To gain fur-ther insight into the function of the supernumerary subunits in the assembly process, we have examined the subunit composition of mitochondrial membranes isolated from yeast
Trang 1Further insights into the assembly of the yeast cytochrome bc1
complex based on analysis of single and double deletion mutants
Vincenzo Zara1, Ilaria Palmisano1, Laura Conte1and Bernard L Trumpower2
1
Dipartimento di Scienze e Tecnologie Biologiche ed Ambientali, Universita` di Lecce, Italy;2Department of Biochemistry,
Dartmouth Medical School, Hanover, NH, USA
The cytochrome bc1 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
composition of mitochondrial membranes isolated from
yeast mutants in which the genes for supernumerary
sub-units and cytochrome b were deleted and from yeast
mutants containing double deletions of supernumerary
subunits Deletion of any one of the genes encoding
cyto-chrome b, subunit 7 or subunit 8 caused the loss of the other
two subunits This is consistent with the crystal structure
of the cytochrome bc1complex that shows that these three
subunits comprise its core, around which the remaining
subunits are assembled Absence of the cytochrome b/sub-unit 7/subb/sub-unit 8 core led to the loss of subb/sub-unit 6, whereas cytochrome c1, iron–sulfur protein, core protein 1, core protein 2 and subunit 9 were still assembled in the mem-brane, although in reduced amounts Parallel changes in the amounts of core protein 1 and core protein 2 in the mito-chondrial membranes of all of the deletion mutants suggest that these can be assembled as a subcomplex in the mito-chondrial membrane, independent of the presence of any other subunits Likewise, evidence of interactions between subunit 6, subunit 9 and cytochrome c1 suggests that a subcomplex between these two supernumerary subunits and the cytochrome might exist
Keywords: cytochrome bc1; assembly; supernumerary sub-units; Saccharomyces cerevisiae
The cytochrome bc1 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 bc1 complex has been
crystallized and analyzed from bovine, chicken and yeast
mitochondria [3–7]
In mitochondria of the yeast Saccharomyces cerevisiae,
the cytochrome bc1 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 c1 and the Rieske iron–sulfur protein (ISP) In
addition, there are seven supernumerary subunits that lack
any cofactors The supernumerary subunits are core 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 masses of about 17, 14, 11, 7.3 and 8.5 kDa, respectively Although the supernumerary subunits of the mitochond-rial bc1complexes were discovered one to two decades ago [16], little is known about their function It is also not known how these peripheral subunits are assembled around the catalytic core of the enzyme to arrive at the three dimen-sional organization revealed by the crystal structures (Fig 1A) The supernumerary subunits and the catalytic subunits of the yeast cytochrome bc1 complex show sequence similarities to those of the bc1complexes of higher eucaryotes [1,2,9] In addition, the crystallographic analysis
of the Saccharomyces cerevisiae cytochrome bc1 complex has revealed an essentially identical overall structure of this complex and that of chicken and beef [6] In yeast and higher eukaryotes, cytochrome b is encoded by mito-chondrial DNA, while the remaining subunits of the bc1 complex are encoded in the nucleus, synthesized by cytosolic polysomes, and then imported into mitochondria, thereby reaching their final location in the inner membrane [17] The similarities of the yeast bc1complex to the bc1complexes
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: vincenzo.zara@unile.it
Abbreviations: DFP, diisopropyl fluorophosphate; ISP, Rieske
iron–sulfur protein.
