Role of Arg-166 in Yeast Cytochrome c1*

Zulfiqar AhmadDagger and Fred Sherman§

From the Department of Biochemistry and Biophysics, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642

Received for publication, January 19, 2001


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

A systematic screen for dominant-negative mutations of the CYT1 gene, which encodes cytochrome c1, revealed seven mutants after testing ~104 Saccharomyces cerevisiae strains transformed with a library of mutagenized multicopy plasmids. DNA sequence analysis revealed multiple nucleotide substitutions with six of the seven altered Cyt1p having a common R166G replacement, either by itself or accompanied with other amino acid replacements. A single R166G replacement produced by site-directed mutagenesis demonstrated that this change produced a nearly nonfunctional cytochrome c1, with diminished growth on glycerol medium and diminished respiration but with the normal or near normal level of cytochrome c1 having an attached heme group. In contrast, R166K, R166M, or R166L replacements resulted in normal or near normal function. Arg-166 is conserved in all cytochromes c1 and lies on the surface of Cyt1p in close proximity to the heme group but does not seem to interact directly with any of the physiological partners of the cytochrome bc1 complex. Thus, the large size of the side chain at position 166 is critical for the function of cytochrome c1 but not for its assembly in the cytochrome bc1 complex.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

The cytochrome bc1 complex, also known as complex III of the respiratory chain or ubiquinol:cytochrome c oxidoreductase, is an oligomeric complex found in the inner mitochondrial membrane of eukaryotes and in the plasma membrane of bacteria (1-4). This complex transfers electrons from ubiquinol to cytochrome c and couples this transfer to a proton gradient across the inner mitochondrial or bacterial plasma membrane by a mechanism known as the proton motive Q cycle (2, 5-7). The prokaryotic and eukaryotic cytochrome bc1 complexes contain three essential catalytic subunits having the following characteristic prosthetic groups: cytochrome b with two b-type hemes; cytochrome c1 with a c-type heme; and the so-called Rieske protein that contains a high potential [2Fe-2S] cluster.

In addition to the three catalytic subunits, mitochondrial cytochrome bc1 complexes from vertebrates (8) and the yeast Saccharomyces cerevisiae (9, 10) contain eight and seven additional subunits, respectively. X-ray crystallographic atomic structures have been determined for the soluble fragment of the Rieske protein at 1.5-Å resolution (11) and for entire mitochondrial cytochrome bc1 complexes from bovine, chicken (12-14), and S. cerevisiae (15) at 2-3-Å resolution. The cytochrome bc1 complexes are dimers with each monomer of the yeast complex consisting of the following 10 protein subunits (with the orthologous bovine subunits or synonyms shown in parentheses): Cyt1p (cytochrome c1); COB (cytochrome b); Rip1p (ISP, Rieske protein); Cor1p (Core 1, SU1); Qcr2p (Core 2, SU2); Qcr6p (SU8); Qcr7p (SU6); Qcr8p (SU7); Qcr9p (SU10); and Qcr10p (SU11). In addition, the subunit SU9 is present in the vertebrate but not in the yeast cytochrome bc1 complex. Furthermore, the protein prepared for determining the atomic structure of the yeast complex lacked Qcr10p (15). (The overall structure and components of the bovine cytochrome bc1 complex are presented in Fig. 1.) SU11 of the bovine complex, orthologous to Qcr10p, forms a transmembrane helix that is bound on the outside of the complex to the helices of the Rieske protein and SU10 (Qcr9p) (14). Removal of SU10 did not affect enzymatic activity (8), but it may be important for the correct assembly of the complex (10). The similar overall structure of the yeast compared with the vertebrate complexes suggests that Qcr9p is associated with the complex in the same way (15). Furthermore, the relative positions and orientations of heme groups and the distances between the iron positions indicate that the yeast and vertebrate complexes are essentially the same, although the exact position, length, and conformation of connecting loops varied (15).


