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The Role of Charged Amino Acids in the α1-β4 Loop of the Iron-Sulfur Protein of the Cytochrome bc 1Complex of Yeast Mitochondria

1998; Elsevier BV; Volume: 273; Issue: 19 Linguagem: Inglês

10.1074/jbc.273.19.11917

1083-351X

Victor H. Obungu, Yudong Wang, Diana S. Beattie,

RNA and protein synthesis mechanisms

Previous experiments using deletion mutants of the iron-sulfur protein had indicated that amino acid residues 138–153 might be involved in the assembly of this protein into the cytochromebc 1 complex. To determine which specific residues might be involved in the assembly process, charged amino acids located in the α1-β4 loop of the iron-sulfur protein were mutated to uncharged residues and tryptophan 152 to phenylalanine. The mutant genes were used to transform yeast cells (JPJ1) lacking the iron-sulfur protein gene. Mutants R146I and W152F had almost undetectable growth in medium containing glycerol/ethanol, whereas mutants D143A, K148I, and D149A grew more slowly than the wild type. Activity of the cytochromebc 1 complex was decreased 50, 90, 67, 89, and 90% in mutants D143A, R146I, K148I, D149A, and W152F, respectively, but unchanged in mutants D139A, Q141I, D145L, and V147S. In all of these mutants except W152F, the cytochrome c 1content, determined by immunoblotting, was comparable with that of wild-type cells. However, immunoblotting revealed that the content of the iron-sulfur protein was decreased proportionately in the five mutants with lowered enzymatic activity and growth suggesting that these amino acids are critical for maintaining the stability of the iron-sulfur protein. The efficiency of assembly in vitrocompared with the wild type determined by selective immunoprecipitation was unchanged in the mutants with the exception of R146I, D149A, and W152F where decreases of 80, 60, and 60%, respectively, were observed suggesting that these amino acids are critical for the proper assembly of the iron-sulfur protein into the bc 1complex. Previous experiments using deletion mutants of the iron-sulfur protein had indicated that amino acid residues 138–153 might be involved in the assembly of this protein into the cytochromebc 1 complex. To determine which specific residues might be involved in the assembly process, charged amino acids located in the α1-β4 loop of the iron-sulfur protein were mutated to uncharged residues and tryptophan 152 to phenylalanine. The mutant genes were used to transform yeast cells (JPJ1) lacking the iron-sulfur protein gene. Mutants R146I and W152F had almost undetectable growth in medium containing glycerol/ethanol, whereas mutants D143A, K148I, and D149A grew more slowly than the wild type. Activity of the cytochromebc 1 complex was decreased 50, 90, 67, 89, and 90% in mutants D143A, R146I, K148I, D149A, and W152F, respectively, but unchanged in mutants D139A, Q141I, D145L, and V147S. In all of these mutants except W152F, the cytochrome c 1content, determined by immunoblotting, was comparable with that of wild-type cells. However, immunoblotting revealed that the content of the iron-sulfur protein was decreased proportionately in the five mutants with lowered enzymatic activity and growth suggesting that these amino acids are critical for maintaining the stability of the iron-sulfur protein. The efficiency of assembly in vitrocompared with the wild type determined by selective immunoprecipitation was unchanged in the mutants with the exception of R146I, D149A, and W152F where decreases of 80, 60, and 60%, respectively, were observed suggesting that these amino acids are critical for the proper assembly of the iron-sulfur protein into the bc 1complex. The cytochrome bc 1complex 1The abbreviations used are: cytochromebc 1 complex, ubiquinol-cytochrome coxidoreductase; DBH2, 2,3-dimethoxy-5-methyl-6-decyl-1,4-benzoquinol; PAGE, polyacrylamide gel electrophoresis; RIP, wild-type iron-sulfur protein gene; rip, mutant iron-sulfur protein gene. is an integral multiprotein complex of the inner mitochondrial membrane which catalyzes the transfer of electrons from ubiquinol to cytochromec coupled to the translocation of protons across the membrane (1Brandt U. Trumpower B. Crit. Rev. Biochem. Mol. Biol. 1994; 29: 