(Received 8 January 2004, revised 23 January 2004,
accepted 6 February 2004)
Trang 2In this study, we have investigated the role of the
supernumerary subunits in the assembly of the bc1complex
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 bc1 subunits present in mitochondrial
membranes using antibodies directed against the various
subunits Yeast mutant strains containing single deletions of
genes for supernumerary subunits were described previously
[8,15,18–20], even though an exhaustive analysis of
cyto-chrome bc1subunit composition in these yeast strains has
not been reported We have also created two yeast strains
in which the mitochondrial gene encoding cytochrome b
has been deleted or truncated and examined the subunit
composition of membranes in which the catalytic and
structural core of the enzyme is absent
Experimental procedures
Materials
Yeast extract and bacto-peptone were purchased from
Difco Yeast nitrogen base without amino acids,
Coomas-sie Brilliant Blue, phenylmethylsulfonyl fluoride, glass
beads, acrylamide, bis-acrylamide,
N,N,N¢N¢-tetramethyl-ethylenediamine, ammonium persulfate, diisopropyl
fluoro-phosphate (DFP), glucose and glycerol were from Sigma
Anti-mouse and anti-rabbit IgG, coupled to peroxidase,
were from Bio-Rad The ECL detection system for
Western blotting was from Amersham Nitrocellulose
was from Pall Life Sciences, New York, NY, USA
Polyclonal and monoclonal antibodies against the various
subunits of the yeast cytochrome bc1 complex were
prepared in the Trumpower laboratory The anti-Tom40
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 previhomolog-ously [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¢- and 3¢-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 His+, His+and Ura+, or His+and Leu+prototrophy, respectively Other yeast genetic methods used were as described in [23] The expected absence of the corresponding protein pro-ducts in mitochondrial membranes from the deletion strains was assessed by Western blot analysis (Results)
The respiratory capacity of the yeast strains was checked
on nonfermentable solid medium containing 1% (w/v) yeast extract, 2% (w/v) bacto-peptone, 2% (w/v) agar, 3% (v/v) glycerol and 2% (v/v) ethanol (YPEG) Viability of the strains on fermentable medium was confirmed on 1% (w/v) yeast extract, 2% (w/v) bacto-peptone, 2% (w/v) agar and
Fig 1 The yeast cytochrome bc 1 complex (A) The structure of the dimeric yeast bc 1 complex with the redox subunits, cytochrome b, cyto-chrome c 1 , 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].
Trang 32% (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 30C,
unless otherwise specified, in 800 mL of YPDuntil
expo-nential growth phase was reached (D600
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 mMmannitol, 50 mMTris/
HCl, 2 mMEDTA, pH 7.4) Acid-washed glass beads were
added up to a final volume of 30 mL to the mixture kept
at 4C and 1 mMDFP was then added Afterwards, the
cells were mixed with a vortex mixer at maximum speed for
10 min at 4C After the further addition of MTE buffer to a
final volume of 50 mL, the mixture was centrifuged at 1000 g
for 10 min at 4C The supernatant was then centrifuged
at 18 500 g for 30 min at 4C in order to pellet the
mitochondrial membranes The pellet was washed with
20–30 mL of MTE and re-isolated by centrifugation as
described above The mitochondrial membranes were then resuspended in 1 mL of MTE buffer, divided in aliquots of
50 lL each, and stored at)80 C for subsequent analysis
by SDS/PAGE and Western blotting
SDS/PAGE and Western blotting Mitochondrial membranes were analyzed by standard SDS/PAGE with 15% (w/v) acrylamide and an acryl-amide/bis-acrylamide ratio of 30 : 0.8 (w/w) [25] The proteins were then stained with Coomassie Blue or transferred to nitrocellulose membranes Immunodetection
of the yeast mitochondrial proteins was carried out with monoclonal and polyclonal antibodies by chemilumines-cence The stained polyacrylamide gels and the fluoro-graphs containing the immunodetected proteins were scanned and quantified using an Imaging Densitometer GS-700 from Bio-Rad
Other methods 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.
