Further insights into the assembly of the yeast cytochrome
bc
1
complex based on analysis of single and double deletion mutants
lacking supernumerary subunits and cytochrome
b
Vincenzo Zara
1
, Ilaria Palmisano
1
, Laura Conte
1
and Bernard L. Trumpower
2
1
Dipartimento di Scienze e Tecnologie Biologiche ed Ambientali, Universita
`
di Lecce, Italy;
2
Department of Biochemistry,
Dartmouth Medical School, Hanover, NH, USA
The cytochrome bc
1
complex of the yeast Saccharomyces
cerevisiae is composed of 10 different subunits that are
assembled as a symmetrical dimer in the inner mitochondrial
membrane. Three of the subunits contain redox centers and
participate in catalysis, whereas little is known about the
function of the seven supernumerary subunits. To gain fur-
ther insight into the function of the supernumerary subunits
in the assembly process, we have examined the subunit

; assembly; supernumerary sub-
units; Saccharomyces cerevisiae.
The cytochrome bc
1
complex is a multisubunit complex
embedded in the inner membrane of mitochondria [1,2].
This respiratory enzyme catalyzes the transfer of electrons
from ubiquinol to cytochrome c and couples the electron
transfer to vectorial proton translocation across the inner
mitochondrial membrane. The bc
1
complex has been
crystallized and analyzed from bovine, chicken and yeast
mitochondria [3–7].
In mitochondria of the yeast Saccharomyces cerevisiae,
the cytochrome bc
1
complex is composed of 10 different
subunits organized in the lipid bilayer as a homo-dimer as
shown in Fig. 1A [8,9]. There are three catalytic subunits
that contain redox prosthetic groups, cytochrome b,cyto-
chrome c
1
and the Rieske iron–sulfur protein (ISP). In
addition, there are seven supernumerary subunits that lack
any cofactors. The supernumerarysubunits arecore protein 1
and core protein 2 [10,11], with apparent molecular masses of
 44 and 40 kDa on SDS/PAGE, respectively, and five
smaller proteins. The latter are Qcr6p [12], Qcr7p [13], Qcr8p
[14], Qcr9p [15] and Qcr10p [8] with apparent molecular

complex to the bc
1
complexes
of higher eukaryotes suggest that the yeast enzyme may
serve as a paradigm to understand how this oligomeric
protein complex is assembled into the inner mitochondrial
membrane.
Correspondence to V. Zara, Dipartimento di Scienze e Tecnologie
Biologiche ed Ambientali, Universita
`
di Lecce, Via Prov.le Lecce-
Monteroni, I-73100 Lecce, Italy. Fax: + 39 0832 298626,
Tel.: + 39 0832 298705, E-mail:
Abbreviations: DFP, diisopropyl fluorophosphate; ISP, Rieske
iron–sulfur protein.
(Received 8 January 2004, revised 23 January 2004,
accepted 6 February 2004)
Eur. J. Biochem. 271, 1209–1218 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04024.x
In this study, we have investigated the role of the
supernumerary subunits in the assembly of the bc
1
complex
in S. cerevisiae mitochondria. To this end we have prepared
single and double deletion yeast mutants in which one or
two nuclear genes encoding the supernumerary subunits
Qcr6p, Qcr7p, Qcr8p, Qcr9p and Qcr10p have been deleted
and analyzed the bc
1
subunits present in mitochondrial
membranes using antibodies directed against the various

Igs were a gift of N. Pfanner
2
(Institute for Biochemistry
and Molecular Biology, Freiburg, Germany). All other
reagents were of analytical grade.
Yeast strains, media and genetic methods
The S. cerevisiae strains used in this study are listed in
Table 1. The construction of the QCR7 deletion strain
(VZ1) was performed following the procedure of homolog-
ous recombination as described previously [21]. A DNA
fragment prepared by PCR and carrying the coding region
for the selectable TRP1 marker, plus the flanking sequences
of the QCR7 open-reading frame at the 5¢-and3¢-regions,
was used to transform yeast cells by treatment with lithium
acetate [22]. The transformants were then selected for
tryptophan prototrophy.
The double deletion strains were constructed as follows.
The haploid strains VZ1 (D7) and MES8 (D6), VZ2 (D7) and
LLD9 (D8), JDP1 (D9) and LLD9 (D8), JDP2 (D9) and
UBL2 (D10), were mated and the resulting diploids were
sporulated to obtain the double deletion strains VZ4
(D6/D7), VZ6 (D7/D8), VZ14 (D8/D9) and VZ9 (D9/D10),
respectively. The selectable markers exhibited a 2 : 2 segre-
gation pattern, and some spores were prototrophic for both
markers. Haploid spores of VZ4, VZ6, VZ14 and VZ9 were
then selected for Trp
+
and Leu
+
,Trp