165-197Crossref PubMed Scopus (295) Google Scholar, 2di Rago J.P. Bruel C. Graham L.A. Slonimski P. Trumpower B. J. Biol. Chem. 1996; 271: 15341-15345Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). The bc 1 complex of yeast mitochondria consists of 10 subunits, of which 3 have prosthetic groups that serve as redox centers: cytochromes b andc 1 and the Rieske iron-sulfur protein. The iron-sulfur protein is an important component of the catalytic reaction center P of the bc 1 complex where it is involved in the transfer of electrons to cytochrome c 1from ubiquinol (2di Rago J.P. Bruel C. Graham L.A. Slonimski P. Trumpower B. J. Biol. Chem. 1996; 271: 15341-15345Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). Consistent with its catalytic function, the iron-sulfur protein is anchored on the outer surface of the inner mitochondrial membrane where it protrudes into the intermembrane space (3Trumpower B.L. Gennis B. Annu. Rev. Biochem. 1994; 63: 675-716Crossref PubMed Scopus (471) Google Scholar, 4Sidhu A. Clejan L. Beattie D.S J. Biol. Chem. 1983; 258: 12308-12314Abstract Full Text PDF PubMed Google Scholar). The recent resolution of the crystal structure of the cytochrome bc 1 complex of beef heart mitochondria has revealed that the complex exists as a dimer with 13 membrane-spanning helices (5Xia D. Yu C.A. Kim H. Xia J.Z. Kachurin A.M. Zhang L. Yu L. Deisenhofer J. Science. 1997; 277: 60-66Crossref PubMed Scopus (876) Google Scholar). With the exception of cytochrome b, the sole mitochondrial gene product of the bc 1 complex, all the subunits of this complex are synthesized on free cytoplasmic ribosomes and in a subsequent step imported into mitochondria where they are assembled into a functional complex in the membrane (6Beckman J.D. Ljungdahl P.O. Lopez J.L. Trumpower B.L. J. Biol. Chem. 1987; 262: 8901-8909PubMed Google Scholar). The iron-sulfur protein of Saccharomyces cerevisiae is synthesized as a precursor protein with a molecular mass of 29 kDa and possesses a 30-amino acid leader sequence at the amino terminus of the protein. The precursor form of the iron- sulfur protein is processedin vivo into the mature form through an intermediate form in two distinct processing events observed both in vitro andin vivo (7Fu W. Japa S. Beattie D.S. J. Biol. Chem. 1990; 265: 16541-16547Abstract Full Text PDF PubMed Google Scholar, 8Graham L.A. Trumpower B.L J. Biol. Chem. 1991; 266: 22485-22492Abstract Full Text PDF PubMed Google Scholar). The mechanism of assembly of the subunits of the cytochromebc 1 complex and, in particular, that of the iron-sulfur protein has been the subject of several studies (9Fu W. Beattie D.S. J. Biol. Chem. 1991; 266: 16212-16218Abstract Full Text PDF PubMed Google Scholar, 10Japa S. Beattie D.S. Arch. Biochem. Biophys. 1989; 268: 716-720Crossref PubMed Scopus (10) Google Scholar, 11Sidhu A. Beattie D.S. J. Biol. Chem. 1983; 258: 10649-10656Abstract Full Text PDF PubMed Google Scholar). These investigations have led to the suggestion that the iron-sulfur protein may be one of the last proteins to become associated with a postulated “core” membrane-bound complex during mitochondrial biogenesis (11Sidhu A. Beattie D.S. J. Biol. Chem. 1983; 258: 10649-10656Abstract Full Text PDF PubMed Google Scholar, 12Crivellone M.D. Wu M.A. Tzagoloff A. J. Biol. Chem. 1988; 263: 14323-14333Abstract Full Text PDF PubMed Google Scholar). In previous studies in our laboratory, the assembly of the iron-sulfur protein into the bc 1complex was investigated in vitro by using selective immunoprecipitation with antiserum against either the iron-sulfur protein or the intact bc 1 complex after import of radiolabeled precursor into mitochondria lacking the iron-sulfur protein (9Fu W. Beattie D.S. J. Biol. Chem. 1991; 266: 16212-16218Abstract Full Text PDF PubMed Google Scholar). More recently, the import and assembly of 8 deletion mutants of the iron-sulfur protein into the bc 1complex was studied using this technique (13Ramabadran R.S. Japa S. Beattie D.S. J. Bioenerg. Biomembr. 