ura3–1, can1–100
Gift from A Tzagoloff, Columbia University, New York
ura3–1, can1–100
Gift from A Tzagoloff, Columbia University, New York
ura3–1, can1–100, qcr6D::LEU2
[37]
ura3–1, can1–100, qcr7D::TRP1
This study
ura3–1, can1–100, qcr7D::TRP1
This study
ura3–1, can1–100, qcr8D::HIS3
Daniels and Trumpower, unpublished data
ura 3–1, can1–100, qcr9D1::URA3
[15]
can1–100, qcr10D2::LEU2
[8]
ura3–1, can1–100, qcr6D::LEU2, qcr7D::TRP1
This study
ura3–1, can1–100, qcr7D::TRP1, qcr8D::HIS3
This study
ura3–1, can1–100, qcr8D::HIS3, qcr9D1::URA3
This study
qcr10D2::LEU2
This study
qcr10D1::HIS3
[8]
(no mtDNA)
Gift from B Meunier, UCL
point mutation in cytochrome b gene)
Gift from B Meunier, UCL
Trang 4transformation of Escherichia coli and isolation of plasmid
DNA from bacterial cells were carried out by standard
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 bc1 complex was
determined by plating the cells on solid media containing
fermentable or nonfermentable carbon sources and then
incubating at 30C 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
grow on the same medium
Cytochromebc1subunit analysis of single deletion
mutants
We sought to determine how the absence of individual
supernumerary subunits affected the composition of bc1
subunits in the mitochondrial membranes For this purpose, mitochondrial membranes were isolated from the single deletion strains grown at 30C in YPD, then transferred
to nitrocellulose and probed with an antiserum against Tom40p, an outer membrane protein belonging to the import machinery of yeast mitochondria (data not shown)
In this way we adjusted the amount of mitochondrial membranes in order to use comparable amounts of protein for the subsequent immunoblot experiments
The blot in Fig 2 shows the cytochrome bc1subunits in the mitochondrial membranes from the mutants in which genes for subunit 6 (MES8), 7 (VZ1), 8 (LLD9), 9 (JDP1) or
10 (UBL2) were deleted Relative amounts of the subunits determined by densitometry scanning of the stained gels are tabulated in Table 3 The relative amounts of cytochrome b and the mature forms of both cytochrome c1and Rieske ISP decreased to 52, 64 and 68%, respectively, in the 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 YPDmedium to the same original
density and subsequently plated on solid media containing
ferment-able (YPD) or nonfermentferment-able carbon sources (YPEG) Normal
growth, +; reduced growth rate (+); no growth, ).
Strain
Lacking subunit(s)
Growth YPDYPEG
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 YPDmedium 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.
Trang 5Interestingly, the absence of subunit 6 also resulted in an
increase in the ratio of intermediate to mature
cyto-chrome c1 and a disappearance of the intermediate form
of the Rieske protein At the same time, the levels of
subunits 7, 8 and 9 significantly decreased in this mutant
strain However, the amounts of core protein 1 and core
protein 2 were relatively unaffected Therefore, the absence
of subunit 6 appeared to alter the rates of processing of two
of the redox subunits and caused minor changes in amounts
of the small supernumerary subunits, but it did not cause
dramatic changes in the cytochrome bc1 composition
Accordingly, this yeast strain was respiratory-competent
Deletion of the gene encoding either subunit 7 or subunit
8 resulted in a more severe phenotype, and the changes in
bc1 subunit composition of the membranes were
compar-able in these two deletion strains, as can be seen from the
blot in Fig 2 In addition, the absence of subunit 7 caused
a strong decrease in subunit 8 and vice versa, suggesting
a correlation between these two subunits In both strains,
cytochrome b and the Rieske protein were almost
unde-tectable, while the amounts of cytochrome c1were similar to
that found in the wild-type strain Subunit 9 decreased to
36% of the wild-type level in both mutant strains, and the
two core proteins decreased in parallel in both mutants, 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 c1 (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 25C 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 bc1subunits (Fig 2 and Table 3) It is also worth noting that the mitochondrial membranes from these mutant cells also showed the same ratio of intermediate to mature form of ISP when compared
to the wild-type membranes (Fig 2) Accordingly, deletion
of QCR10 did not affect mitochondrial respiration, even though bc1activity was significantly reduced [8] This is due
to the fact that activity of the bc1complex in wild-type yeast
is significantly greater than what is required to support normal rates of respiration
Cytochromebc1subunit analysis of double deletion mutants
Mitochondrial membranes were isolated from the double deletion strains and processed by SDS/PAGE and Western blotting using Tom40p to normalize the protein load in the same manner as for the single deletion strains The immuno-detection of bc1subunits in the mitochondrial membranes isolated from the double deletion mutants is shown in Fig 3, and the corresponding quantifications are reported
in Table 4 A comparison of the immunoblots in Figs 3 and
2 reveals that the double deletions of genes encoding bc1 subunits had more marked effects on the composition of
bc1 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 bc1 complex This strain showed only 18% and 6% of the wild-type levels of iron– sulfur protein and cytochrome b, respectively, while mature cytochrome c1 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 c1 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 c1decreased by 50%, as did both core proteins There was also a strong reduction in the amounts of both subunit 6 and subunit 9 in this strain The highest amount of cytochrome c1, approximately 80% of the normal amount, was found in the mitochondrial membranes from the D8/D9 double deletion strain (VZ14) However, there was a strong defect in both cytochrome b and ISP in this strain, similar to what was observed in the other double deletion strains Core proteins 1 and 2 were reduced to approximately half of the wild-type levels, while subunits 6 and 7 were present only in small amounts (18% and 8%, respectively)
The mitochondrial membranes from the D9/D10 double deletion strain (VZ9), which is one of the two respiratory
Table 3 Cytochrome bc 1 subunit analysis of single deletion mutants.