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Fig. 1.   The bovine cytochrome bc1 complex (14) shown in two orientations (A and B) and the position of Arg-166 (R166) (C) and R166G (D) in cytochrome c1. The three-dimensional structure of cytochrome bc1 complex from S. cerevisiae has been published (15), but the coordinates will not be deposited until May 12, 2001 (1EZV, Protein Data Bank). We therefore have used the related bovine complex to depict the position of Arg-166 relative to the heme group and other components of the complex. Subunit 11 is not found in the yeast cytochrome bc1 complex, and subunit 10 was not in the preparation that was used to determine the atomic structure of the yeast complex (15). The figure was generated with the molecular graphics program RasMol using the bovine cytochrome bc1 coordinates (14).

Cytochrome c1 is responsible directly for the electron transfer reaction with cytochrome c by catalyzing the oxidation of ubiquinol and reduction of cytochrome c (16, 17). Like all other physiological partners, cytochromes c and c1 interact with each other through electrostatic forces (18).

Although cytochrome c1 is a mitochondrial protein, it is encoded by a nuclear gene, CYT1, translated in the cytosol, and subsequently imported in mitochondria, a process involving cleavage of a leader sequence (19, 20). In yeast, the cleavage of a 61-amino acid amino-terminal region from the 309-amino acid-long precursor results in a 248-amino acid-long mature form.

We have undertaken a genetic investigation of the functional requirements of amino acid residues of yeast cytochrome c1 and of possible critical interactions between cytochromes c1 and c. Although mutational analysis of cytochromes c1 from yeast (17, 21-23)1 and Rhodobacter sphaeroides and Rhodobacter capsulatus (7, 24-27) has been used in several studies, we have elected to isolate and characterize dominant-negative mutants. The characterization of altered cytochromes c1 generated by "random" mutagenesis may reveal functional requirements that are difficult to predict even with a detailed knowledge of the structure of the protein. However, the vast majority of nonfunctional proteins generated by random mutagenesis are defective for trivial reasons such as missense mutations that affect folding or assembly and nonsense mutations that produce truncated proteins. On the other hand, interesting nonfunctional but stable proteins can be detected by the dominant-negative genetic test (28). For example, if the overexpression of a cyt1-x mutation inhibits the function of the normal CYT1+ chromosomal gene, then the cyt1-x allele most likely encodes a nonfunctional cytochrome c1, which is competing with the normal form.

The systematic screen for dominant-negative mutations of the CYT1 gene carried out in this study revealed that the R166G replacement caused a nearly nonfunctional cytochrome c1, with diminished growth on glycerol medium and diminished respiration but with the normal or near normal level of cytochrome c1 having an attached heme group. Furthermore, the small size of the side chain was responsible for the defect because R166K, R166M, or R166L replacements resulted in normal or near normal function of cytochromes c1.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

Numbering of Amino Acid Positions of Cytochrome c1-- The amino acid positions of yeast cytochrome c1 are assigned in this paper according to the full-length precursor having 309 amino acid residues. For example, Arg-166 of yeast cytochrome c1 corresponds to Arg-102 of bovine cytochrome c1.

Media and General Methods-- Standard YPD2 (1% Bacto-yeast extract, 2% Bacto-peptone, and 2% glucose) and YPG (1% Bacto-yeast extract, 2% Bacto-peptone, and 2% (v/v) glycerol) and synthetic media for the growth of yeast and the normal cultivation and manipulation of yeast strains have been described by Sherman (29). SG - uracil and SD - uracil designate synthetic media containing 3% glycerol and 2% glucose, respectively, and 12 other supplements (29) without uracil.

The relative growth of the strains was estimated by inoculating a dilute suspension of cells on the surface of YPD and YPG plates, incubating the plates at 23, 30, and 37 °C, and examining the plates daily for up to 5 days.

Yeast cells were transformed by the lithium-acetate method (30).