, and the Rieske ISP colored blue, red and yellow, respectively. The supernumerary subunits are colored gray. The structure is oriented as
it would appear in the inner mitochondrial membrane, with the mitochondrial matrix at the bottom. (B) The structure of cytochrome b and
supernumerary subunits 7 and 8 in one monomer (the Ôcytochrome b, subunit 7, subunit 8 coreÕ). Cytochrome b is colored blue, subunit 7 is colored
pink, and subunit 8 is colored green. The arrow labeled (a) points to the N-terminus of cytochrome b where it is enveloped by subunit 7. The arrows
labeled (b) and (c) point to the areas of interaction between the transmembrane helix of subunit 8 and helices G and H1 of cytochrome b and
between the N-terminus of subunit 8 and helix a of cytochrome b. The figure was constructed from the crystal structure of the yeast bc
1
complex [6].
1210 V. Zara et al. (Eur. J. Biochem. 271) Ó FEBS 2004
2% (w/v) glucose (YPD). For the isolation of mitochondrial
membranes, the yeast strains were grown in liquid YPD
medium containing 1% (w/v) yeast extract, 2% (w/v) bacto-
peptone and 2% (w/v) glucose, pH 5.0.
Isolation of mitochondrial membranes
Mitochondrial membranes were isolated from the various
yeast strains by a modification of a previously described
method [24]. Yeast cells were grown overnight at 30 °C,
unless otherwise specified, in 800 mL of YPD until expo-
nential growth phase was reached (D
600
3
of 1–2). Cells were
recovered by centrifugation at 3200 g for 15 min and then
washed once with distilled water. The pellet was resuspended
in 25 mL of MTE buffer (400 m
M
mannitol, 50 m
M
Tris/
HCl, 2 m

Protein concentrations were determined by the Bradford
method [26] or the modified Lowry method [27]. Electro-
phoretic analysis of DNA on agarose gels, restriction
endonuclease analysis, ligation of DNA fragments,
Table 1. Yeast strains used in this study.
Strain Genotype Reference
W303–1 A (WT) MATa, ade2–1, his3–11,15, trp1–1, leu2–3,112,
ura3–1, can1–100
Gift from A. Tzagoloff, Columbia University,
New York
W303–1B (WT) MATa, ade2–1, his3–11,15, trp1–1, leu2–3,112,
ura3–1, can1–100
Gift from A. Tzagoloff, Columbia University,
New York
MES8 (D6) MATa, ade2–1, his3–11,15, trp1–1, leu2–3,112,
ura3–1, can1–100, qcr6D::LEU2
[37]
VZ1 (D7) MATa, ade2–1, his3–11,15, trp1–1, leu2–3,112,
ura3–1, can1–100, qcr7D::TRP1
This study
VZ2 (D7) MATa, ade2–1, his3–11,15, trp1–1, leu2–3,112,
ura3–1, can1–100, qcr7D::TRP1
This study
LLD9 (D8) MATa, ade2–1, his3–11,15, trp1–1, leu2–3,112,
ura3–1, can1–100, qcr8D::HIS3
Daniels and Trumpower, unpublished data
JDP1 (D9) MATa, ade2–1, his3–11,15, trp1–1, leu2–3,112,
ura 3–1, can1–100, qcr9D1::URA3
[15]
JDP2 (D9) MATa, leu2–3,112, his3, can 1–11, qcr9D2::HIS3 [15]

procedures [28].
Results
Growth phenotype of single and double deletion
mutants
The growth phenotype of the yeast strains with deletions of
genes encoding various subunits of the bc
1
complex was
determined by plating the cells on solid media containing
fermentable or nonfermentable carbon sources and then
incubating at 30 °C. The results are summarized in Table 2.
Among the single deletion mutants, only the subunit 6
(MES8) and subunit 10 (UBL2) deletion strains were able to
grow on nonfermentable carbon source at a rate compar-
able to the wild-type strain (W303). Under the same
conditions, the strain JDP1, in which the nuclear gene
encoding subunit 9 had been deleted, exhibited a reduced
growth rate with respect to the wild-type strain as reported
previously [15,29]. The yeast mutants with deletions for the
genes encoding subunit 7 (VZ1) or subunit 8 (LLD9) failed
to grow on the nonfermentable YPEG medium.
Among the double deletion mutants, the strain with the
genes encoding subunits 9 and 10 deleted (VZ9) and that
with the genes encoding subunits 6 and 10 deleted (SUY
106-a) grew on nonfermentable medium, although at a
reduced rate compared to the wild-type strain. In the case
of the VZ9 strain, this was to be expected, based on the
reduced growth rate of the single deletion strain lacking
subunit 9. The remaining double deletion mutants, VZ4
(D6/D7), VZ6 (D7/D8) and VZ14 (D8/D9), were unable to