The values represent the percentages of the amounts of the individual
subunits present in the yeast mutant strains with respect to the
amounts present in the wild-type strain W303, which were set to 100%.
The numbers are the averages of at least three independent
experi-ments.
Subunits
Yeast mutant strains MES8
(D6)
VZ1 (D7)
LLD9 (D8)
JDP1 (D9)
ULB2 (D10)
Trang 6competent double deletion strains characterized here,
showed decreased levels of all three catalytic subunits,
cytochrome b, ISP and cytochrome c1 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,
cytochrome b and ISP were reduced to about 12% and
27% of the original levels Cytochrome c1and core proteins
1 and 2 decreased by about 50%, whereas a greater decrease
was found in the case of subunits 7, 8 and 9
Cytochromebc1subunit analysis of cytochromeb deletion mutants
Crystal structures of the bc1 complexes indicate that cytochrome b is the organizing component of the bc1 complex, providing eight transmembrane helices that form the central core of the complex [6] This central core is surrounded by four additional transmembranes helices contributed by cytochrome c1, the Rieske protein, and subunits 8 and 9 It is therefore clear that cytochrome b plays a fundamental role in organizing and stabilizing the structure of the entire complex in the inner mitochondrial membrane For this reason, we investigated the composition
of cytochrome bc1 complex subunits in mitochondrial 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 YPDat 30C 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 c1and the ISP, were reduced
by about 70–80% (Fig 4A,C) In the case of the strain
Table 4 Cytochrome bc 1 subunit analysis of double deletion mutants The values represent the percentages of the amounts of individual subunits present in the yeast mutant strains with respect to the amounts present in the wild-type strain W303, which were set to 100% The numbers are the averages of at least three independent experi-ments.
Subunits
Yeast mutant strains VZ4
(D6/D7)
VZ6 (D7/D8)
VZ14 (D8/D9)
VZ9 (D9/D10)
SUY 106-a (D6/D10)
Fig 3 Subunit composition of mitochondrial membranes from yeast
mutants with double deletions of genes for nuclear encoded
super-numerary subunits Yeast strains were grown on YPDmedium 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.
Trang 7CKL57 the amount of ISP was 40%, while the
cyto-chrome c1content 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
YPDat 25C 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 c1increased from 29 to 73%, and the amounts
of core proteins 1 and 2 increased from 33 to 74% and 29 to
81%, respectively Likewise, the relative amount of
sub-unit 9 increased from 22 to 48% The amount of ISP
changed only slightly at the lower growth temperature, from
23 to 32% In the CKL57 mutant strain, the amounts of all
subunits increased by about 10–20% Subunit 9, as already
seen at 30C, was present in wild-type amounts Subunit 6
was present in only small amounts in the W303–1B q strain
(22%) and in considerably greater amounts (80%) in the
CKL57 strain Interestingly, subunits 7 and 8 remained
undetectable, even at the lower growth temperature, in both
cytochrome b mutants
Discussion
We have analyzed the composition of cytochrome bc1
subunits in mitochondrial membranes of yeast mutants in which genes for individual and pairs of bc1 subunits have been deleted As far as we know, this is the first time that such a large collection of single and double deletion mutants
of the yeast cytochrome bc1complex has been characterized simultaneously Our results add to and extend previous work on the assembly of the yeast bc1complex from the laboratories of Berden [30] and Tzagoloff [31] It has been demonstrated previously that gene expression, import of proteins into mitochondria and sorting to the inner membrane are not influenced by the absence of subunits
of the bc1 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 bc1complex assembly Defects in the mitochondrial membrane composition of bc1subunits in the deletion strains can be ascribed to an altered process of assembly of the multisubunit complex in the inner mito-chondrial membrane The bc1subunits 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 YPDmedium 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 blots of the q and CKL57 membranes are shown in panels C and D, respectively.
