(Received for publication, June 21, 1994; and in revised form, November 28, 1994)
From the
A cDNA encoding a dihydrolipoamide acetyltransferase (E2) subunit of the pyruvate dehydrogenase complex has been isolated from Arabidopsis thaliana. A cell culture cDNA expression library was screened with a monoclonal antibody (JIM 63) raised against nuclear matrix proteins, and four clones were isolated. One of these was 2175 base pairs in length, and it contained an open reading frame with an amino acid sequence and domain structure with strong similarity to the E2s of other eukaryotic and prokaryotic organisms. The organization and number of functional domains within the Arabidopsis protein are identical to those of the human E2, although the amino acid sequences within these domains are equally similar to those of the yeast and human proteins. The predicted amino acid sequence reveals the presence of a putative amino-terminal leader sequence with characteristics similar to those of other proteins, which are targeted to the plant mitochondrial matrix. The cross-reactivities of plant mitochondrial matrix proteins with JIM 63 and antibodies raised against the E2 and protein X components of eukaryotic pyruvate dehydrogenase complexes are consistent with the clone encoding a mitochondrial form of E2 and not the smaller protein X. The E2 mRNA of 2.2 kilobases was expressed in a range of Arabidopsis and Brassica napus tissues.
The pyruvate dehydrogenase complex (PDC), ()in all
organisms studied to date, is a large multi-enzyme complex that
catalyzes the oxidative decarboxylation of pyruvate, the transfer of
the acetyl unit to coenzyme A, and ultimately the reduction of
NAD
(Miernyk et al., 1985, 1987). The complex
is composed of three enzymes that act sequentially: pyruvate
dehydrogenase (named E1) (EC 1.2.4.1), dihydrolipoamide S-acetyltransferase (named E2) (EC 2.3.1.12), and
dihydrolipoamide dehydrogenase (named E3) (EC 1.8.1.4). The E1 subunit
catalyzes decarboxylation of pyruvate and the subsequent reductive
acylation of the lipoyl moiety, which is covalently bound to E2. E2
catalyzes the acyl transfer step, and E3 catalyzes the reoxidation of
the oxidized form of lipoamide (for reviews, see Reed and Hackert,
1990; Perham and Packman, 1989). Pyruvate dehydrogenase complexes are
organized as a core of E2 subunits, 24 copies arranged octahedrally in E. coli and other Gram-negative bacteria or 60 copies arranged
icosahedrally in Gram-positive bacteria, and all eukaryotic species so
far studied, around which are organized multiple copies of E1 and E3.
The mammalian and yeast PDC complexes also contain a protein designated
as protein X, with a molecular mass of about 50 kDa (De
Marcucci and Lindsay, 1985; Jilka et al., 1986). Protein X binds E3 and positions it to the E2 core of PDC (Lawson et
al., 1991; Rahmatullah et al., 1989) while E1 binds to
the core E2 proteins directly (Rahmatullah et al., 1989). The
binding of E1 and E3 to E2 and protein X, respectively,
involves analogous domains in the latter proteins (Lawson et
al., 1991). These domains are present in the somewhat similar
amino-terminal regions of protein X and E2 (Behal et
al., 1989; Rahmatullah et al., 1989). This suggests that
the two proteins evolved from a common ancestor but now represent
functionally distinct components.
In mammals, PDC is located exclusively in the mitochondria (Reed, 1974), whereas in plants PDC is present in both the mitochondria and the plastids. By comparison with animals and fungi, very little is known about the size, organization, and subunit structure of plant PDCs. The plant mitochondrial PDC has been purified (Randall et al., 1977; Rubin and Randall, 1977), although only the E3 subunit has been purified to homogeneity (Bourguignon et al., 1988). It is believed that a single dihydrolipoamide dehydrogenase is common to the pyruvate dehydrogenase, 2-oxoglutarate dehydrogenase, and glycine decarboxylase complexes of the mitochondria from leaves of higher plants (Bourguignon et al., 1992; Turner et al., 1992b). Isolation of a cDNA encoding the E3 of pea leaf mitochondria has provided the only sequence information on any of the plant PDC proteins to date (Bourguignon et al., 1992; Turner et al., 1992b). Plastidial PDC activity has been demonstrated in both green (Williams and Randall, 1979; Elias and Givan, 1979) and other plant tissues (Reid et al., 1977; Denyer and Smith, 1988; Fan and Rawsthorne, 1994). The subunit composition of chloroplast and mitochondrial PDC from pea has been examined immunologically by probing Western blots with antibodies raised against the whole PDC of mitochondria from broccoli florets. This study showed differences in the banding pattern of the subunits of the PDC from the two organelles (Camp and Randall, 1985). Most recently, E2 and protein X from pea mitochondria and chloroplasts have been investigated by the use of antibodies raised against the mammalian E2 and protein X (Taylor et al., 1992). It was reported that the mitochondrial and chloroplast E2s have a molecular mass of 50 kDa. Protein X in mitochondrial PDC was reported as 67 kDa, whereas in chloroplasts it was 48 kDa. In this study, we report the first isolation and characterization of a cDNAclone encoding the E2 subunit from a plant (Arabidopsis thaliana).
