A multisubunit acetyl coenzyme A carboxylase from soybean

Abstract

A multisubunit form of acetyl coenzyme A (CoA) carboxylase (ACCase) from soybean (Glycine max) was characterized. The enzyme catalyzes the formation of malonyl CoA from acetyl CoA, a rate-limiting step in fatty acid biosynthesis. The four known components that constitute plastid ACCase are biotin carboxylase (BC), biotin carboxyl carrier protein (BCCP), and the alpha- and beta-subunits of carboxyltransferase (alpha- and beta-CT). At least three different cDNAs were isolated from germinating soybean seeds that encode BC, two that encode BCCP, and four that encode alpha-CT. Whereas BC, BCCP, and alpha-CT are products of nuclear genes, the DNA that encodes soybean beta-CT is located in chloroplasts. Translation products from cDNAs for BC, BCCP, and alpha-CT were imported into isolated pea (Pisum sativum) chloroplasts and became integrated into ACCase. Edman microsequence analysis of the subunits after import permitted the identification of the amino-terminal sequence of the mature protein after removal of the transit sequences. Antibodies specific for each of the chloroplast ACCase subunits were generated against products from the cDNAs expressed in bacteria. The antibodies permitted components of ACCase to be followed during fractionation of the chloroplast stroma. Even in the presence of 0.5 M KCl, a complex that contained BC plus BCCP emerged from Sephacryl 400 with an apparent molecular mass greater than about 800 kD. A second complex, which contained alpha- and beta-CT, was also recovered from the column, and it had an apparent molecular mass of greater than about 600 kD. By mixing the two complexes together at appropriate ratios, ACCase enzymatic activity was restored. Even higher ACCase activities were recovered by mixing complexes from pea and soybean. The results demonstrate that the active form of ACCase can be reassembled and that it could form a high-molecular-mass complex.

Figure 1

DNA gel-blot analysis of soybean genomic DNA. Blots were probed with coding parts of soybean BCCP (A), α-CT (B), and BC (C) cDNAs at high stringency (65°C; see Methods for details). DNA was digested overnight; the restriction enzymes are indicated above each lane. The complexity of the restriction patterns suggests multiple gene copies for each of these ACCase subunits. Positions of λ/HindIII DNA size markers are indicated in thousands of bp.

Figure 2

Alignment of the 5′-UTR (A) and 3′-UTR (B) regions of soybean accA cDNAs. The ATG start codons are boxed and the terminator codons are underlined. Note that the deletion in clones 17a and 21a (marked with arrowheads) result in a frame shift in the coding region (shown by uppercase letters) such that the C terminus of the protein encoded by these clones is different from the one encoded in clones 1b and 8a. C, Alignment of a segment of ACCase α-CT subunits from different organisms. The region considered to be involved in acetyl CoA binding (Li and Cronan, 1992b) is underlined. All sequences are aligned to the E. coli protein.

Figure 3

Alignment of predicted secondary structures for subunits from soybean chloroplast and E. coli ACCases. Plots represent potential α-helix rods and β-sheets. The homologous regions of soybean and E. coli proteins are indicated.

Figure 4

A, Coelution of pea ACCase and [³H]BCCP. [³H]BCCP precursors were synthesized in vitro and imported into isolated pea chloroplasts. Plastid ACCase activity in the stromal fractions was determined after chromatography with a Sephacryl S300 column. Identical results obtained with α-CT precursors produced from accA were used in the same type of experiment (Reverdatto et al., 1997). B and C, Processing of the in vitro-synthesized soybean BC precursor by pea chloroplasts. Lanes 1, Translation with [³⁵S]Met. Lanes 2, Intact pea chloroplasts incubated with the BC precursor and reisolated through a Percoll cushion (total chloroplast protein). Lanes 3, BC precursor incubated with pea stromal preparation that has CPE activity. Lanes 4, Same as for lanes 3, but 5× concentrated (by ultrafiltration through Microcon-30) pea stroma. Note that lanes 2 and 4 contain sizable amounts of ribulose bisphosphate carboxylase, which causes the band attributable to mature BC to be distorted, whereas in lanes 3 there is no distortion. B, Coomassie blue stain for total protein. C, Autoradiogram of the gel from B. These data show that the soybean BC precursors processed after import into chloroplasts, and by cleavage with CPE, are of the same apparent size.

Figure 5

Microsequence analysis of the N termini of soybean α-CT2, BCCP, and BC after import into isolated pea chloroplasts. Proteins synthesized in vitro as precursors were labeled with [³H]Leu. The lower horizontal axis in each plot records the deduced amino acid sequences of the corresponding precursor, and the upper horizontal axis in each plot records the sequencer cycle. Vertical bars indicate the [³H]Leu recovered from each sequencer cycle.