Trang 8cytochrome b, subunit 7 and subunit 8 Deletion of the gene
for any one of these proteins caused a strong decrease or the
disappearance of the other two components Accordingly,
the double deletion mutant VZ6, in which both QCR7 and
QCR8 had been deleted, showed no cytochrome b This
agrees with the crystal structures that show that these two
supernumerary subunits both interact with cytochrome b
As shown in Fig 1B, subunit 7 envelopes the N-terminus of
cytochrome b within the membrane near the inner
mem-brane surface Subunit 8 exhibits a single transmemmem-brane
helix that spans the membrane parallel to cytochrome b,
interacting extensively with transmembrane helices G and
H1 and also interacting with helix a of cytochrome b
parallel to the inner membrane surface This structural
relationship and the coincidental behavior of these three
subunits in the deletion strains lend support to previous
suggestions [13,30,31] that cytochrome b, subunit 7 and
subunit 8 may form a nucleating subcomplex in the lipid
bilayer of the inner mitochondrial membrane, around which
the other subunits are assembled (Fig 5)
Subunit 8 interacts with several other subunits of the
complex in addition to cytochrome b [6] Our results with
the single deletion mutant lacking subunit 8 extend the
previous findings of Maarse coworkers [19] In addition
to the strong decrease or disappearance of subunit 7,
cytochrome b and ISP as reported previously [19], we
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 c1, were observed
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 bc1complex It was proposed previously that
the N-terminus of subunit 7 plays an important role during
the assembly of the cytochrome bc1 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 c1 appears to be the cytochrome bc1
com-ponent least influenced by the absence of other subunits of
the complex In fact, only marginal variations in
cyto-chrome c1 were observed in the single deletion mutants
tested, except for the increase of the intermediate form of
c1 in the strain lacking subunit 6 Subunit 6 is an acidic
protein that interacts with cytochrome c1on the cytosolic
surface of the membrane The retardation in c1maturation
in the absence of subunit 6 suggests that the
apo-cytochrome must associate with this subunit before the
c-type heme can be inserted The formation of a
subcom-plex between cytochrome c1and subunit 6 has previously
been proposed on the basis of biochemical [35] and genetic
evidence [18]
Interestingly, the D6/D7 double deletion strain is the only one showing a complete lack of mature cytochrome c1and also showed only a small amount of the cytochrome c1 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 c1along 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 c1 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 c1maturation 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
prior to insertion of ISP and subunit 10 (Qcr10p) in the inner mito-chondrial membrane is currently not known.