1997; 29: 45-54Crossref PubMed Scopus (5) Google Scholar, 14Obungu V. Yu L.P. Japa S. Beattie D.S. Biochim. Biophys. Acta. 1997; 1321: 229-237Crossref PubMed Scopus (3) Google Scholar). The results obtained in these studies indicated that the amino acids located in the extramembranous regions of the iron-sulfur protein might be involved in assembly with other subunits of the bc 1 complex. By contrast, the amino acid residues in the membranous region of the protein are apparently not required for the efficient assembly of the iron-sulfur protein into the bc 1 complex (14Obungu V. Yu L.P. Japa S. Beattie D.S. Biochim. Biophys. Acta. 1997; 1321: 229-237Crossref PubMed Scopus (3) Google Scholar). Further examination of one of these extramembranous regions containing amino acid residues 138–153 revealed the presence of 6 charged amino acids located in a loop connecting the α1 helix and the β4 sheet of a soluble fragment of the iron-sulfur protein from beef heart mitochondria (15Iwata S. Saynovtis M. Link T.A. Michel H. Structure. 1996; 4: 567-579Abstract Full Text Full Text PDF PubMed Scopus (281) Google Scholar). To determine the possible role of these charged amino acids in this region of the yeast iron-sulfur protein, which we assume has a similar if not identical structure, we performed site-directed mutagenesis of the charged amino acids present in this region. The effect of these mutations on the activity and the assembly of the iron-sulfur protein into the bc 1 complex has been examined. Site-directed mutagenesis was performed using the Stratagene Quick ChangeTM site-directed mutagenesis kit. The codons for the selected amino acids in the predominantly charged α1-β4 loop of the iron-sulfur protein iron-sulfur protein were altered to encode the desired amino acids according to the manufacturer's instructions. Mutations were performed using theRIP gene inserted in the single copy plasmid, pRG415; however, in some instances the mutations were performed in the high copy vector pSP64. When this plasmid was used, the mutantrip genes were subsequently subcloned into the pRG415 vector that was used to transform yeast cells. The mutant DNA, thus constructed, was analyzed by restriction enzyme analysis and by sequencing the mutated gene using the Cy5TM AutoCycle (or AutoRead) DNA sequencing kit for use with the Alf express automated sequencer (Amersham Pharmacia Biotech). This sequencing kit is based on the dideoxy chain termination method (16Sambrook J.E. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989: 13.3-13.10Google Scholar, 17Sanger F. Science. 1981; 214: 1205-1210Crossref PubMed Scopus (448) Google Scholar). DNA containing the wild-type RIP gene or the mutant rip genes was used to transform yeast cells (JPJ1), in which the RIP gene had been deleted, by the lithium acetate method (18Ito H. Fukuda Y. Murata K. Kimura A. J. Bacteriol. 1983; 153: 163-168Crossref PubMed Google Scholar) as modified (19Dunn B. Szauter P. Pardue L. Szostak J.W. Cell. 1984; 39: 191-201Abstract Full Text PDF PubMed Scopus (94) Google Scholar). Mutant yeast cells were selected by their ability to grow on a medium lacking uracil. To test for respiratory competence, the transformed JPJI colonies from the uracil minus plates were streaked on plates containing 1% yeast extract, 2% peptone, 3% glycerol, and 4% ethanol, pH 5.0, in 1.5% agar. The plates were incubated at both permissive (30 °C) and non-permissive (37 °C) temperatures. Subsequently, the transformed yeast cells were grown in the same liquid medium at 30 °C, and the rate of growth was monitored by measuring the absorbance at 650 nm over several hours. JPJI, the yeast strain lacking the RIP gene, was grown aerobically at 30 °C in a semi-synthetic medium as described previously (8Graham L.A. Trumpower B.L J. Biol. Chem. 1991; 266: 22485-22492Abstract Full Text PDF PubMed Google Scholar). The transformed cells were grown in a medium containing 1% yeast extract, 2% peptone, and 2% galactose prior to mitochondrial isolation for enzymatic studies. In order to prepare mitochondria, yeast cells were grown to early logarithmic phase (A 650 = 0.9–1.2), harvested by centrifugation at 1500 × g for 5 min at room temperature, and washed once with distilled water prior to mitochondrial isolation. For enzymatic and spectral analyses, yeast cells were broken using the modified glass bead method (2di Rago J.P. Bruel C. Graham L.A. Slonimski P. Trumpower B. J. Biol. Chem. 1996; 271: 