Methods used in the construction of plasmids including restriction enzyme digests, separation of plasmid DNA and restriction fragments on agarose gels, ligation of DNA fragments, and the isolation of plasmid DNA are described by Maniatis et al. (31). Escherichia coli transformations were performed with the CaCl2 method (32). The polymerase chain reaction (PCR) was carried out as described by Saiki et al. (33).

The enzymes used in this study, BamHI, SalI, DNA polymerases, polynucleotide kinase, etc., were purchased from either New England Biolabs, Amersham Pharmacia Biotech, or U. S. Biochemical Corp. The media constituents were obtained from Difco or Roche Molecular Biochemicals. All other chemicals used were from Sigma. Agarose was obtained from Roche Molecular Biochemicals.

DNA sequencing of segments containing CYT1 was carried out with the oligonucleotides OL.ZA01-OL.ZA04 (Table I) using the ABI PRISM dye Terminator Cycle Sequencing Ready Reaction kit with AmpliTaq DNA polymerase (Big Dye).

                              
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Table I
List of oligonucleotides

Yeast Strains and Plasmids-- The yeast strains used in this study along with their complete and partial genotypes are presented in Table II. The normal CYT1 strain B-7553 described by Dumont et al. (34) served as the parental strain for generating the cyt1-Delta ::TRP1 mutant B-9737 by the one-step gene replacement procedure. A 1.1-kilobase KpnI-SpeI segment containing the CYT1 gene in the plasmid pAB1192 was replaced with an 829-base pair KpnI-SpeI segment containing the TRP1 gene, resulting in a plasmid denoted pAB1193. B-7553 was transformed with a fragment from pAB1193 encompassing cyt1-Delta ::TRP1, and the desired disruptant was confirmed by PCR analysis.1

                              
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Table II
Yeast strains

The plasmids used in this study are listed in Table III, and some are described below.

                              
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Table III
Description of plasmids

Dominant-negative Mutants-- A library of 1.5-kilobase CYT1 segments containing BamHI and SalI sites and random alterations was generated from pAB1097 by error-prone PCR with oligonucleotides OL.ZA01 and OL.ZA03. PCRs were carried out separately with 0.05, 0.10, 0.15, and 0.20 mM MnCl2. All reactions contained the appropriate amounts of DNA (200-600 ng), MgCl2 (1.5-6.0 mM), oligos (4.0-6.0 pmol), dNTPs, buffer, and AmpliTaq DNA polymerase. The libraries of PCR segments were inserted in pAB1198, and the resulting plasmids were amplified in XL1-Blue. The plasmid libraries, obtained by different error-prone PCR conditions, were pooled and used to transform the B-7553 strain; the resulting transformants were plated on SD - uracil plates. Approximately 104 colonies from the SD - uracil plates were replica-plated on SG - uracil, SD - uracil, YPG, and YPD plates for the detection of dominant-negative mutants. A transformant was considered to be a dominant-negative mutant if it exhibited diminished growth on SG - uracil, if the corresponding Ura- strain lacking the plasmid had normal growth on YPG, and if reintroduction of the plasmid in pAB1198 again resulted in a transformant with diminished growth on SG - uracil medium.

Oligonucleotide-directed Mutagenesis-- Oligonucleotide-directed mutagenesis was carried out by the procedure described by Kunkel et al. (37) using the plasmid pAB2306 (Table III), E. coli strain CJ236 (dut1 ung1 thi1 relA1/pCJ105[CmR]) (38), and the oligonucleotides OL.Z05-OL.Z14 (Table I). The E. coli strain XL1-Blue (supE44 hsd17 recA1 endA1 gyrA46 thi1 relA1 lac-) was used for the amplification and storage of plasmids. The site-directed change was confirmed by DNA sequencing of the CYT1 region.

