlings, roots, or light-adapted leaves was fractionated
on an agarose formaldehyde gel, transferred to a Nytran membrane, and
probed with the entire E1 or E1 cDNA. RNA marker sizes are
indicated. As a gel-loading control, rRNA is included for comparison in
the bottom panel. B, Oligonucleotides specific for the E1 cDNA were
used for RT-PCR analysis with maize RNA isolated from dark-grown
seedlings, roots, or light-adapted leaves as the template. The top
panel displays the product obtained with primers DDR212 and DDR229. The
product in the bottom panel was obtained with primers DDR206 and DDR229
(Fig. 2). C, In vivo PDH specific activity was determined from maize
organs (dark-grown seedlings, roots, or light-adapted leaves) using a
radioisotopic assay. Values are the means of five independent
reactions. Error bars indicate SD.
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Immunoblot Analysis and Protein Microsequencing
Monoclonal antibodies to maize mitochondrial E1 recognized a
43-kD protein in mitochondria isolated from maize, pea, Arabidopsis, and K. daigremontianum, representing
C4 monocot (maize), C3
dicot (pea and Arabidopsis), and CAM plants (K. daigremontranum; Fig. 6). Antibodies
raised against recombinant Arabidopsis E1 recognized a 37-kD protein
from all plants; however, they primarily recognized a 40-kD polypeptide
from maize. On two-dimensional electrophoresis the maize E1 from
purified PDC preparations appeared predominantly as a single
isoelectric species with a mass of 40 kD (Thelen et al., 1998a).
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| Figure 6.
Immunoblot analysis of mitochondria purified from
various plants. Approximately 10 µg of purified mitochondrial protein
was loaded in each lane. Maize mitochondria were obtained from
etiolated shoots, whereas pea, Arabidopsis, and K. daigremontianum mitochondria were isolated from light-grown
leaves. Molecular masses of polypeptides are indicated.
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The 40-kD maize E1-subunit was subjected to N-terminal polypeptide
sequencing (Fig. 7A) and yielded 20 residues nearly identical to the deduced amino acid sequence of all
three E1 cDNAs, corresponding to Ser-35 through Glu-54 (Fig. 7B).
The only difference between the sequenced polypeptide and the deduced
sequence of isoforms 2 and 3 (both derived from B73 inbreds) occurred
at residue 49, where a Thr instead of a Ser was present. This
discrepancy could be the result of a protein-sequencing error or
multiple residues at this cycle, some of which went undetected.
Comparing the deduced sequence of all three isoforms showed that
isoform 1 has an Ile, whereas isoforms 2 and 3 have Met at position 41, indicating that the microsequenced polypeptide was likely a
mixture of isoforms.
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| Figure 7.
N-terminal microsequencing of PDH subunits. A,
Coomassie-blue-stained two-dimensional gel electrophoresis of highly
purified maize mitochondrial PDC from etiolated shoots. The pI is
indicated at the top and the size in kilodaltons is indicated to the
right. The circled polypeptide was microsequenced from a replica-blot
transferred to a PVDF membrane. At cycles 7 and 12 two residues were
obtained (indicated by the slash). B, Comparison of the deduced amino
acid sequence for the plant E1 subunits. Zm, Z. mays;
At, Arabidopsis; Ps, P. sativum. Shading indicates amino
acid identity. Gaps denoted by dashes were inserted to maximize
homology. The N termini of the mature maize and pea polypeptides are
underlined. Conserved Arg residues involved with peptide processing are
indicated in bold type.
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Organ-Specific Expression of PDH Subunits
The transcripts for both subunits are more abundant in roots than
in etiolated shoots or in light-adapted leaves (Fig. 5, A and B). This
expression pattern is also supported by PDH-specific activity
measurements showing a 5- and 7-fold higher specific activity in root
compared with etiolated shoot and light-adapted leaves, respectively
(Fig. 5C). The relative amount of each subunit, determined by
immunoblot analysis, revealed that E1 and E1 proteins are
abundant in roots, particularly compared with light-adapted leaves
(Fig. 8). Both PDH subunits were
expressed in similar amounts from all organs examined (Fig. 8).
Overall, the PDH subunits are most abundant in nonphotosynthetic organs
such as etiolated shoots, roots, flower silks, immature anthers, ear
shoots, and, particularly, pollen. Neither subunit was detectable in
the endosperm from 2-d-imbibed seeds.
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| Figure 8.