15341-15345Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar); however, for import studies mitochondria was prepared from yeast digested with Zymolyase as described previously (14Obungu V. Yu L.P. Japa S. Beattie D.S. Biochim. Biophys. Acta. 1997; 1321: 229-237Crossref PubMed Scopus (3) Google Scholar). The activity of the cytochromebc 1 complex was determined by measuring the reduction of 40 μm horse heart ferricytochromec at 550–540 nm using the ubiquinol analog decylbenzoquinol (DBH2) as electron donor. The assay was performed at 25 °C in 50 mm Tris, pH 7.4, 1 mm EDTA, 250 mm sucrose, and 2 mm KCN. The non-enzymatic rate of cytochrome c reduction was determined by adding 33 μm DBH2 and allowing the reaction to proceed for 5 s after which the enzymatic reaction was initiated by addition of 0.1 mg of mitochondrial protein. The inhibition of enzymatic activity by antimycin A and myxothiazol was determined for each mutant. Optical spectra were recorded using the dual wavelength mode of the Aminco DW-2A spectrophotometer coupled to a recorder with reference beam set at 539 nm. Mitochondrial membranes were suspended at 2 mg/ml in 25 mmpotassium phosphate buffer, pH 7.4, 1 mm EDTA, and 1% dodecyl maltoside, oxidized by ferricyanide, and then reduced by adding a few grains of sodium dithionite. The spectra of the oxidized cytochromes was subtracted from that of the reduced cytochromes to obtain the reduced versus oxidized difference spectra. The concentration of cytochromes was determined by using the following absorption coefficients and wavelength pairs for the reduced minus oxidized proteins; 20.9 mm−1 cm−1at 553–539 for cytochrome c-c 1 and 25.6 mm−1 cm−1 at 562–575 for cytochrome b (20Sen K. Beattie D.S. Arch. Biochem. Biophys. 1985; 242: 393-401Crossref PubMed Scopus (22) Google Scholar). The wild-type and mutant rip genes, inserted in the expression vector pSP64, were transcribed and translated in vitro in the presence of [35S]methionine using the TnT-coupled reticulocyte lysate system from Promega. The expressed radiolabeled proteins were imported into mitochondria and immunoprecipitated with antibodies against the iron-sulfur protein and the intact cytochrome bc 1 complex as described previously (9Fu W. Beattie D.S. J. Biol. Chem. 1991; 266: 16212-16218Abstract Full Text PDF PubMed Google Scholar, 14Obungu V. Yu L.P. Japa S. Beattie D.S. Biochim. Biophys. Acta. 1997; 1321: 229-237Crossref PubMed Scopus (3) Google Scholar). The mitochondria were then reisolated and the proteins separated by SDS-PAGE. The gels were scanned and quantitated using a PhosphorImager (Molecular Dynamics) as described previously (14Obungu V. Yu L.P. Japa S. Beattie D.S. Biochim. Biophys. Acta. 1997; 1321: 229-237Crossref PubMed Scopus (3) Google Scholar). Immunoblotting was performed using the ECL Western blotting system from Amersham Pharmacia Biotech. Mitochondria were isolated from yeast cells and the proteins separated by SDS-PAGE prior to transfer onto polyvinylidene difluoride or nitrocellulose membranes. Primary antibodies raised against the iron-sulfur protein were used to locate the proteins immobilized on the membrane. These antibodies were subsequently detected by goat anti-rabbit secondary antibodies labeled with horseradish peroxidase that catalyzes the oxidation of luminol, thereby emitting small but sustained quantities of light. The chemiluminescence was then specifically enhanced allowing an image to be recorded on a photosensitive film (Amersham ECL bulletin). Previous studies in our laboratory involving deletion mutants of the iron-sulfur protein had indicated that amino acid residues 138–153 might be involved in the interaction of this protein with other subunits during the assembly of the cytochromebc 1 complex (13Ramabadran R.S. Japa S. Beattie D.S. J. Bioenerg. Biomembr. 1997; 29: 45-54Crossref PubMed Scopus (5) Google Scholar, 14Obungu V. Yu L.P. Japa S. Beattie D.S. Biochim. Biophys. Acta. 1997; 1321: 229-237Crossref PubMed Scopus (3) Google Scholar). The crystal structure of a water-soluble fragment of the iron-sulfur protein from beef heart mitochondria had revealed that these residues are located in the predominantly charged α1-β4 loop on the surface of the protein (15Iwata S. Saynovtis M. Link T.A. Michel H. Structure. 