After many unsuccessful attempts to detect cDNA encoding the catalytic subunit of bovine pyruvate dehydrogenase phosphatase (PDPc) in bovine cDNA libraries, an approach based on the polymerase chain reaction (PCR) was undertaken. Overlapping DNA fragments were generated by PCR from bovine genomic DNA and from cDNA synthesized from total RNA with synthetic oligonucleotide primers on the basis of experimentally determined amino acid sequences. The DNA fragments were subcloned and sequenced. The complete cDNA is 1900 base pairs in length and contains an open reading frame of 1614 nucleotides encoding a putative presequence of 71 amino acid residues and a mature protein of 467 residues with a calculated M(r) of 52,625. Hybridization analysis showed a single mRNA transcript of about 2.0 kilobases. Comparison of the deduced amino acid sequences of the mitochondrial PDPc and the rat cytosolic protein phosphatase 2C indicates that these protein serine/threonine phosphatases evolved from a common ancestor. The mature form of PDPc was coexpressed in Escherichia coli with the chaperonin proteins groEL and groES. The recombinant protein (rPDPc) was purified to near homogeneity. Its activity toward the bovine 32P-labeled pyruvate dehydrogenase complex was Mg(2+)-dependent and Ca(2+)-stimulated and comparable to that of native bovine PDP. An active, truncated form of rPDPc, with M(r) approximately 45,000, was produced in variable amounts during growth of cells and/or during the purification procedure.
Disruption of the PDX1 gene encoding the protein X component of the mitochondrial pyruvate dehydrogenase (PDH) complex in Saccharomyces cerevisiae did not affect viability of the cells. However, extracts of mitochondria from the mutant, in contrast to extracts of wild-type mitochondria, did not catalyze a CoA- and NAD(+)-linked oxidation of pyruvate. The PDH complex isolated from the mutant cells contained pyruvate dehydrogenase (E1 alpha + E1 beta) and dihydrolipoamide acetyltransferase (E2) but lacked protein X and dihydrolipoamide dehydrogenase (E3). Mutant cells transformed with the gene for protein X on a unit-copy plasmid produced a PDH complex that contained protein X and E3, as well as E1 alpha, E1 beta, and E2, and exhibited overall activity similar to that of the wild-type PDH complex. These observations indicate that protein X is not involved in assembly of the E2 core nor is it an integral part of the E2 core. Rather, protein X apparently plays a structural role in the PDH complex; i.e., it binds and positions E3 to the E2 core, and this specific binding is essential for a functional PDH complex. Additional evidence for this conclusion was obtained with deletion mutations. Deletion of most of the lipoyl domain (residues 6-80) of protein X had little effect on the overall activity of the PDH complex. This observation indicates that the lipoyl domain, and its covalently bound lipoyl moiety, is not essential for protein X function. However, deletion of the putative subunit binding domain (residues approximately 144-180) of protein X resulted in loss of high-affinity binding of E3 and concomitant loss of overall activity of the PDH complex.(ABSTRACT TRUNCATED AT 250 WORDS)
Structural studies by three-dimensional electron microscopy of the Saccharomyces cerevisiae truncated dihydrolipoamide acetyltransferase (tE 2 ) component of the pyruvate dehydrogenase complex reveal an extraordinary example of protein dynamics. The tE 2 forms a 60-subunit core with the morphology of a pentagonal dodecahedron and consists of 20 cone-shaped trimers interconnected by 30 bridges. Frozen-hydrated and stained molecules of tE 2 in the same field vary in size ϳ20%. Analyses of the data show that the size distribution is bell-shaped, and there is an approximately 40-Å difference in the diameter of the smallest and largest structures that corresponds to ϳ14 Å of variation in the length of the bridge between interconnected trimers. Companion studies of mature E 2 show that the complex of the intact subunit exhibits a similar size variation. The x-ray structure of Bacillus stearothermophilus tE 2 shows that there is an ϳ10-Å gap between adjacent trimers and that the trimers are interconnected by the potentially flexible C-terminal ends of two adjacent subunits. We propose that this springlike feature is involved in a thermally driven expansion and contraction of the core and, since it appears to be a common feature in the phylogeny of pyruvate dehydrogenase complexes, protein dynamics is an integral component of the function of these multienzyme complexes.The pyruvate dehydrogenase complexes (PDCs) 1 are among the largest (M r ϳ10 6 to 10 7 ) and most complex multienzyme structures known. A central feature of these complexes is a 24-mer (Escherichia coli) or 60-mer (eukaryotes and some Gram-positive bacteria) dihydrolipoamide acetyltransferase (E 2 ) core with the morphologies of a cube or a pentagonal dodecahedron, respectively (1-4). The cores have both functional and structural roles in organizing the multienzyme complex; the E 2 activity is associated with the scaffold to which the other components are attached. These include the pyruvate dehydrogenase (E 1 ) and dihydrolipoamide dehydrogenase (E 3 ), which requires a binding protein (BP) to anchor it to the core of the yeast and mammalian PDCs, although, in E. coli and Bacillus stearothermophilus PDCs, BP is not required (1-4).The E 2 subunits have multidomain structures consisting of one, two, or three amino-terminal lipoyl domains, followed by an E 1 and/or E 3 binding domain, and a carboxyl-terminal catalytic domain (1-4). X-ray crystallography (5-9) and threedimensional electron microscopy (10, 11) show that the E 2 catalytic domains are arranged in cone-shaped trimers at each of the 8 or 20 vertices of the cubic or dodecahedral structures, respectively (7,8,10,11). The trimers are interconnected by bridges to form an empty cage-like complex with the tip of the trimer directed toward the center of the structure.Examination of the 4-Å resolution crystal structures of dodecahedral truncated E 2 (tE 2 ) cores from Enterococcus faecalis and B. stearothermophilus and the 2-Å resolution crystal structure of a cubic tE 2 core from Azotobacter vinelandi...
