Proc. Nad. Acad. Sci. USA Vol. 88, pp. 5001-5005, June 1991

Biochemistry

From one gene to two proteins: The biogenesis of cytochromes b and cl in Bradyrhizobium japonicum (heme c binding/polyprotein/protein translocation/signal peptide/ubiquinol-cytochrome-c reductase)

LINDA THONY-MEYER*t, PETER JAMESO§, AND HAUKE HENNECKE*¶ *Mikrobiologisches Institut and 4Laboratonum fir Biochemie, Eidgen6ssische Technische Hochschule, ETH-Zentrum, CH-8092 Zarich, Switzerland Communicated by Werner Arber, February 7, 1991 (received for review December 12, 1990)

Genes coding for polyproteins that are ABSTRACT cleaved posttranslationally into two or more functional proteins are rarely found in prokaryotes. One example concerns the biogenesis of the Bradyrhizobiumjaponicum cytochromes b and cl, two of the three constituent subunits of ubiquinolcytochrome-c reductase (ubiquinol:ferricytochrome-c oxidoreductase, EC 1.10.2.2); the respective apoproteins for these subunits are encoded by the 5' and 3' halves of a single gene, fbcH. These two halves are linked by an extra piece of DNA encoding a characteristic signal peptide for protein translocation across the cytoplasmic membrane. Processing of thejbcH gene product is shown to occur at a typical signal peptidase recognition site. This reaction is reminiscent of that catalyzed by the regular bacterial signal peptidase that normally cleaves off presequences from the N termini of translocated proteins. Mutational alteration of the signal peptidase recognition site within FbcH results in the appearance of an uncleaved bec fusion protein in the membrane. Additionally, a functional heme-binding site in the apocytochrome cl section of FbcH is shown to be a necessary prerequisite for the formation of the be1 complex.

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FIG. 1. Primary structure characteristics of the B. japonicum FbcH protein. The large, horizontal arrow symbolizes the fbcHgene product with its N-terminal cytochrome b part (cyt b), the C-terminal cytochrome cl part (cyt cl), and an internal signal peptide (ISP) that connects them. At top, positions and amino acid sequences of peptides used to make antibodies are shown. Below FbcH are the amino acid sequences of the internal signal peptide (hydrophobic central part is underlined), of the N-terminus of mature cl after the signal peptidase cleavage site (bold vertical arrow), and of the heme-binding site in cl. Mutational alterations in the signal peptidase recognition site and the heme-binding site are also indicated (A = deletion). e, Characteristic amino acids in signal peptides (see text); 3, net positive charges.

Many species of the aerobic bacteria possess a respiratory chain similar to that in mitochondria (1). In this chain, ubiquinol-cytochrome-c reductase (ubiquinol:ferricytochrome-c oxidoreductase, EC 1.10.2.2; also called cytochrome bc, complex or complex III in mitochondria) oxidizes ubiquinol and reduces cytochrome c, which, in turn, transfers electrons to the terminal oxidase cytochrome aa3. In its simplest (bacterial) form, the cytoplasmic membranebound bc1 complex consists of the Rieske iron-sulfur protein, cytochrome b, and cytochrome c1, the apoproteins of which are encoded by the fbcF, fbcB, and fbcC genes, respectively (2). The genetic organization is different, however, in Bradyrhizobium japoqicum, the nitrogen-fixing, microaerobic endosymbiont in soybean root nodules. In this bacterium, the three electron-transfer subunits of the bc, complex are uniquely encoded by only two genes, .fbcF and fbcH, which form the JbcFH operon (3). The characteristics of the predicted FbcH protein, as derived from the nucleotide sequence of thejbcH gene, are shown in Fig. 1. The N-terminal sequence of =400 amino acids resembles the cytochrome b primary structures from many different organisms, whereas the C-terminal segment of -250 amino acids is similar to cytochrome c1. The two pieces are connected by =30, mostly hydrophobic, amino acids. The 687-amino-acid FbcH protein (Fig. 1) with its predicted Mr of 76,489 is a hypothetical protein that has never been detected in B. japonicum cells. Instead, we had previously obtained evidence for a cytochrome cl of Mr 28,000, which obviously must be a processed derivative of thejbcH gene product (3). The question

tPresent address: Departments of Biochemistry and Developmental Biology, Beckman Center, Stanford University School of Medicine,