Immunoblot analyses showing the expression of PDH
subunits from various organs. Maize kernels were allowed to soak for
2 d prior to isolating the endosperm and scutellum. Etiolated
shoots were grown for 5 d in complete darkness. The remaining
samples were obtained from light-grown plants, grown in either a growth
chamber (four-leaf stage) or a greenhouse (adult). As a control, the
blots were reprobed with antibodies specific to other mitochondrial
proteins, HSP70 and the -subunit to ATP synthase. Approximately 25 µg of a total protein preparation was loaded per lane. Molecular
masses of polypeptides are indicated on the left (in
kilodaltons).
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DISCUSSION |
Three unique cDNAs encoding three isoforms of the -subunit of
PDH and at least three hybridized bands on Southern analyses indicate
that the maize -subunit is encoded by a multigene family. Although
the amino acid identity among the three isoforms is high, the
percentage of identity at the nucleotide level is 65%, 70%, and 89%.
Since the cDNAs are quite different, it is unlikely that the
differences are due to sequencing errors or artifacts of library construction. The present data indicate that the maize genome is also
multigenic for E1. This multigenic nature suggests the possibility
for complex regulation of PDH expression in maize.
The insertion within the ORF of the E1 cDNA can be parsimoniously
explained as an unspliced intron. In favor of this is the higher AU
content in this region (51%) compared with the coding region (42%)
and an AU-UG splice sequence at the 3 end of the intron, both
characteristic of introns (Brown, 1986; Goodall et al., 1989).
Furthermore, the transcript size for E1 suggests that this putative
intron is normally excised. To confirm this, RT-PCR was performed with
primers that overlap the region containing the putative intron to
determine whether the intron sequence is typically removed. The PCR
products shown in Figure 5B are the proper size amplicons provided that
the E1 transcript lacks this 285-bp intron. Perhaps the excision
efficiency of this intron is reduced because of the lack of a canonical
splice site at the 5 end.
The first 40 amino acids of the maize E1 and E1 isoforms are
enriched with Ala, Arg, and Val residues (about 45%), typical of
mitochondrial targeting peptides (von Heijne, 1989; Sjöling and
Glaser, 1998). When the first 40 amino acids are modeled as an
-helix, the positively charged Arg residues cluster to one side,
whereas the hydrophobic residues cluster on the other side, forming an
amphipathic helix (data not shown), another characteristic of
mitochondrial targeting peptides (von Heijne, 1989; Sjöling and
Glaser, 1998). The mature N terminus of the maize E1 polypeptide was
determined by sequencing the purified polypeptide (Fig. 7). Processing
of the E1 polypeptides occurs between Tyr-32,34 and Ser-33,35 for
maize, between Tyr-29 and Ala-30 (by analogy) for Arabidopsis
(accession no. U09137), and between Phe-20 and Ser-21 for pea
(accession no. U56697; N.R. David, unpublished results; Fig. 7). The
plant E1 polypeptides all have a conserved Arg three residues
upstream from the cleavage site, a characteristic of mitochondrial
precursor processing (von Heijne, 1989).
We reported previously (Thelen et al., 1998a) that the kinetic
properties of PDH from maize are similar to those of other plant
species, except for the Km values for TPP
and divalent cations. In light of the high amino acid conservation
among plant PDH subunits, the discrepancy in
Km values might be explained by differences in enzyme preparations (i.e. purity, buffer composition). However, the
10-fold higher Km for TPP is intriguing and
might be due to nonconserved substitutions within the TPP-binding site.
One particular substitution (Asp-215 [pea] to Lys-218 [maize]; Fig.
2, ) in the TPP-binding domain of E1 is adjacent to an essential
hydrophobic residue (Trp-217). Perhaps a basic residue in place of an
acidic residue within this conserved region weakens the association
with the TPP moeity. Direct determination will require site-directed mutagenesis of the recombinantly expressed E1 subunit.
The E1 and E1 transcripts are most abundant in roots, followed by
etiolated shoots and light-adapted leaves (Fig. 5, A and B). This order
is the same as the abundance of E1 and E1 transcripts in
Arabidopsis organs, i.e. roots contain the most (Luethy et al., 1994,
1995a). The order of the relative abundance for E1 and E1 protein
(Fig. 8) follows that of their transcripts, roots > etiolated
shoots >> leaves. The specific activities of PDH (Fig. 5C) from roots
and leaves also correlate with transcript and protein levels. The
specific activity in etiolated shoots was slightly lower than predicted
by transcript and immunoblot analysis. However, caution must be used
when interpreting PDH activity for three reasons. At present, the
contribution of plastidial PDH to the total activity is difficult to
estimate. Therefore, to keep plastid activity to a minimum, the assay
was performed at pH 7.4 with 0.5 mM
MgCl2, which is optimal for the mitochondrial but
not for the plastidial PDH (Camp and Randall, 1985). Second, PDH
is regulated by reversible phosphorylation. The maize PDH kinase is
more abundant in leaves than in roots (Thelen et al., 1998b), which
might confound the interpretation of PDH activity. Also, cell breakage
necessary for enzyme release might alter the phosphorylation status of
PDH due to mixing of subcompartmental ATP. To control for the latter, the time between organ disruption and assay initiation was kept to a
minimum of about 15 s.