Low Temperature Spectroscopic and Spectrophotometric Analysis of Intact Cells-- The yeast strains were grown on the surface of YP1%S (1% Bacto-yeast extract, 2% Bacto-peptone, and 1% sucrose) plates at 23 °C for 4 days, 30 °C for 3 days, or 37 °C for 2 days, which are slightly modified conditions of our standard procedure (39). The levels of cytochromes aa3, b, c, and c1 were estimated in intact cells at -196 °C by spectroscopic visual examination (40) and by absorbance recordings using an Aviv model 14 spectrophotometer as described by Hickey et al. (41).

Rates of Respiration-- Oxygen uptake was measured polarographically with a commercially available Teflon-covered Clark electrode, the Yellow Stone Instruments oxygen monitor (Yellow Springs, OH), as described previously (42), using 3-ml solutions of 44 mM KH2PO4, 1 mM glucose, and various amounts of washed yeast cells obtained from cultures grown to late stationary phase in YPD medium. [QO2 is expressed as microliters of oxygen consumed per hour per milligram of yeast, dry weight.

    RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

Dominant-negative Mutants-- We undertook an extensive screen for dominant-negative mutations of the CYT1 gene that encodes cytochrome c1 with the aim of identifying amino acid residues that are critical for function but not for stability or incorporation into the cytochrome bc1 complex. For example, residues that are on the surface of the complex and are required for interaction with their physiological partners would be expected to be revealed with dominant-negative mutations. Because such altered cytochromes c1 are expected to be stable, they should be particularly amenable to biochemical studies.

In this study, we have used a library of multicopy plasmids containing a CYT1 segment that was mutated randomly by error-prone PCR. The plasmids, which are derivatives of pAB1198 (Table III), are maintained at a high copy number in the strain B-7553 (Table II) because of the URA3 marker and the 2 µ origin of replication. In addition, the LEU2-d markers can be used to produce an even higher copy number on medium lacking leucine.

The screen is based on the lack of utilizing a nonfermentable carbon source, glycerol, because of competition of an altered nonfunctional form for the wild-type cytochrome c1. Because manifestation of dominant-negative mutations depends on the presence of the plasmid, the desired mutants can be differentiated conveniently from other glycerol-negative mutants such as commonly occurring rho - mutations.

Approximately 104 transformants containing the library of mutagenized plasmids were screened for diminished growth on synthetic medium lacking uracil in a plasmid-dependent manner. A total of 181 colonies with some degree of diminished growth was uncovered, but only 12 were almost completely negative. The CYT1 region of seven of these was subjected to DNA sequencing. The seven strains were designated B-13412-B-13418; the corresponding plasmids were designated pAB2657-pAB2663; and the corresponding altered alleles were designated cyt1-101-cyt1-107 (Tables IV and V). The growth of the strains under various conditions is presented in Table IV, and as an example, the growth of B-13415 on SG - uracil medium is presented in Fig. 2.

                              
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Table IV
Growth of dominant-negative (B-13412-B-13418) and site-directed (B-13445-B-13452) mutants

                              
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Table V
Amino acid replacements and codon changes in the dominant-negative mutations of CYT1


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Fig. 2.   Growth of the following strains on SG medium lacking uracil, demonstrating the dominant-negative effect of the R166G replacement: B-12705 (CYT1 ura3 p[2 µ URA3]), the CYT1 normal strain; B-13415 (CYT1 ura3 p[2 µ URA3 cyt1-104]), the R166G dominant-negative mutant; and B-13435 (cyt1-Delta ura3 p[CEN6 URA3]), a cyt1-Delta -deficient mutant.

The sequences of the CYT1 region of the pAB2657-pAB2663 plasmids, presented in Table V, revealed multiple base pair substitutions with many of the changes common to more than one plasmid. The two plasmids pAB2658 and pAB2659 were identical, and all seven plasmids contained the neutral change A168A (GCCright-arrowGCT). Two different sets (six each) of the seven plasmids contained the neutral change L36L (CTCright-arrowCTA) and the radical change R166G (AGAright-arrowGGA), respectively. The plasmid pAB2663 contained 14 base pair substitutions including the formation of a UAG nonsense codon at amino acid position 227. The multiple and common base pair substitutions suggest that the altered PCR products may be related clonally, a result that would be expected if the DNA fragments were derived from common molecules because of low amounts of starting material. Thus, it is unclear which if any of the multiple directed changes occurred independently.