Trang 9cytochrome b, subunit 7 and subunit 8) hinders the insertion
of mature cytochrome c1into the complex However, when
the cytochrome b subcomplex is missing, but the gene
encoding subunit 6 is not deleted, as in several of the single
and double deletion strains, mature cytochrome c1 is
present in the mitochondrial membranes in considerable
amount
As reported previously [18,37], the strain lacking the gene
for subunit 6 showed only moderate defects in the levels of
most of the other subunits of the bc1complex when grown
at permissive temperatures However, we found that subunit
9 was present in this deletion strain at about only one-third
of the normal level, which suggests that subunit 6 stabilizes
subunit 9, although the crystal structure shows that these
two subunits do not interact directly [6] Deletion of the gene
encoding subunit 9 resulted in a respiratory deficient yeast
strain with very low bc1 complex activity, particularly at
high temperatures [15,29] In this strain we found a
significant decrease of both cytochrome c1and subunit 6
Interestingly, previous studies suggested an interaction
between subunit 9 and cytochrome c1 [24,38,39] Taken
together, these results suggest that a subcomplex between
cytochrome c1and 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 c1[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, D8 and D9 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 bc1complex, especially at high temperatures In this
regard, it is noteworthy that the level of cytochrome b
increased in the strain lacking the gene for subunit 9 when
the cells were grown at 25C instead of 30 C In addition,
less dramatic changes in subunit composition were found in
cytochrome b mutant strains grown at 25C instead of
30C (Results) A critical effect of the temperature on the
level of various subunits of cytochrome bc1 complex is
therefore evident in the yeast strains in which the genes for
subunit 6, subunit 9 and cytochrome b had been deleted
Core protein 1 and core protein 2 interact with each other
and with the membrane-embedded subunits of the bc1
complex and protrude, almost completely, into the
mito-chondrial matrix [6] In contrast to previous results with
several yeast bc1 complex mutants [31], the amounts of
core 1 and core 2 proteins were significantly influenced by
the absence of other subunits of the bc1complex Deletion
of the genes for subunit 7, subunit 8 or subunit 9 caused a
strong reduction of the two core proteins in the
mito-chondrial membranes (Fig 2 and Table 3) These results
were confirmed by those obtained with the double deletion
strains (Fig 3 and Table 4) Furthermore, deletion of the
gene for cytochrome b caused a decrease of both core
proteins (Fig 4) The low levels of both core proteins found
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 bc1complex Two of the supernumerary subunits, 7 and 8, along with cytochrome b, appear to play
an important role in the structural organization of the
bc1complex 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 bc1complex Deletion of the QCR10 gene has no effect on the composition of bc1
subunits in the mitochondrial membrane This subunit is readily lost during purification and is not present in the crystal structure of the bc1complex [6] This suggests that subunit 10 is in a peripheral location on the bc1complex and that it is added late in the assembly pathway In general, our results agree with and extend the model for the assembly of the yeast bc1complex proposed by Berden and coworkers [30] In fact, these authors proposed the existence of three distinct subcomplexes, essentially con-firmed by the present data (Fig 5) In addition, our results revealed a strict interdependence between the cytochrome b subcomplex and the supernumerary subunit Qcr6p It is also evident that Qcr9p plays an important role in the temperature-sensitive stabilization of the yeast bc1complex
At present it is not possible to deduce the sequence in which subunits of the bc1 complex are assembled into subcom-plexes or the sequence in which the putative subcomsubcom-plexes are assembled to form the bc1 complex The sequence in which the subunits and subcomplexes are assembled is under investigation
Acknowledgements
This study was supported by the Ministero dell’Istruzione, dell’Uni-versita` e della Ricerca (MIUR), PRIN 2002, and by NIH Grant GM
20379 to B L T.
References
1 Trumpower, B.L (1990) Cytochrome bc 1 complexes of micro-organisms Microbiol Rev 54, 101–129.
2 Berry, E.A., Guergova-Kuras, M., Huang, L.S & Crofts, A.R (2000) Structure and function of cytochrome bc complexes Annu Rev Biochem 69, 1005–1075.
3 Xia, D., Yu, C.-A., Kim, H., Xia, J.-Z., Kachurin, A.M., Zhang, L., Yu, L & Deinsenhofer, J (1997) Crystal structure of the cytochrome bc 1 complex of bovine heart mitochondria Science
277, 60–66.
4 Zhang, Z., Huang, L., Shulmeister, V.M., Chi, Y., Kim, K.K., Hung, L., Crofts, A.R., Berry, E.A & Kim, S (1998) Electron transfer by domain movement in cytochrome bc 1 Nature 392, 677–684.
5 Iwata, S., Lee, J.W., Okada, K., Lee, J.K., Iwata, M., Rasmussen, B., Link, T.A., Ramaswamy, S & Jap, B.K (1998) Complete structure of the 11-subunit bovine mitochondrial bc 1 complex Science 281, 64–71.
Trang 106 Hunte, C., Koepke, J., Lange, C., Rossmanith, T & Michel, H.
(2000) Structure at 2.3 A˚ resolution of the cytochrome bc 1 complex
from the yeast Saccharomyces cerevisiae co-crystallized with an
antibody fragment Structure 8, 669–684.