Figure 6

A, Expression of soybean chloroplast ACCase subunits in E. coli. Total cell lysates were separated on a 9% SDS-PAGE gel. Gels were stained with Coomassie blue and photographed. Lane 1, Construct pB-1x; lane 2, pC-1x; lane 3, pD-1x; lane 4, pD-3x; lane 5, pA1-3x; lane 6, pA1-4x; lane 7, pB-2x. (See Table II for overview of expression constructs.) B, Refolding of E. coli-expressed BCCP subunit precursor. Soluble protein was run on SDS-PAGE and then probed with streptavidin (lane 1) or stained with Coomassie blue for total protein (lane 2). A substantial proportion of the bacterially expressed BCCP became soluble after refolding. In addition, the BCCP synthesized in E. coli is biotinylated.

Figure 7

SDS-PAGE gel blots probed with antibodies against soybean MS ACCase subunits. Soybean total seed (A) and pea leaf chloroplast stroma (B) extracts were probed. Lanes 1, Anti-BCCP; lanes 2, anti-BC; lanes 3, anti-β-CT (anti-pD-1x); lanes 4, anti-β-CT (anti-pD-3x); lanes 5, anti-α-CT (anti-pA1-3x); and lanes 6, anti-α-CT (anti-pA1-4x). (See Table II for overview of expression constructs.) Membranes were treated with both immune (lanes a) and preimmune (lanes b) sera. Whole membranes were cut into strips and each was incubated with a different antibody. Primary antibodies were used at 1:10,000 dilutions.

Figure 8

Analysis of column fractions from total soybean seed extracts after chromatography through antibody-affinity columns. Lanes 1, Protein-gel blot probed with streptavidin-alkaline phosphatase conjugate; lanes 2, anti-BCCP probe; lanes 3, anti-BC probe; lanes 4, anti-α-CT probe; lanes 5, anti-β-CT probe. Gly eluates from each column (indicated above the lanes) were separated on 9% SDS-PAGE and transferred to PVDF membranes. Each membrane was subsequently cut into strips and probed with each ACCase antibody.

Figure 9

Separation of soybean seed protein extract by gel filtration on Sephacryl S400. The column was eluted with buffer A (see Methods) at 0.05 mL/min. Fractions (0.75 mL) were collected, and a high-molecular-mass gel-filtration calibration kit (Pharmacia) was used to calibrate the column. Eluted protein fractions were separated on SDS-PAGE, blotted to nylon membranes, and analyzed with different antibodies as indicated. strept, Streptavidin-alkaline phosphatase conjugate. The results from the protein-gel blot are aligned with the chromatographic profile. Approximate positions of electrophoretic molecular-mass markers are shown at the right of the protein-blot panels. The position of Blue Dextran 2000 (BD) and other markers used to calibrate the column are shown above the elution profile. The markers were thryoglobulin (667 kD), ferritin (440 kD), catalase (232 kD), and aldolase (158 kD).

Figure 10

The same experiment as in Figure 9, except that buffer A contained 0.5 m KCl. Each fraction was concentrated and desalted with Microcon-30 concentrators. Other conditions are as in Figure 9. Note that the BC/BCCP and α-CT/β-CT complexes were resolved from one another and from the MF ACCase more completely at 0.5 m KCl than they were at lower salt, and that the salt did not cause disassembly of the two complexes.

Figure 11

Anion-exchange chromatography (Q-Sepharose column) of a soybean seed extract. After loading, the column was washed with buffer A (see Methods) at 0.5 mL/min, and then a KCl gradient (0–0.5 m) was applied. Final wash was with 2 m KCl. Fractions of 2 mL were collected, desalted, and concentrated with Microcon-10 concentrators. Proteins were separated on 9% SDS-PAGE, transferred to PVDF membranes, and probed with antibodies as indicated. strept, Streptavidin-alkaline phosphatase conjugate. The central part of the chromatographic profile is expanded to align with the lanes on the protein-blot panels. The positions of electrophoretic protein molecular-mass markers are shown at the right side of the panels for the protein blots.

Figure 12

Reconstitution of ACCase activity. The BC/BCCP and α-CT/β-CT components of soybean and pea ACCase from chloroplasts were each purified by ion-exchange chromatography as illustrated in Figure 11. Aliquots from fractions that contained either the BC/BCCP or α-CT/β-CT complexes were assayed for ACCase activity both individually and when combined with one another in various ratios (v/v). The expected activity refers to the sum of residual ACCase activity from each fraction from the column. Observed activity refers to that actually recovered after combining fractions at various ratios. In each case combining the BC/BCCP and α-CT/β-CT complexes stimulated ACCase activity. Remarkably higher activities than expected were observed when the soybean BC/BCCP and pea α-CT/β-CT complexes were combined.