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

§Present address: Department of Pharmaceutical Chemistry, Mass Spectrometry Facility, University of California, San Francisco, CA 94143-0446. ITo whom reprint requests should be addressed. 5001

of how this postulated processing event occurs is addressed in this report. This type of analysis is additionally complicated by the topology of the JbcH gene products in the cytoplasmic membrane. The cytochrome b is an integral membrane protein containing eight helical, membrane-spanning domains (2). The transmembrane helices II and IV each contain two histidine residues, the imidazole nitrogens of which function as ligands to the central iron atoms of the two b-type heme molecules.- By contrast, the bulk of cytochrome c1, in which a c-type heme is bound covalently by thioether linkage to two cysteines, faces the periplasmic side, and only one transmembrane domain at the very C-terminal end of the protein anchors it in the cytoplasmic membrane (2). To reach its destination, cytochrome c1 is translocated through the cytoplasmic membrane. Analogously, mitochondrial cytochrome c1 is transported from the matrix through the inner mitochondrial membrane so that the major part of c1 faces the intermembrane space. To this end, bacterial and mitochondrial c1 proteins use characteristic N-terminal signal sequences for vectorial membrane translocation (4-7). A closer examination of the hydrophobic domain linking the b and c1 parts in the B. japonicum FbcH protein now reveals that it has all the characteristics of a signal peptide (Fig. 1) with the notable exception that it is located in the

Stanford CA 94305-5427.

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Proc. NatL Acad. Sci. USA 88 (1991)

Biochemistry: Thony-Meyer et al.

center of the putative precursor protein (3) and not at the N as is usual for exported proteins (8-11). This observation led to two predictions as to how the singlefjbcH gene might direct the synthesis of the two cytochromes. (i) An FbcH precursor may be made first and then cleaved proteolytically near the end of the apocytochrome b section, thus releasing a pre-apocytochrome c1 with the signal peptide attached to its N terminus. Cytochromes b and c1 could then insert separately into the membrane, and cytochrome cl would lose its presequence by a second cleavage event. (it) Alternatively, the large FbcH precursor could insert into the membrane first, thereby using the centrally located signal peptide to direct the c1 part across the membrane. This part would then be cleaved off by a signal peptidase. To test these possibilities, two experimental strategies were followed. (i) We isolated the mature cytochrome c1 to determine its N-terminal amino acid sequence. (it) We altered the putative signal peptidase recognition site (Ala-Arg-Ala; see Fig. 1), predicting that this would lead to the formation of either an uncleaved precytochrome c1 or an uncleaved FbcH precursor, according to the aforementioned options i or ii, respectively. In a separate aspect of this work, the heme c-binding site in cytochrome c1 (Cys-Ala-Ser-Cys-His) was also altered by mutation. The results had important implications in the context of the cytochrome b- and c1-maturation pathway.

1

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Bacterial Strains and Growth Conditions. The wild-type strain was B. japonicum 110spc4 (12). For complementation experiments theJbcF: :l mutant 3067 was used as a recipient (3). B. japonicum cells were grown aerobically in PSY medium (12) with spectinomycin (wild type) at 100 ,ug/ml and streptomycin (3067) at 100 ,g/ml. Plasmid-containing B. japonicum strains were cultivated selectively using tetracycline at 100 ,ug/ml in the medium. Escherichia coli strains JM101 (13) and TG1 (Amersham) were used as recipients for transfections with M13 derivatives. E. coli RR28 (14) was used for transformations in cloning experiments. E. coli HB101 carrying pRK2013 served as a helper strain in triparental matings (15, 16). Recombinant DNA Work. Clonings and nucleotide sequence analyses were done by using routine protocols (17). Point mutagenesis was done according to the method of Eckstein and coworkers (18); Nci I was used for nicking the nonmutant DNA strand. The following oligonucleotides were used to create point mutations in fbcH: 413fbc8 (5'CTTGCAGGACGATCGCGCCAACG-3'), replacing Ala432 by Asp-432; 393fbc (5'-GGCAGCTTGCAGGACAAC-

GAAGGCAGCGAC-3'), deleting Ala-432-Ala-434; 430fbc7 (5'-CAAGGAAGTCAGCGCCAGCAGCCACGGCC-3'), replacing Cys-471 and Cys-474 by Ser-471 and Ser-474, respectively. Oligonucleotides were synthesized in a DNA synthesizer (model 380A; Applied Biosystems). Construction of Mutant Clones for Complementation Analysis. The strategy is outlined in Fig. 2. Plasmids pRJ3099 and pRJ3234, both containing the complete fbcFH operon in different vectors, served as starting material. A 1775-basepair (bp) BamHI-Xho I fragment of pRJ3234 was first cloned in M13mpl8. For this purpose, M13mpl8 (13) was cut with Sma I and BamHI, and an Xho I linker was attached at the blunt-end Sma I site. The shortened JbcH region in the resulting M13 derivative was the target for site-directed mutagenesis (18). The successful creation of the correct mutations was confirmed by sequencing. As a precaution against second-site mutations and to avoid siblings, two to three

independent clones were isolated

for

each mutation.