1996; 4: 567-579Abstract Full Text Full Text PDF PubMed Scopus (281) Google Scholar). In order to determine which of these residues might be specifically involved in the proposed interactions of the iron-sulfur protein with other proteins, site-directed mutagenesis of the following amino acids was performed as described under “Experimental Procedures.” The acidic amino acids, Asp-139, -143, and -149, were mutated to alanine (D139A, D143A, D149A), and Asp-145 was mutated to leucine (D145L), whereas the basic amino acids, Arg-146 and Lys-148, were mutated to isoleucine (R146I, K148I). As a control, two uncharged amino acid residues (Gln-141 and Val-147) were mutated to isoleucine and serine, respectively (Q141I, V147S). In addition, Trp-152 was mutated to phenylalanine (W152F), as a previous study had reported that the W152R mutation resulted in a loss of enzymatic activity (21Graham L.A. Brandt U. Sargent J.S. Trumpower B.L. J. Bioenerg. Biomembr. 1993; 25: 245-257Crossref PubMed Scopus (58) Google Scholar). The mutations were confirmed by DNA sequencing. The wild-type RIP gene and each of the mutant ripgenes were used to transform the JPJ1 strain of yeast cells, and growth was monitored on the non-fermentable carbon source glycerol/ethanol. After the initial screening on plates, the rate of growth of the transformed yeast cells was determined in liquid medium with the same carbon sources at 30 °C. JPJ1 cells transformed with the wild-typeRIP gene, indicated as wild type in TableI, grew with a doubling time of 2 h. Two of the mutants, R146I and W152F, had an almost undetectable growth rate calculated as doubling times of >12 and >10 h, respectively, whereas mutants D143A, K148I, and D149A grew more slowly than the wild type with doubling times of 3.5, 4, and 5 h, respectively. Growth of the remaining mutants, D139A, Q141I, D145L, and V147S, was identical to that of the wild-type cells.Table IGrowth characteristics, enzymatic activities, and cytochrome b and c 1 content in JPJ1 yeast cells transformed with the wild-type RIP and mutant rip genesMutantsDoubling timeEnzymatic activityConcentrationCytochromebCytochrome c-c 1hμmol/min/mg proteinnmol/mg proteinWild type2.00.930.0880.095D139A2.50.820.0740.086Q141I2.00.840.0730.095D143A3.50.470.0870.095D145L2.50.900.0860.090R146I>120.060.0600.090V147S2.00.940.0900.107K148I4.00.310.0800.092D149A5.00.100.0740.083W152F>100.0790.0520.067JPJ1No growth00.0410.074 Open table in a new tab The enzymatic activity of the cytochrome bc 1 complex was determined as DBH2 cytochrome c reductase to determine whether a lowered activity of the complex correlated with the decreased growth rates observed for some mutant cells. For these experiments, mitochondria were isolated from the transformed mutant yeast cells that had been grown in a medium containing galactose as carbon source. To confirm the enzymatic nature of the observed cytochrome creductase activity, the inhibitory effects of the specific inhibitors of the cytochrome bc 1 complex, antimycin A and myxothiazol, were determined. The cytochrome c reductase activity of the two mutants with very low rates of growth, R146I and W152F, was less than 10% of that observed with mitochondria from the wild-type cells (Table I). In addition, the enzymatic activity of the mutants D143A, K148I, and D149A was reduced approximately 50, 67, and 89%, respectively, compared with the wild type. The remaining mutants D139A, Q141I, D145L, and V147S retained enzymatic activity at wild-type levels. In general, these results demonstrate a good correlation between the observed reduction in the growth rate of these mutants in the glycerol/ethanol medium and the enzymatic activity of the cytochrome bc 1 complex. Our next approach was to investigate whether changes in the spectral properties or content of cytochromes b and c-c 1had occurred as a result of the mutations introduced into the iron-sulfur protein. Spectral analysis of the wild-type mitochondria, JPJ1 cells transformed with the wild-type RIP, is presented for comparison with several of the iron-sulfur protein mutants (Fig.1). The concentration of cytochromesb and c-c 1 in the wild-type mitochondria was determined to be 0.088 and 0.095 nmol/mg of protein, respectively (Table I). Examination of the spectra of the mutants revealed the presence of cytochromes c-c 1 at the same level as that of the wild-type cells with the exception of W152F in which the cytochrome c-c 1 content was diminished by 30%. By contrast, spectral analysis of mitochondria obtained from several of the mutants revealed significant changes in the spectra of cytochrome b leading to calculated decreases in the amount of cytochrome b present in the mitochondria (Fig. 1). For example, the iron-sulfur mutants D139A, Q141I, and D149A had 16–17% less cytochrome b heme than the wild type whereas the cytochrome b heme content was diminished by 32% in R146I and 41% in W152F. These values can be compared with the 53% decrease in cytochrome b heme observed in mitochondria isolated from JPJ1, the strain lacking the iron-sulfur protein. These reductions in the cytochrome b levels may reflect damage to the environment of the b hemes due either to the absence of the iron-sulfur protein or to a change in its conformation as a result of the specific amino acid mutation (8Graham L.A. Trumpower B.L J. Biol. Chem. 1991; 266: 22485-22492Abstract Full Text PDF PubMed Google Scholar). The decreased levels of cytochrome b observed in several of these mutants rather than the actual mutation in the iron-sulfur protein may be responsible for the observed lowered enzymatic activity of the bc 1 complex in these mutants. To make this determination, the turnover numbers of thebc 1 complex in the various mutants were calculated as enzymatic activity (nmol of cytochrome creduced mg−1 of mitochondrial protein min−1)/the content of cytochrome b (nmol mg−1of mitochondrial protein) (Fig. 2). A similar pattern in the loss of enzymatic activity determined as turnover numbers was observed for the mutants in which the enzymatic activity was lowered. For example, the decrease in the turnover numbers was as follows: 50% for D143A, 90% for R146I, 60% for K148I, and 90% for D149A and W152F. The turnover numbers for the remaining mutants were not affected. These results indicate that the loss of enzymatic activity observed in these five mutants resulted from the mutations introduced into the iron-sulfur protein and not from a decreased level of cytochrome b. The observation that site-directed mutagenesis of several charged amino acids plus Trp-152 in the region containing residues 139–152 of the iron-sulfur protein resulted in a reduction in the enzymatic activity of the cytochromebc 1 complex prompted an investigation of the level of expression of the iron-sulfur protein in these mutants. The presence of the iron-sulfur protein in mitochondria isolated from these mutants was determined by Western blotting with an antibody against the iron-sulfur protein. As a control, the expression of cytochromec 1 in these mitochondria was compared on the same gel. Immunoblot analysis revealed the presence of the iron-sulfur protein in all of the mutants (Fig. 3,A and C); however, the levels of expression of the iron-sulfur protein were considerably lower in the mutants D143A, R146I, K148I, and D149A when compared with the wild-type levels. By contrast, the levels of cytochrome c 1 determined by immunoblotting with the antibody against cytochromec 1 were unaffected in the mutants (Fig. 3,B and D). These results indicate that mutating two acidic amino acids, Asp-143 and Asp-149, two basic amino acids, Arg-146 and Lys-148, and the aromatic residue, Trp-152, results in varying decreases in the levels of the iron-sulfur protein observedin vivo, perhaps resulting from a decreased expression of the protein or from an increased instability of the protein. For example, removal of the charges in this region of the iron-sulfur protein or changing the tryptophan moiety through mutagenesis may result in the formation of an unstable protein that is degraded soon after translation. The decreased amounts of the iron-sulfur protein observed in several of the mutants prompted us to investigate whether the import into mitochondria of these mutant proteins and their subsequent assembly into thebc 1 complex were affected by the mutations. The import and assembly of the iron-sulfur protein were determined in vitro after transcription and translation of these ripgenes inserted