subunit 6 deletion strain compared to the wild-type strain.
Table 2. Growth phenotype of single and double deletion mutants. All
the strains were first grown in liquid YPD medium to the same original
density and subsequently plated on solid media containing ferment-
able (YPD) or nonfermentable carbon sources (YPEG). Normal
growth, +; reduced growth rate (+); no growth, ).
Strain
Lacking
subunit(s)
Growth
YPD YPEG
W303 – + +
MES8 Qcr6p + +
VZ1 Qcr7p + –
LLD9 Qcr8p + –
JDP1 Qcr9p (+) (+)
UBL2 Qcr10p + +
VZ4 Qcr6p/Qcr7p + –
VZ6 Qcr7p/Qcr8p + –
VZ14 Qcr8p/Qcr9p + –
VZ9 Qcr9p/Qcr10p + (+)
SUY 106-a Qcr6p/Qcr10p (+) (+)
Fig. 2. Subunit composition of mitochondrial membranes from yeast
mutants with single deletions of genes for each of the nuclear encoded
supernumerary subunits. Yeast strains were grown on YPD medium
and mitochondrial membranes were analyzed by SDS/PAGE and
Western blotting with antibodies to the subunits of the yeast bc
1
complex indicated on the left side of the blots.
1212 V. Zara et al. (Eur. J. Biochem. 271) Ó FEBS 2004

36% of the wild-type level in both mutant strains, and the
twocoreproteinsdecreasedinparallelinbothmutants,with
lower amounts found in the subunit 8 deletion strain. The
only difference between the two strains was that subunit 6
was present in small amounts in the subunit 7 deletion strain
but completely absent in the subunit 8 deletion strain.
In mitochondrial membranes from the strain JDP1, in
which the gene encoding subunit 9 had been deleted, there
was a significant decrease in cytochrome c
1
(45% of the
wild-type content), a barely detectable amount of Rieske
protein and low levels of cytochrome b (12% of the wild-
type content). Core protein 1 and core protein 2 decreased
significantly to about 40% of the wild-type levels. Subunit 8
decreased to the same extent as the core proteins, whereas a
greater decrease was seen in the case of both subunits 6 and
7. Interestingly, a higher amount of cytochrome b,almost
equivalent to that of wild-type cells, was detected in the
JDP1 (D9) mitochondrial membranes when this mutant
strain was grown at 25 °C instead of 30 °C (results not
shown). This effect of temperature on cytochrome b content
was not observed in the case of the other single deletion
mutants.
Among the single deletion strains tested, UBL2, in which
the gene for subunit 10 was deleted, was the only one
showing wild-type levels of all of the bc
1
subunits (Fig. 2
and Table 3). It is also worth noting that the mitochondrial

bc
1
subunits in the mitochondrial membranes than was
observed with the single deletion mutants.
The membranes from the D6/D7 double deletion strain
(VZ4) exhibited the strongest defect in the assembly of the
catalytic subunits of cytochrome bc
1
complex. This strain
showed only 18% and 6% of the wild-type levels of iron–
sulfur protein and cytochrome b, respectively, while mature
cytochrome c
1
disappeared completely and only a small
amount of the intermediate form was visible. Subunits 8 and
9 were reduced to about one third of the original levels.
However, the core proteins were only slightly diminished.
The most notable difference between this double deletion
strain and the others (see below) was the complete absence
of mature cytochrome c
1
.
The mitochondrial membranes from the D7/D8 double
deletion strain (VZ6) showed no cytochrome b and only a
negligible amount of ISP, as expected on the basis of the
results obtained with the single deletion strains. The relative
amount of cytochrome c
1
decreased by 50%, as did both
core proteins. There was also a strong reduction in the