The steady-state level of both PDH subunits is highest in dry pollen
(Fig. 8). To investigate whether PDH is highly expressed in pollen of
other species, germinating tobacco pollen was also analyzed (provided
by Dr. Stefano Caveniscini, University of Missouri) and the result was
the same for both PDH subunits. From these data it is evident that PDH
is highly expressed in both dry and germinating pollen. This finding is
quite surprising in light of a recent observation that tobacco pollen
was able to germinate in the presence of a mitochondrial PDC inhibitor
(op den Camp and Kuhlemeier, 1997), which prompted this group to
propose the absence of PDC and that an alternative pathway for
acetyl-CoA production from pyruvate was operational. Our results
suggest that both PDH subunits are abundant in pollen, which points to multiple routes to acetyl-CoA or to an intriguing regulatory difference in pollen PDC.
Immunoblot analysis was used to examine whether PDH polypeptides were
differentially expressed in various organs relative to other
mitochondrial proteins (Fig. 8). The results show that PDH expression
does not correlate with the overall abundance of mitochondria in these
organs, which indicates that PDH and perhaps PDC are spatially
expressed in a manner different from total mitochondria. Why is PDH
spatially regulated, and, in particular, why is it so low in green leaf
tissue? Pyruvate is a potent inhibitor of PDH kinase, and in maize
total pyruvate levels are relatively high (3-4 mM in
leaves; Stitt and Heldt, 1985), which is favorable for PDH
activity. Pyruvate generated from the decarboxylation of
malate in bundle-sheath cells is recycled to PEP in mesophyll cells for
another round of carboxylation (Hatch, 1987, and refs. therein). If the
pyruvate intermediate were consumed in either cell type by PDH,
pyruvate would need to be synthesized de novo. Since all indications
are that pyruvate is recycled, PDH from maize must either have unique
properties or be down-regulated in leaves. Except for the unusually
high Km for TPP and the lower Km for Mg, the properties of the maize PDH
are similar to PDHs from other C3 plants (Thelen
et al., 1998a). Perhaps down-regulation of the mitochondrial PDH in
light-adapted leaves allows the photosynthetic C3-C4-shuttling mechanism
to proceed without consuming the pyruvate intermediate.
The apparent absence of PDH in scutellum and yet the relative abundance
of mitochondrial ATPase suggests that carbon for oxidative phosphorylation may be supplied in a form other than through pyruvate. This is surprising in that mobilization of starch from the endosperm would seemingly proceed through glycolysis and the Kreb's cycle. The
Kreb's cycle cannot function without acetyl-CoA input. Perhaps ATP
needs are met by the oxidative pentose pathway supplying reducing equivalents to the electron transport chain, or acetyl-CoA for the
Kreb's cycle is provided by -oxidation of storage lipids.
In conclusion, we have identified cDNAs encoding both PDH subunits
from maize. Both subunits are encoded by multigene families in maize,
which may contribute to the surprisingly complex spatial regulation
observed by immunoblot analysis. Transcript and protein levels
indicate that the two subunits for PDH are coordinately regulated.
Based on transcript, protein, and activity measurements, PDH is least
abundant in light-adapted leaves, which might be advantageous for
C4 metabolic function.
| |
FOOTNOTES |
1
This research was supported by National Science
Foundation grant no. IBN-9419489 and by a Maize Training Grant
Fellowship awarded to J.J.T. This is journal report 12,744 from the
Missouri Agricultural Experiment Station.
*
Corresponding author; e-mail bchemdr{at}showme.missouri.edu; fax
1-573-882-5635.
Received June 12, 1998;
accepted November 2, 1998.
| |
ABBREVIATIONS |
Abbreviations:
EST, expressed sequence tag.
ORF, open reading
frame.
PDC, pyruvate dehydrogenase complex.
PDH, pyruvate
dehydrogenase.
RT, reverse transcriptase, TPP, thiamin PPi.
| |
ACKNOWLEDGMENTS |
The authors are grateful to Dr. Michael Muszynski for
his assistance with the database searches and also to Pioneer Hi-Bred International for supplying the cDNA clones. The authors thank Nancy R. David for isolating mitochondria from the various plant species. We
also thank Professor Thomas E. Elthon and Dr. Gautum Sarath for the
protein microsequencing performed at the Protein Core Facility
(University of Nebraska, Lincoln).
| |
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Abstract
Introduction