Figure 1: Nucleotide sequence of PDC-E2 cDNA and its deduced amino acid sequence. Nucleotides are numbered 5` to 3`. Amino acid residues (one letter code) of the putative mature form and its putative leader sequence are separately numbered with the mature peptide beginning at +1 (see Fig. 2for assignment of +1). The two lipoyl binding domains are underlined. The subunit binding domain is highlighted with roundbrackets. The catalytic domain is indicated by squarebrackets. Potential polyadenylation signals are indicated by underlineditalics.
Figure 2: Alignment of the amino acid sequences derived from clone 121 with that of the yeast, human, and E. coli PDC-E2 subunits. Asterisks represent residues from Arabidopsis, which are identical in at least one other PDC-E2 subunit. The deduced amino terminus of mature human E2 is based upon the homology to the directly determined amino acid sequence of bovine E2 and is indicated by the underlining of identical residues in the two mammalian proteins.
The sequence of the putative Arabidopsis PDC-E2 (Fig. 1) contains two highly conserved repeats of 18 amino acids (83% identity between residues 43-61 and 170-188) that are situated in larger repeating units (amino acid residues 1-79 and 129-206). Similar observations have been reported for human PDC-E2 (Thekkumkara et al., 1988) in which the two repeating units have been identified as the lipoyl domains and the conserved repeats as lipoyl binding sites. Comparison of one of the repeating units from putative Arabidopsis PDC-E2 with one of the lipoyl domains of human PDC-E2 exhibited 79% similarity (Table 1). The two repeating units are therefore postulated to be lipoyl domains in the putative Arabidopsis protein.
A sequence of 33 amino acids (residues 253-284) of Arabidopsis PDC-E2 is highly homologous to the subunit binding domain of PDC-E2 for human, yeast, and Escherichia coli (Table 1) and is probably the E1 binding site for the Arabidopsis enzyme.
The COOH-terminal region of putative Arabidopsis PDC-E2 has very good homology with the COOH-terminal catalytic domain of the human, yeast, and E. coli enzymes (Table 1). This COOH-terminal domain of the putative Arabidopsis E2 also contains the highly conserved sequence His-Xaa-Xaa-Xaa-Asp-Gly (residues 531-536), which is thought to be part of the catalytic site of all dihydrolipoamide acyltransferases (Guest, 1987). This motif is absent from yeast protein X in which the COOH-terminal part is less similar to that of yeast PDC-E2 than the amino-terminal part (Behal et al., 1989).
Figure 3: Immunoblots of Pisum sativum (pea) mitochondrial matrix. Lane1, JIM 63; lane2, PDC-E2/protein X autoantibody; lane3, anti-mammalian PDC-E2; lane4, anti-broccoli PDC; lane5, ant-pea PDC-E3. Positions of molecular weight markers are shown at the side. JIM 63 strongly labeled a band at 80 kDa, which was also labeled by antibodies to PDC-E2 (lane3) and PDC-E2/protein X (lane2).
Figure 4: Electron micrograph of Arabidopsis leaf cell immunogold labeled with JIM 63. Both chloroplast (C) and mitochondrial (M) labeling is shown. Bar = 200 nm.
Figure 5: Northern blot of total RNA from A. thaliana probed with clone 121. A single band of about 2.2 kilobases (kb) is shown.
We conclude that clone 121 encodes one of the PDC-E2 subunits from Arabidopsis and represents the first E2 sequence from any plant. This conclusion is based on the following observations. First, the amino acid sequence predicted from clone 121 is very similar to those of the PDC-E2s from E. coli, mammals, and fungi, and the defined domain structure of PDC-E2 is also highly conserved (Fig. 2, Table 1). Second, rescreening of the positive plaques identified by JIM 63 with an antibody to plant mitochondrial PDC also showed positive signals, indicating that the expressed protein was recognized by the anti-PDC antibody. Finally, Western blotting of plant mitochondrial matrix proteins was used to compare the proteins recognized by JIM 63 and by a range of antibodies with PDC components (Fig. 3). A protein of 80 kDa was specifically recognized by antibodies to mammalian PDC-E2 and was very strongly recognized by JIM 63. Although JIM 63 was raised against plant nuclear matrix, it must be concluded that it recognizes an epitope common to different proteins.
Although no detailed physical and chemical data are available for PDC-E2 from Arabidopsis, its extensive homology with PDC-E2 from human, yeast, and E. coli clearly demonstrates that the Arabidopsis enzyme has a highly defined structure similar to those of well studied E. coli acetyl- and succinyltransferases (Miles and Guest, 1988). The three characteristic domains that correspond to the lipoyl, E3 binding, and the catalytic domains are all well conserved in the Arabidopsis PDC-E2 sequence. The Arabidopsis PDC-E2 contains two lipoyl domains, and in this respect, it resembles the human counterpart. A comparison of the amino acid sequences suggests that plant PDC-E2 is as closely related to human as it is to yeast. This symmetrical relationship suggests that these three proteins were derived from a common ancestor and diverged early during evolution.