JDP1
(D9)
ULB2
(D10)
Cytochrome b 52 5 2 12 109
Cytochrome c
1
64 90 90 45 106
ISP 68 10 9 3 113
Core 1 103 57 30 38 101
Core 2 104 58 28 45 116
Qcr6p – 18 – 10 120
Qcr7p 64 – 5 28 111
Qcr8p 43 12 – 40 113
Qcr9p 36 36 36 – 100
Ó FEBS 2004 Assembly of the yeast cytochrome bc
1
complex (Eur. J. Biochem. 271) 1213
competent double deletion strains characterized here,
showed decreased levels of all three catalytic subunits,
cytochrome b, ISP and cytochrome c
1
. In addition, core
proteins 1 and 2 and subunit 6 were reduced to about half of
their original levels, while subunits 7 and 8 were reduced to
about one quarter of their original levels. The deletion of
both genes encoding subunit 6 and 10 in the strain SUY
106-a caused significant changes in the amount of catalytic
subunits not observed previously with the single deletion
strains lacking either subunit 6 or subunit 10. In fact,

membranes from yeast strains in which the gene encoding
cytochrome b had been deleted or truncated.
To this end, we used the yeast strain W303–1B q°,devoid
of mitochondrial DNA, and therefore without the gene
encoding cytochrome b. We performed similar experiments
with the strain CKL57 that contains a point mutation
(L263-STOP) in the cytochrome b gene that results in a
nonfunctional, truncated protein (Table 1). Both of these
yeast strains were respiratory-deficient.
Figure 4 shows the subunit composition of the mito-
chondrial membranes from the W303–1B q° and CKL57
strains and from the corresponding wild-type cells grown in
YPD at 30 °C. In general, the pattern of subunits present in
the mitochondrial membranes was identical for these two
mutant strains, although the decrease in amounts of the
subunits was more severe in the q° strain. As expected,
cytochrome b was absent from the W303–1B q° strain.
Likewise, no cytochrome b protein was detectable in the
CKL57 strain. We do not know whether the lack of
immunoreactivity in the latter strain was due to the inability
of the truncated protein to insert into and be stable in the
inner mitochondrial membrane or lack of detection of the
truncated protein by the antibodies.
In the W303–1B q° strain the amounts of the other two
catalytic subunits, cytochrome c
1
and the ISP, were reduced
by about 70–80% (Fig. 4A,C). In the case of the strain
Table 4. Cytochrome bc
1

mutants with double deletions of genes for nuclear encoded super-
numerary subunits. Yeast strains were grown on YPD medium and
mitochondrial membranes were analyzed by SDS/PAGE and Western
blotting with antibodies to the subunits of the yeast bc
1
complex
indicated on the left side of the blots.
1214 V. Zara et al. (Eur. J. Biochem. 271) Ó FEBS 2004
CKL57 the amount of ISP was 40%, while the cyto-
chrome c
1
content was almost unaffected (Fig. 4B,D). Core
1 and core 2 proteins were significantly reduced in both
mutant strains, being 33 and 29%, respectively, in the
W303–1B q° strain (Fig. 4A,C) and 57 and 53%, respect-
ively, in the CKL57 strain (Fig. 4B,D). Interestingly, the
small subunits 6, 7 and 8 were totally absent in both mutant
strains. Only a small amount (22%) of subunit 9 was present
in the W303–1B q° strain, Fig. 4A,C, while essentially
normal amounts of this subunit were present in the CKL57
strain.
When the cytochrome b mutant strains were grown in
YPD at 25 °C instead of 30 °C, the defects in subunit
composition appeared less evident, especially in the case of
the W303–1B q° strain (results not shown). In the mito-
chondrial membranes from this strain, the content of
cytochrome c
1
increased from 29 to 73%, and the amounts
of core proteins 1 and 2 increased from 33 to 74% and 29 to

membrane are not influenced by the absence of subunits
of the bc
1
complex [19,20,30]. Thus, this experimental
strategy allows the determination of which subunits are
present in the inner mitochondrial membrane independent
of previous steps in bc
1
complex assembly. Defects in the
mitochondrial membrane composition of bc
1
subunits in the
deletion strains can be ascribed to an altered process of
assembly of the multisubunit complex in the inner mito-
chondrial membrane. The bc
1
subunits that are imported
but not assembled into the multisubunit complex or
subcomplexes thereof are probably more susceptible to
proteolysis, as previously proposed [19,20,30,32]. This is
reflected in decreased amounts or absence of the non-
assembled subunits in the mitochondrial membranes.
With all of the single and double deletion mutants there
appeared to be a strict correlation in the amounts of
Fig. 4. Subunit composition of mitochondrial membranes from a yeast mutant lacking mitochondrial DNA and a yeast mutant with a truncated
cytochrome b gene. The wild-type (WT), q°, and CKL57 yeast strains were grown on YPD medium and mitochondrial membranes were analyzed
by SDS/PAGE and Western blotting with antibodies to the subunits of the yeast bc
1
complex indicated on the left side of the blots. The Western
blots are shown in panels A and B and the relative amounts of each of the subunits determined by densitometry scanning of the stained Western