7 Lange, C & Hunte, C (2002) Crystal structure of the yeast
cytochrome bc 1 complex with its bound substrate cytochrome c.
Proc Natl Acad Sci USA 99, 2800–2805.
8 Brandt, U., Uribe, S., Scha¨gger, H & Trumpower, B.L (1994)
Isolation and characterization of QCR10, the nuclear gene
encoding the 8.5-kDa subunit 10 of the Saccharomyces cerevisiae
cytochrome bc 1 complex J Biol Chem 269, 12947–12953.
9 Tzagoloff, A (1995) Ubiquinol-cytochrome-c oxidoreductase
from Saccharomyces cerevisiae Methods Enzymol 260, 51–63.
10 Tzagoloff, A., Wu, M & Crivellone, M (1986) Assembly of the
mitochondrial membrane system Characterization of COR1, the
structural gene for the 44-kilodalton core protein of yeast
coen-zyme QH2-cytochrome c reductase J Biol Chem 261, 17163–
17169.
11 Oudshoorn, P., Van Steeg, H., Swinkels, B.W., Schoppink, P &
Grivell, L.A (1987) Subunit II of yeast QH2: cytochrome-c
oxidoreductase Nucleotide sequence of the gene and features of
the protein Eur J Biochem 163, 97–103.
12 Van Loon, A.P., De Groot, R.J., Haan, M., Dekker, A & Grivell,
L.A (1984) The DNA sequence of the nuclear gene coding for the
17-kd subunit VI of the yeast ubiquinol-cytochrome c reductase: a
protein with an extremely high content of acidic amino acids.
EMBO J 3, 1039–1043.
13 De Haan, M., Van Loon, A.P.G.M., Kreike, J., Vaessen,
R.T.M.J & Grivell, L.A (1984) The biosynthesis of the
ubiquinol-cytochrome c reductase complex in yeast DNA sequence analysis
of the nuclear gene coding for the 14 kDa subunit Eur J
Bio-chem 138, 169–177.
14 Maarse, A.C & Grivell, L.A (1987) Nucleotide sequence of the
gene encoding the 11-kDa subunit of the ubiquinol-cytochrome c
oxidoreductase in S cerevisiae Eur J Biochem 165, 419–425.
15 Phillips, J.D., Schmitt, M.E., Brown, T.A., Beckmann, J.D &
Trumpower, B.L (1990) Isolation and characterization of QCR9,
a nuclear gene encoding the 7.3-kDa subunit 9 of the
Saccharo-myces cerevisiae ubiquinol-cytochrome c oxidoreductase complex.
J Biol Chem 265, 20813–20821.
16 Yang, X & Trumpower, B.L (1988) Protonmotive Q cycle
pathway of electron transfer and energy transduction in the
three-subunit ubiquinol-cytochrome c oxidoreductase complex of
Paracoccus denitrificans J Biol Chem 263, 11962–11970.
17 Pfanner, N & Geissler, A (2001) Versatility of the mitochondrial
protein import machinery Nat Rev Mol Cell Biol 2, 339–349.
18 Schoppink, P.J., Hemrika, W., Reynen, J.M., Grivell, L.A &
Berden, J.A (1988) Yeast ubiquinol: cytochrome c oxidoreductase
is still active after inactivation of the gene encoding the 17-kDa
subunit VI Eur J Biochem 173, 115–122.
19 Maarse, A.C., De Haan, M., Schoppink, P.J., Berden, J.A &
Grivell, L.A (1988) Inactivation of the gene encoding the 11-kDa
subunit VIII of the ubiquinol-cytochrome-c oxidoreductase in
Saccharomyces cerevisiae Eur J Biochem 172, 179–184.
20 Schoppink, P.J., Berden, J.A & Grivell, L.A (1989) Inactivation
of the gene encoding the 14-kDa subunit VII of yeast ubiquinol
cytochrome c oxidoreductase and analysis of the resulting mutant.
Eur J Biochem 181, 475–483.
21 Baudin, A., Ozier-Kalogeropoulos, O., Denouel, A., Lacroute, F.
& Cullin, C (1993) A simple and efficient method for direct gene
deletion in Saccharomyces cerevisiae Nucleic Acids Res 21,
3329–3330.