The results clearly established that at least the R166G replacement was responsible for the dominant-negative phenotype. This amino acid replacement occurred in six of the seven sequenced plasmids, pAB2657-pAB2662, and was the only amino acid change in the two plasmids pAB2660 and pAB2661. Furthermore, the only plasmid lacking the R166G replacement, pAB2663, contained the UAG nonsense mutation at amino acid position 227. The drastic nature of premature chain termination suggests that the R227End change in pAB2663 is responsible for the dominant-negative effect.

Site-directed Mutants-- To confirm and extend these findings and to determine whether other replacements may confer a dominant-negative phenotype, the following changes were introduced in the single-copy CEN6 plasmid by oligonucleotide-directed mutagenesis (Table VI): R166G; R166M; R166L; R166K; S49L; T63A; S49L and T63A; and R227End. Strain B-9737 (cyt1-Delta ura3) was transformed with each of the plasmids, and the transformants, B-13445-B-13452, were examined for growth on a variety of media at various temperatures and for the levels of the cytochromes aa3, b, c1, and c. The growth of B-13445 (R166G) and B-13452 (R227End) was diminished greatly on YPG medium, and the growth of B-13446 (R166L), B-13448 (R166K), B-13449 (T63A), and B-13451 (S49L, T63A) was diminished only slightly on SG - uracil medium (Tables IV and VI, Fig. 3). These results confirm that the R166G and R227End changes are responsible for the cytochrome c1 defects and presumably for the dominant-negative phenotypes. Furthermore, the T63A replacement in pAB2657, pAB2658, and pAB2659 and the S49L replacement in pAB2657 are apparently innocuous. The detrimental effect of the R166G replacement was substantiated further from the diminished respiratory rate of strain B-13445, which was equivalent to the strain B-13435 lacking cytochrome c1 (Table VI). Nevertheless, B-13445 (R166G) contained the nearly normal level of cytochrome c1 as indicated by the alpha -peak in the low temperature (-196 °C) spectrophotometric recording (Fig. 4).

                              
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Table VI
Properties of mutants constructed by site-directed mutagenesis


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Fig. 3.   Growth of 1:10 serial dilution of the following strains on YPD and YPG: B-7553 (CYT1 ura3); B-9737 (cyt1-Delta ura3); B-13435 (cyt1-Delta ura3 p[CEN6 URA3]); B-13436 (cyt1-Delta ura3 p[CEN6 URA3 CYT1]); B-13445 (cyt1-Delta ura3 p[CEN6 URA3 cyt1-201] (R166G)); B-13446 (cyt1-Delta ura3 p[CEN6 URA3 cyt1-202] (R166L)); B-13447 (cyt1-Delta ura3 p[CEN6 URA3 cyt1-203] (R166M)); B-13448 (cyt1-Delta ura3 p[CEN6 URA3 cyt1-204] (R166K)); B-13449 (cyt1-Delta ura3 p[CEN6 URA3 cyt1-205] (T63A)); B-13452 (cyt1-Delta ura3 p[CEN6 URA3 cyt1-208] (R227End)); and B-7553 (CYT1 ura3).


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Fig. 4.   Low temperature (-196 °C) spectrophotometric recordings of intact cells of the following isogenic strains: A, B-7553, CYT1 (normal); B, B-13445, cyt1-201 (R166G); and C, B-9737, cyt1-Delta (deficient mutant). The alpha -peaks of cytochromes a·a3, b, c1, and c are located at 602.5, 558.5, 553.3, and 547.3 nm, respectively. The peak at ~575 nm in B-9737 (C) is caused by zinc protoporphyrin.