into the high copy pSP64 plasmid. After coupled transcription/translation in vitro, the radiolabeled precursors of the mutant and the wild-type iron-sulfur protein were all imported efficiently into the mitochondria isolated from yeast strain JPJ1, where they underwent the identical two-step cleavage process (Fig. 4). These results indicate that the mutations introduced into the iron-sulfur protein had no effect on the import and processing of the respective precursor proteins. The assembly of the wild-type and mutant iron-sulfur proteins into the cytochrome bc 1 complex was investigated in vitro using selective immunoprecipitation with specific antibodies. The procedure is based on the principle that radiolabeled precursor and mature forms of the iron-sulfur protein can be immunoprecipitated with antiserum against the wild-type iron-sulfur protein; however, the precursor form of the iron-sulfur protein cannot be immunoprecipitated by the antiserum raised against the intact complex III (13Ramabadran R.S. Japa S. Beattie D.S. J. Bioenerg. Biomembr. 1997; 29: 45-54Crossref PubMed Scopus (5) Google Scholar, 14Obungu V. Yu L.P. Japa S. Beattie D.S. Biochim. Biophys. Acta. 1997; 1321: 229-237Crossref PubMed Scopus (3) Google Scholar). Moreover, this antibody does not recognize the iron-sulfur proteins in immunoblots of mitochondrial membranes. Consequently, the immunoprecipitation of radiolabeled iron-sulfur protein with the antiserum against complex III after import reflects the assembly of the iron-sulfur protein with other subunits of thebc 1 complex recognized by the complex III antiserum. The efficiency of assembly of the wild-type and mutant iron-sulfur proteins was determined by dividing the radioactivity immunoprecipitated by the antiserum against complex III by that immunoprecipitated by the antiserum against the iron-sulfur protein, assumed to represent the total amount of the protein imported into mitochondria (Fig. 5). Slight variations in the efficiency of assembly of the various mutants were observed (Fig. 6); however, the most significant effects were noted with the following mutants: an 80% decrease in R146I and a 60% decrease in both D149A and W152F. These results suggest that certain amino acids, especially Arg-146, Asp-149, and Trp-152, present in the α1-β4 loop of the iron-sulfur protein, may be critical for the efficient assembly of this protein into thebc 1 complex.Figure 6Efficiency of assembly of the mutant iron-sulfur proteins into the cytochrome bc 1 complex of mitochondria from JPJ1. The relative radioactivities of the mature forms of the mutant and wild-type iron-sulfur proteins assembled into the bc 1complex were quantified by PhosphorImager analysis. The percentages indicate the value of normalized immunoprecipitates divided by that of the wild type.View Large Image Figure ViewerDownload (PPT) In the current study, we have investigated the role of charged amino acids located in the α1-β4 loop of the iron-sulfur protein on the activity and assembly of the yeast cytochromebc 1 complex. These parameters were examined in mutants in which six charged amino acids in this region were changed to uncharged residues, and the tryptophan at position 152 at the base of the β4 sheet was changed to a phenylalanine. Four of these mutants, D143A, R146I, K148I, and D149A, as well as the W152F mutant grew more slowly than the wild type, whereas growth of two of the mutants, D139A and D145L, was unaffected. A corresponding decrease in the enzymatic activity of the bc 1 complex was observed in all of these mutants suggesting that the slower rate of growth reflected a lowered activity of the bc 1 complex. For example, the two mutants with a barely detectable growth rate, R146I and W152F, had enzymatic activities less than 10% that of the wild type (Table I). Spectral analysis of cytochrome b andc-c 1 in the mutant mitochondria and the subsequent determination of turnover numbers indicated that the reduction in the activity of the bc 1 complex did not result from a decrease in the levels of the two cytochromes. Indeed, Western blot analysis of mitochondrial proteins from these mutants revealed that the loss of enzymatic activity of thebc 1 complex was correlated with a decreased level of expression