22 Ito, H., Fukuda, Y., Murata, K & Kimura, A (1983)
Transfor-mation of intact yeast cells treated with alkali cations J Bacteriol.
153, 163–168.
23 Guthrie, C & Fink, G.R (1991) Guide to Yeast Genetics and Molecular Biology Academic Press, San D iego, CA.
24 Schmitt, M.E & Trumpower, B.L (1991) The petite phenotype resulting from a truncated copy of subunit 6 results from loss of assembly of the cytochrome bc 1 complex and can be suppressed
by overexpression of subunit 9 J Biol Chem 266, 14958–14963.
25 Laemmli, U.K (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4 Nature 227, 680–685.
26 Bradford, M.M (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding Anal Biochem 72, 248–254.
27 Dulley, J.R & Grieve, P.A (1975) A simple technique for elimi-nating interference by detergents in the Lowry method of protein determination Anal Biochem 64, 136–141.
28 Sambrook, J., Fritsch, E.F & Maniatis, T (1989) Molecular Cloning: a Laboratory Manual, 2nd edn Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.
29 Phillips, J.D., Graham, L.A & Trumpower, B.L (1993) Subunit 9
of the Saccharomyces cerevisiae cytochrome bc 1 complex is required for insertion of EPR-detectable iron-sulfur cluster into the Rieske iron-sulfur protein J Biol Chem 268, 11727–11736.
30 Berden, J.A., Schoppink, P.J & Grivell, L.A (1988) A model for the assembly of ubiquinol: cytochrome c oxidoreductase in yeast.
In Molecular Basis of Biomembrane Transport (Palmieri, F & Quagliariello, E., eds), pp 195–208 Elsevier, Amsterdam.
31 Crivellone, M.D , Wu, M & Tzagoloff, A (1988) Assembly of the mitochondrial membrane system J Biol Chem 263, 14323– 14333.
32 Rep, M & Grivell, L.A (1996) The role of protein degradation
in mitochondrial function and biogenesis Curr Genet 30, 367– 380.
33 Malaney, S., Trumpower, B.L., Debers, C.M & Robinson, B.H (1997) The N terminus of the Qcr7 protein of the cytochrome bc 1
complex is not essential for import into mitochondria in Saccharomyces cerevisiae but is essential for assembly of the complex J Biol Chem 272, 17495–17501.
34 Lee, S.Y., Hunte, C., Malaney, S & Robinson, B.H (2001) The N-terminus of the Qcr7 protein of the cytochrome bc 1 complex in
S cerevisiae may be involved in facilitating stability of the sub-complex with the Qcr8 protein and cytochrome b Arch Biochem Biophys 393, 215–221.
35 Kim, C.H., Balny, C & King, T.E (1987) Role of the hinge protein in the electron transfer between cardiac cytochrome c 1 and
c Equilibrium constants and kinetic probes J Biol Chem 262, 8103–8108.
36 Yang, M & Trumpower, B.L (1994) Deletion of QCR6, the gene encoding subunit six of the mitochondrial cytochrome bc 1 com-plex, blocks maturation of cytochrome c 1 , and causes tempera-ture-sensitive petite growth in Saccharomyces cerevisiae J Biol Chem 269, 1270–1275.
37 Schmitt, M.E & Trumpower, B.L (1990) Subunit 6 regulates half-of-the-sites reactivity of the dimeric cytochrome bc 1
complex in Saccharomyces cerevisiae J Biol Chem 265, 17005– 17011.
38 Gonzales-Halphen, D., Lindorfer, M.A & Capaldi, R.A (1988) Subunit arrangement in beef heart complex III Biochemistry 27, 7021–7031.
39 Scha¨gger, H., Link, T.A., Engel, W.D & von Jagow, G (1986) Isolation of the eleven protein subunits of the bc 1 complex from beef heart Methods Enzymol 126, 224–237.
40 Saint-Georges, Y., Bonnefoy, N., di Rago, J.P., Chiron, S & Dujardin, G (2002) A pathogenic cytochrome b mutation reveals new interactions between subunits of the mitochondrial bc 1 com-plex J Biol Chem 277, 49397–49402.