The reported homology between protein X and PDC-E2 raises the question of whether clone 121 represents protein X rather than the E2 subunit. Taylor et al.(1992) have reported that the pea mitochondrial and chloroplast E2s have apparent molecular masses of 50 kDa. Although a 50-kDa protein is very weakly recognized by JIM 63, we think it most likely that the 80-kDa protein, which is recognized by JIM 63 and the mammalian PDC-E2/protein X antibody, is the plant E2, and the 50-kDa protein may be protein X. This idea is supported by a number of lines of evidence. First, the highly conserved acyltransferase active site of dihydrolipoamide acyltransferases (Guest, 1987) is present in the COOH-terminal domain of the deduced protein sequence from clone 121. This acyltransferase active site is absent from yeast protein X (Behal et al., 1989). Second, the mammalian PDC-E2 has an apparent molecular mass of 74 kDa (Fussey et al., 1991), which is similar to that of the 80-kDa protein that is strongly recognized by JIM 63 in pea mitochondria. This observation is consistent with the similarity in domain structure between the plant and human E2s. Third, the 50-kDa protein of pea mitochondria is both similar in size to the protein X of mammalian and yeast PDCs (De Marcucci and Lindsay, 1985; Jilka et al., 1986) and is more weakly recognized by both JIM 63 and the mammalian PDC-E2 antibody. Taylor et al.(1992) had previously suggested that the apparent molecular mass of the plant E2s of 50 kDa was consistent with the presence of only a single lipoyl domain in the plant E2. The predicted amino acid sequence from clone 121 clearly reveals the presence of two lipoyl domains in the Arabidopsis E2. Based on these observations, we conclude that the 80-kDa protein in pea mitochondria is the E2 subunit of PDC while the 50-kDa protein may well be protein X.
Although the length and composition of the
interdomain linker segments vary among the acetyltransferases, these
segments are similar in that they are rich in the conservatively
substituted residues alanine, proline, serine, and threonine and in
charged amino acid residues. This is also true for the PDC-E2 of Arabidopsis (Fig. 1). These interdomain linker segments
are thought to contribute, at least in part, to the slow migration of
these proteins in SDS-polyacrylamide gel electrophoresis, resulting in
an aberrantly high M value (Guest et al.,
1985; Spencer et al., 1984). Thus, the calculated molecular
mass of the deduced amino acid sequence of the predicted mature Arabidopsis E2 is 60 kDa, whereas the apparent molecular mass
estimated by SDS-polyacrylamide gel electrophoresis is approximately 80
kDa. Another possibility for this difference in size may come from the
cDNA length and the prediction of the mature amino terminus. As the
cDNA does not contain an in frame stop codon upstream from the first
methionine, it may not be a full-length clone, and the predicted mature
protein could therefore be larger than 60 kDa. However, the mRNA
present in a range of organs of both Arabidopsis and B.
napus has an estimated size of slightly less than 2.2 kilobases,
which suggests that the clone is either full-length or at least very
close to full-length. Furthermore, the most upstream methionine
(residue -19) shows a good match to the plant consensus
translation initiation site (Lutcke et al., 1987). Further
confirmation of the initiation site could be obtained by sequencing the
5`-end of a longer clone or by primer extension studies.
It will be
necessary to identify definitively whether clone 121 encodes a
mitochondrial or a chloroplast enzyme. Comparison of the amino acid
sequences of PDC-E2 from Arabidopsis with that from human and
bovine PDC-E2s (Thekkumkara et al., 1988; Rahmatullah et
al., 1989) suggests that the threonine residue at +1 might be
the first amino-terminal residue of the mature protein. If the most
upstream methionine is the translation initiation site, a 19-amino acid
leader sequence would be predicted. If the entire predicted sequence is
translated, a leader sequence of 52 amino acids would be predicted,
which would be similar in size to the predicted leader sequence from
human PDC-E2 (Fig. 2). In either case, these putative leader
sequences are rich in serine (21%), with regions of hydroxylated amino
acids interspersed with positively charged amino acids (Rosie et
al., 1986), and they are predicted to form an amphipathic
-helix. These structural features are characteristic of a
mitochondrial targeting sequence, which is necessary for the transport
of nuclear-encoded proteins into mitochondria (von Heijne, 1986). Thus,
clone 121 may encode a mitochondrial enzyme. The ubiquitous expression
of the mRNA encoded by clone 121 across all the plant organs examined
also supports the possibility that it encodes the mitochondrial E2. It
has been shown that some plant tissues lack the plastidial form of PDC
(Rapp and Randall, 1980). The evidence from gold labeling and Western
blotting presented here suggest that the use of JIM 63 is unlikely to
resolve whether clone 121 might encode a plastidial PDC-E2. Indeed,
given that the antibody to the mammalian PDC-E2 (mitochondrial protein)
has been previously shown to recognize equally a similar sized protein
in both mitochondria and chloroplasts purified from pea leaves (Taylor et al., 1992), it appears that an immunological approach using
the antibodies that are currently available will not be conclusive.
Definitive proof of which organelle-specific form of E2 has been
identified in this study will require partial peptide sequence of the
purified E2 proteins from the mitochondrial matrix and the plastid.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) Z46230[GenBank].