of the iron-sulfur protein in these mutants. For example, three of the slowest growing mutants, R146I, D149A, and W152F, with corresponding very low enzymatic activity had the lowest levels of the iron-sulfur protein; however, those mutants with moderately reduced growth rates and enzymatic activities, D143A and K148I, had a corresponding greater level of immunodetectable iron-sulfur protein. The lowered levels of the iron-sulfur protein in these mutants indicate that four of the charged amino acids in the α1-β4 loop of the protein plus the tryptophan at residue 152 are necessary for expression of the iron-sulfur protein in vivo. These charged residues and Trp-152 may be crucial in maintaining the overall stability of the protein such that when these amino acids are mutated, the resulting iron-sulfur protein may become unstable. Such an unstable protein might be degraded soon after translation in the cytosol or during its import into mitochondria. Alternately, the mutated protein might not assemble efficiently into the bc 1 complex resulting in its subsequent degradation in the mitochondria. To distinguish among these possibilities, the effect of these mutations on the import and assembly of the mutated iron-sulfur proteins into the cytochromebc 1 complex was investigated in vitro. None of these mutations of the iron-sulfur protein had any effect on the import of the mutated proteins into mitochondria in vitro or on the subsequent two-step processing of the precursor to the mature form in the mitochondrial matrix. By contrast, differences in the efficiency of assembly in vitro of the five mutations that affect growth and activity were observed. Three of the mutants, R146I, D149A, and W152F, were assembled into thebc 1 complex with very low efficiency when compared with the wild type. It should also be noted that these three mutations resulted in the most severe effects on growth and enzymatic activity of the cytochrome bc 1 complex. We suggest that Arg-146, Asp-149, and Trp-152 may be involved in establishing the conformation of the iron-sulfur protein necessary for its efficient assembly with the other proteins in thebc 1 complex. The charged amino acids may form hydrogen bonds or salt bridges with other charged amino acids to maintain the α1-β4 loop in its extended conformation, whereas the location of Trp-152 at the base of the β4 sheet may be critical for the overall conformation of the protein. Recent reports have indicated that mutations of the iron-sulfur protein of Rhodobacter capsulatus also affect the conformation of the iron-sulfur protein and consequently its stability (22Liebl U. Sled V. Brasseur G. Ohnishi T. Daldal F. Biochemistry. 1997; 36: 11675-11684Crossref PubMed Scopus (37) Google Scholar). By contrast, two of the mutants with lower enzymatic activity and decreased levels of the iron-sulfur protein in vivo, D143A and K148I, assembled into the bc 1 complexin vitro almost as efficiently as the wild-type protein. It should be noted that the effects of these mutations on the enzymatic activity of the bc 1 complex and the mitochondrial content of the iron-sulfur protein in vivowere moderate in comparison with the mutations in Arg-146, Asp-149, and Trp-152. We suggest that the charged amino acids Asp-143 and Lys-148 may not affect the conformation of the protein and its assembly into the bc 1 complex; however, these charged amino acids may be necessary for maintaining the stability of the iron-sulfur protein. Consequently, mutating them to uncharged amino acids may have led to a partial degradation of the mutated iron-sulfur protein during or soon after its translation on cytosolic ribosomes. In conclusion, we suggest that several charged amino acids and Trp-152 located in the α1-β4 loop of the iron-sulfur protein of the yeast cytochrome bc 1 complex are required to maintain the stability of the protein in vivo. As a consequence of this instability, lowered levels of the iron-sulfur protein were observed in vivo resulting in a decreased activity of thebc 1 complex and slower growth of the cells. Currently, we are extending these investigations to mutations of charged amino acids located in the α1 helix of the iron-sulfur protein to obtain further evidence in support of this suggestion.