Bovine pyruvate dehydrogenase phosphatase (PDP) is a Mg2+-dependent and Ca2+-stimulated heterodimer that is a member of the protein phosphatase 2C family and is localized to mitochondria. Insight into the function of the regulatory subunit of PDP (PDPr) has been gained. It Both phosphorylated El (P-E1) and PDP must be bound to the 60-mer icosahedral dihydrolipoamide acetyltransferase (E2) component of PDC to obtain a maximum rate of dephosphorylation. Ca>2 apparently mediates the specific binding of PDP to E2 in juxtaposition to P-El (7). This orientation decreases the apparent Km of PDP for P-El (7) and for Mg2> (6). There appears to be two Ca2+-binding sites, one intrinsic to PDPc and a second produced by association of PDP with E2 (2).At subsaturating concentrations of Mg2>, PDP activity is stimulated by polyamines, particularly spermine (8). Like Ca2+, spermine decreases the apparent Km of PDP for Mg2+ but by a different but hitherto unknown mechanism (6,8,9). This paper reports that PDPr decreases the sensitivity of PDPc to Mg2+ and that this effect is reversed by spermine, which apparently interacts with PDPr. These observations are potentially significant in understanding the molecular basis of the insulin-induced activation of the mitochondrial PDP.MATERIALS AND METHODS Materials. Highly purified PDC and PDP were prepared from bovine kidney mitochondria (10, 11). Recombinant PDPc was expressed in Escherichia coli and purified to near homogeneity (3). EB and E2 were obtained by resolution of the PDC (11). The E2 contained small amounts of tightly bound E3-binding protein (protein X) and PDH kinase (E2-X-K complex). The peptides RRASVA and RRATVA were prepared manually by solid-phase synthesis using Boc-Ala-Pam resin and Boc-protected amino acids (Peptides International 32P-labeled P-El was prepared by incubating a solution containing 10 mg of E1, 50 ,ug of E2-X-K complex, and 0.1 mM [,y-32P]ATP in 2 ml of buffer A for 1 h at 30°C. The solution was adjusted to pH 5.8 to precipitate P-El, which was dissolved in a small volume of buffer A, the solution was dialyzed against three changes of buffer A, and then centrifuged at 35,000 rpm and 4°C for 1-5 h in a Beckman model Optima TLX ultracentrifuge. The P-El contained 9-12 nmol of phosphate groups per mg of protein. P-El was also prepared by resolution of P-PDC (11).The peptides RRASVA and RRATVA were phosphorylated by incubating for 8-10 h at 30°C a solution containing 10 mg peptide, 25 mM Tris HCl (pH 7.3), 2.5 mM MgCl2, 0.1% 2-mercaptoethanol, 10% glycerol, 0.1 mM [,y-32P]ATP, and 40 units of protein kinase A catalytic subunit in a final volume of 2 ml (12). The reaction was terminated by adding 2 ml of 60% acetic acid, and the solution was passed through an anionexchange column (1.0 x 4 cm) of Dowex 2X8-100 equilibrated with 30% acetic acid. The flowthrough, which contained the phosphopeptide, was lyophilized. The peptide was dissolved in 1 ml of 25 mM Tris-HCl (pH 7.3), 0.1% 2-mercaptoethanol, and the pH was adjusted to 7.3 with 1 M Tris (pH 10...
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