In contrast to the R166G replacement, the R166M, R166L, and R166K replacements at most only caused minor diminution of function as indicated by the normal or nearly normal level of growth on YPG medium (Table IV). This finding suggests that the large size but not the charge of the Arg-166 side chain is critical for maintaining the normal function of cytochrome c1.

Yeast Arg-166 (Vertebrate Arg-102)-- The importance of the yeast Arg-166 (or vertebrate Arg-102) along with the adjacent Ala-165 residue is reflected by the phylogenetic conservation of these residues in cytochrome c1 from all species including higher and lower eukaryotes and prokaryotes. As shown in Fig. 5, of the almost 250 residues, Ala-165 and Arg-166 represent two of the 32 absolutely conserved residues in all cytochromes c1.


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Fig. 5.   Amino acid sequence alignment of cytochromes c1 from S. cerevisiae, Neurospora crassa, Solanium tuberosum (potato), Homo sapiens, Blastochloris viridis, Rhodobacter capsulatus, and Paracoccus denitrificans. Highly conserved residues are highlighted in black, where I designates I or V; L designates L or M; Y designates Y or F; and # designates N, D, Q, E, B, or Z. Moderately conserved residues are highlighted in gray. The residue positions are numbered starting with the P. denitrificans sequence. Thus, residue position 1 of S. cerevisiae corresponds to residue position 144 of P. denitrificans. The conserved Arg-166 of S. cerevisiae is denoted at position 308 (black-down-triangle ).

Insight into the function of Arg-166 is provided by considering its position in the cytochrome bc1 complex and the proposed models of electron transfer. As shown in Fig. 1, Arg-166 is located on the surface of cytochrome c1 in close proximity to the exposed pyrrole C corner of the heme group but not adjacent to any of the other components of the complex. The surface location of Arg-166 is consistent with proper assembly and stability of the altered R166G cytochrome c1. Zhang et al. (13) suggested that electron transfer into cytochrome c1 occurs through the D propionate and out of cytochrome c1 through the C corner of the heme to cytochrome c. On the other hand, in vitro protection and cross-linking experiments suggested that at least two different regions of cytochrome c1, encompassed by vertebrate positions 63-81 and 167-174, are folded together to form the cytochrome c binding site (43, 44). It remains to be seen if the R166G cytochrome c1 still binds cytochrome c but is unable to transfer electrons. In this regard, an R166G replacement is not expected to disrupt the alpha -helical structure in this region, and it is unclear how R166G but not R166K, R166M, or R166L replacements could effect binding to cytochrome c.

    ACKNOWLEDGEMENTS

We thank Thomas S. Cardillo for assistance in the low temperature spectrophotometric analysis and in the determination of respiration rates. We also thank Drs. David A. Pearce, Anaul Kabir, and Bogdan Polevoda for useful suggestions and discussions.

    FOOTNOTES

* This work was supported by National Science Foundation Grant 130-6726 and National Institutes of Health Grant R01 GM12702.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger On leave from: Dept. of Biochemistry, Hamdard University, Hamdard Nagar, New Delhi 10062, India.

§ To whom correspondence should be addressed: Dept. of Biochemistry and Biophysics, P. O. Box 712, University of Rochester Medical School, Rochester, NY 14642. Tel.: 716-275-6647; Fax: 716-275-6007; E-mail: Fred_Sherman@urmc.rochester.edu.

Published, JBC Papers in Press, February 26, 2001, DOI 10.1074/jbc.M100550200

1 S. L. Hatch, D. A. Pearce, F. Sherman, and G. McLendon, unpublished result.

    ABBREVIATIONS

The abbreviations used are: YPD, yeast extract peptone dextrose medium; YPG, yeast extract peptone glycerol medium; PCR, polymerase chain reaction.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

