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
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ABSTRACT |
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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.
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).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
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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.
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EXPERIMENTAL PROCEDURES |
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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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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-::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-
::TRP1, and the desired
disruptant was confirmed by PCR analysis.1
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The plasmids used in this study are listed in Table III, and some are described below.
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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.
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RESULTS AND DISCUSSION |
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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 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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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 (GCCGCT). Two different sets (six each) of the seven plasmids contained the neutral change L36L
(CTC
CTA) and the radical change R166G (AGA
GGA), 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-
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
-peak in the low
temperature (
196 °C) spectrophotometric recording (Fig.
4).
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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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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 -helical
structure in this region, and it is unclear how R166G but not R166K,
R166M, or R166L replacements could effect binding to cytochrome
c.
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ACKNOWLEDGEMENTS |
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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.
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FOOTNOTES |
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* 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.
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.
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ABBREVIATIONS |
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The abbreviations used are: YPD, yeast extract peptone dextrose medium; YPG, yeast extract peptone glycerol medium; PCR, polymerase chain reaction.
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