observed the disappearance of subunit 6 and a strong
decrease of subunit 9 and both core proteins. Accordingly,
the QCR8 gene deletion resulted in the most severe
phenotype among the single deletion strains tested.
In the deletion mutant lacking subunit 7 we found an
almost complete lack of cytochrome b, subunit 8 and ISP,
in agreement with previous studies [20]. However, unlike
previous results [20], we also found a significant decrease of
both core proteins, and low levels of subunits 6 and 9. In
fact, concomitant and significant decreases of almost all
remaining subunits, except cytochrome c
1
,wereobserved.
These results were confirmed by those obtained with the
double deletion strain VZ6 (D7/D8). The results with
the deletion mutant lacking subunit 7 further corroborate
the interdependence among subunits 7, 8 and cytochrome b
and the role of this core subcomplex in organizing the
cytochrome bc
1
complex. It was proposed previously that
the N-terminus of subunit 7 plays an important role during
the assembly of the cytochrome bc
1
complex [33,34]. In
support of this proposal, it is the N-terminal 30 amino acids
of subunit 7 that envelopes the N-terminus of cytochrome b
near the matrix side of the inner membrane (Fig. 1B).
Cytochrome c
1

also showed only a small amount of the cytochrome c
1
intermediate form. This is probably due to the combination
of two phenomena, the maturation delay caused by the
absence of subunit 6, and the pleiotropic effects due to the
deletion of QCR7, including almost complete disappearance
of cytochrome b and subunit 8. Similar effects, including the
presence of the intermediate form of cytochrome c
1
along
with a complete lack of the mature form, were previously
seen in the QCR6 deletion strain grown at nonpermissive
temperatures [36]. In that study, also a complete block of
cytochrome c
1
maturation was found together with a
simultaneous lack of both subunits 6 and 8 and low levels
of cytochrome b. Together, these results suggest that the
absence of subunit 6 delays cytochrome c
1
maturation while
the absence of the cytochrome b subcomplex (formed by
Fig. 5. Schematic model summarizing the putative cytochrome bc
1
subcomplexes involved in bc
1
complex assembly. The double arrows
indicate that the sequence of events by which the three subcomplexes
associate to form a subcomplex containing both cytochromes b and c
1

1
and subunit 6.
Interestingly, previous studies suggested an interaction
between subunit 9 and cytochrome c
1
[24,38,39]. Taken
together, these results suggest that a subcomplex between
cytochrome c
1
and the two supernumerary subunits 6 and 9
is possible (Fig. 5). This would be consistent with the crystal
structure, which shows that these two supernumerary
subunits interact with cytochrome c
1
[6].
The level of ISP was significantly influenced in almost all
of the deletion strains. This catalytic subunit was present in
very low amounts in the D7, D8andD9 single deletion
mutants, and in all of the double deletion mutants prepared
in this study. The extensive loss of ISP in the yeast strain
lacking the gene for subunit 9 is in agreement with previous
results indicating that this catalytic subunit is protease-
sensitive in the absence of subunit 9 [29]. In addition, recent
findings show a synergistic interaction between cyto-
chrome b and subunit 9 in yeast mitochondria [40]. These
authors proposed a stabilizing role of subunit 9 on the
interactions among the catalytic subunits of the cyto-
chrome bc
1
complex, especially at high temperatures. In this

in this study may be due to the fact that we examined
mitochondrial membranes instead of mitochondria. Using
mitochondria there is still the possibility to detect proteins in
transit and not yet inserted into the inner mitochondrial
membrane. The fact that both core proteins decreased by
the same extent in the various deletion strains suggests that
they probably form a subcomplex as hypothesized previ-
ously [30,31] (Fig. 5).
Our results allow some insight into the sequence of events
in assembly of the bc
1
complex. Two of the supernumerary
subunits, 7 and 8, along with cytochrome b,appeartoplay
an important role in the structural organization of the
bc
1
complex. This suggests that these subunits associate at
an early step in the assembly pathway. In contrast, the
supernumerary subunit 10 seems to play only a minor role
in the overall structure of the bc
1
complex. Deletion of the
QCR10 gene has no effect on the composition of bc
1
subunits in the mitochondrial membrane. This subunit is
readily lost during purification and is not present in the
crystal structure of the bc
1
complex [6]. This suggests that
subunit 10 is in a peripheral location on the bc

e della Ricerca (MIUR), PRIN 2002, and by NIH Grant GM
20379 to B. L. T.
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