1. Hatefi, Y. (1985) Annu. Rev. Biochem. 54, 1015-1069[CrossRef][Medline] [Order article via Infotrieve]
2. Trumpower, B. L. (1990) Microbiol. Rev. 54, 101-129
3. Brandt, U., and Trumpower, B. L. (1994) CRC Crit. Rev. Biochem. 29, 165-197
4. Berry, E. A., Mariana, G.-K., Huang, L., and Croft, A. R. (2000) Annu. Rev. Biochem. 69, 1005-1075[CrossRef][Medline] [Order article via Infotrieve]
5. Mitchell, P. (1976) J. Theor. Biol. 62, 327-367[Medline] [Order article via Infotrieve]
6. Crofts, A. R. (1985) in The Enzymes of Biological Membranes (Martonosi, A. N., ed), Vol. 4 , pp. 347-382, Plenum Publishing Corp., New York
7. Gennis, R. B., Barquera, B., Hacker, B., Van Doren, S. R., Arnaud, S., Crofts, A. R., Davidson, E., Gray, K. A., and Daldal, F. (1993) J. Bioenerg. Biomembr. 25, 195-209[Medline] [Order article via Infotrieve]
8. Schägger, H., Link, T. A., Engel, W. D., and von Jagow, G. (1986) Methods Enzymol. 126, 224-237[Medline] [Order article via Infotrieve]
9. de Vries, S., and Marres, S. A. M. (1987) Biochim. Biophys. Acta 895, 205-239[Medline] [Order article via Infotrieve]
10. Brandt, U., Uribe, S., Schägger, H., and Trumpower, B. L. (1994) J. Biol. Chem. 269, 12947-12957[Abstract/Free Full Text]
11. Iwata, S., Saynovits, M., Link, T. A., and Michel, H. (1996) Structure 4, 567-579[Abstract]
12. Xia, D., Yu, C. A., Kim, H., Xia, J. Z., Kachurin, A. M., Zhang, L., Yu, L., and Deisenhofer, J. (1997) Science 277, 60-66[Abstract/Free Full Text]
13. Zhang, Z., Huang, L., Shulmeister, V. M., Chi, Y. I., Kim, K. K., Hung, L. W., Crofts, A. R., Berry, E. A., and Kim, S. H. (1998) Nature 392, 677-684[CrossRef][Medline] [Order article via Infotrieve]
14. Iwata, S., Lee, J. W., Okada, K., Lee, J. K., Iwata, M., Rasmussen, B., Link, T. A., Ramaswamy, S., and Jap, B. K. (1998) Science 281, 64-71[Abstract/Free Full Text]
15. Hunte, C., Koepke, J., Lange, C., Rossmanith, T., and Michel, H. (2000) Structure 8, 669-684[CrossRef][Medline] [Order article via Infotrieve]
16. Bosshard, H. R., Zurrer, M., Schagger, H., and von Jagow, G. (1979) Biochem. Biophys. Res. Commun. 89, 250-258[Medline] [Order article via Infotrieve]
17. Nakai, M., Endo, T., Hase, T., Tanaka, Y., Trumpower, B. L., Ishiwatari, H., Asada, A., Bogaki, M., and Matsubara, H. (1993) J. Biochem. (Tokyo) 114, 919-925[Abstract]
18. Koppenol, W. H., and Margoliash, E. (1982) J. Biol. Chem. 257, 4426-4437[Abstract/Free Full Text]
19. van Loon, A. P., and Schatz, G. (1987) EMBO J. 6, 2441-2448[Abstract]
20. Arnold, I., Folsch, H., Neupert, W., and Stuart, R. A. (1998) J. Biol. Chem. 273, 1469-1476[Abstract/Free Full Text]
21. Hase, T., Harabayashi, M., Kawai, K., and Matsubara, H. (1987) J. Biochem. (Tokyo) 102, 401-410[Abstract]
22. Nakai, M., Harabayashi, M., Hase, T., and Matsubara, H. (1989) J. Biochem. (Tokyo) 106, 181-187[Abstract]
23. Nakai, M., Ishiwatari, H., Asada, A., Bogaki, M., Kawai, K., Tanaka, Y., and Matsubara, H. (1990) J. Biochem. (Tokyo) 108, 798-803[Abstract]