The mutant BamHI-Xho I fragment was then fused back to the remainder of the fbcH gene, thus completing the jbcFH operon (Fig. 2). Plasmid pRJ3239 served as an intermediate

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deleted

MATERIALS AND METHODS

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FIG. 2. Cloning strategy to create mobilizable plasmids carrying fbcFH operons mutated in JbcH. Details on construction are described in text. Vectors of respective plasmids are in parentheses. The kanamycin-resistance (KmR) cassette in pRJ3239 is shown as a hatched bar. The star symbolizes the mutant fbcH derivative. At bottom is a description of all mutant clones; numbers refer to nucleotide and amino acid positions as given in ref. 3. B, BamHI; E, EcoRI; H, HindIll; X, Xho I.

in this procedure; it was a derivative of pRJ3099, in which the 1775-bp BamHI-Xho I fragment was replaced by a 1637-bp BamHI-Xho I kanamycin-resistance cassette from pKmB6 (ref. 19; originally derived from TnS). Starting from pRJ3239, restoration of the fbcFH operon was possible by screening for kanamycin-sensitive clones in which the kanamycinresistance cassette was replaced by the mutantjbcH portion (Fig. 2). This way it was also convenient to have the resulting constructs located on the broad-host-range vector pRK29OX (20, 21): they could finally be mobilized into the B. japonicum strain 3067 (JbcF::Ql) for complementation assays (3). A list of all mutant clones obtained is given in Fig. 2. Protein Purification for N-Terinal Sequencing. Purification of the bc, complex from aerobically grown B.japonicum cells (92.7 g) was done by the protocol of Ljungdahl et al. (22) with modifications: The membrane fraction was solubilized by using 0.5 g of dodecyl maltoside (Boehringer Mannheim) per g of protein; the yield was 160 mg of solubilized protein. Proteins were fractionated in a 45-70%o ammonium sulfate precipitation. The precipitate was dialyzed for 30 min against buffer A (10 mM potassium phosphate, pH 8.0/1 mM MgSO4/0.1% dodecyl maltoside) and loaded onto a DEAE Bio-GelA column (Bio-Rad) of 100-ml bed volume. The column was washed with 300 ml of buffer A, and proteins were eluted with 500 ml of a 0-300 mM NaCI gradient in buffer A. Fractions (5 ml) were collected and assayed for protein content (A2BO) and cytochromes (A415). The cytochrome-containing fractions were pooled (17 mg of protein)

Biochemistry: Th6ny-Meyer et al. and analyzed on SDS/polyacrylamide gels for heme staining and Coomassie blue-staining bands. The 28-kDa hemoprotein (cytochrome cl) was cut out of a preparative gel and electroblotted onto a hydrophobic poly(vinylidene difluoride) membrane (Millipore) (23-25). To derivatize any cysteines present, the protein was pyridylethylated according to Amons (26). Filter pieces were used directly for N-terminal sequence analysis in a protein sequencer (model 475A; Applied Biosystems) with on-line phenylthiohydantoin detection on the Applied Biosystems model 120 HPLC. Biochemical and Immunologial Analyses. Cytochrome c oxidase activity, crude membrane preparations, heme stains, and immunoblots were done as described (3). The hydrophilic membrane Hybond-N proved more suitable for detection of cytochrome b and bc, precursor, whereas Hybond-C was advantageous for detection of cytochrome cl (both membranes from Amersham). Synthetic peptides representing domains of cytochrome b (amino acids 219-235 of FbcH) and cytochrome cl (amino acids 581-596 of FbcH) (see Fig. 1) and antibodies against them were custom-synthesized by Cambridge Research Biochemicals (Cambridge, U.K.). Immunoglobulins cross-reacting to proteins on immunoblots were detected by using alkaline phosphatase-conjugated antirabbit IgG and a detection kit from Bio-Rad. RESULTS N-Terminal Sequence of Cytochrome cl. A first step in the elucidation of the biogenesis of the B. japonicum cytochromes b and cl was the determination of the N-terminal amino acids of the mature cl protein. Because apo-cl was encoded by the distal (3') part of the JbcH gene (Fig. 1), this analysis was expected to yield information about where in the FbcH protein a proteolytic cleavage might occur. To this end, we attempted to purify ubiquinol-cytochrome c reductase from the membrane fraction of aerobically grown B. japonicum. With a well-established purification protocol (22) the bc, complex appeared to copurify in a tightly associated "supercomplex" together with a membrane-bound 20-kDa cytochrome c (27) and the aa3-type terminal oxidase (27). A similar supercomplex was reported for Paracoccus denitrificans (28), which, however, could be separated more easily into its individual components. We finally succeeded in isolating microgram quantities of the B. japonicum cl protein that, despite a contaminating second protein (