24. Konishi, K., Van Doren, S. R., Kramer, D. M., Crofts, A. R., and Gennis, R. B. (1991) J. Biol. Chem. 266, 14270-14276[Abstract/Free Full Text]
25. Gray, K. A., Davidson, E., and Daldal, F. (1992) Biochemistry 31, 11864-11873[Medline] [Order article via Infotrieve]
26. Darrouzet, E., Mandaci, S., Li, J., Qin, H., Knaff, D. B., and Daldal, F. (1999) Biochemistry 38, 7908-7917[CrossRef][Medline] [Order article via Infotrieve]
27. Gao, F., Qin, H., Knaff, D. B., Zhang, L., Yu, L., Yu, C. A., Gray, K. A., Daldal, F., and Ondrias, M. R. (1999) Biochim. Biophys. Acta 1430, 203-213[Medline] [Order article via Infotrieve]
28. Sherman, F. (1997) in The Encyclopedia of Molecular Biology and Molecular Medicine (Myers, R. A., ed), Vol. 6 , pp. 302-325, VCH Publishers, Inc., New York
29. Sherman, F. (1991) Methods Enzymol. 194, 3-21[Medline] [Order article via Infotrieve]
30. Ito, H., Fukuda, Y., Murata, K., and Kimura, A. (1983) J. Bacteriol. 153, 163-168[Medline] [Order article via Infotrieve]
31. Maniatis, T., Fritsch, E. F., and Sambrook, J. (1982) Molecular Cloning: A Laboratory Manual , Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
32. Cohen, S. N., Chang, A. C. Y., and Hsu, L. (1972) Proc. Natl. Acad. Sci. U. S. A. 69, 2110-2114[Abstract]
33. Saiki, R. K., Gelfand, D. H., Stoffel, S., Scharf, S. J., Horn, G. T., Mullis, K. B., and Erlich, H. A. (1988) Science 239, 487-491[Medline] [Order article via Infotrieve]
34. Dumont, M. E., Schlichter, J. B., Cardillo, T. S., Hayes, M. K., Bethlendy, G., and Sherman, F. (1993) Mol. Cell. Biol. 13, 6442-6451[Abstract]
35. Sikorski, R. S., and Hieter, P. (1989) Genetics 122, 19-27[Abstract/Free Full Text]
36. Ma, H., Kunes, S., Schatz, P. J., and Botstein, D. (1987) Gene (Amst.) 58, 201-216[CrossRef][Medline] [Order article via Infotrieve]
37. Kunkel, T. A., Bebenek, K., and McClary, J. (1991) Methods Enzymol. 204, 125-139[Medline] [Order article via Infotrieve]
38. Kunkel, T. A., Roberts, J. D., and Zakour, R. A. (1987) Methods Enzymol. 154, 367-382[Medline] [Order article via Infotrieve]
39. Sherman, F., Stewart, J. W., Jackson, M., Gilmore, R. A., and Parker, J. H. (1974) Genetics 77, 255-284[Abstract/Free Full Text]
40. Sherman, F., and Slonimski, P. P. (1964) Biochim. Biophys. Acta 90, 1-15
41. Hickey, D. R., Jayaraman, K., Goodhue, C. T., Shah, J., Fingar, S. A., Clements, J. M., Hosokawa, Y., Tsunasawa, S., and Sherman, F. (1991) Gene (Amst.) 105, 73-81[CrossRef][Medline] [Order article via Infotrieve]
42. Sherman, F., Fink, G. R., and Hicks, J. B. (1987) Methods in Yeast Genetics , Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
43. Broger, C., Salardi, S., and Azzi, A. (1983) Eur. J. Biochem. 131, 349-352[Abstract]
44. Stonehuerner, J., O'Brien, P., Geren, L., Millett, F., Steidl, J., Yu, L., and Yu, C. A. (1985) J. Biol. Chem. 260, 5392-5398[Abstract]


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