(Received for publication, February 12, 1997, and in revised form, April 23, 1997)
From the Departments of Subunit 7 of the yeast cytochrome
bc1 complex is encoded by the nuclear
QCR7 gene and is essential for respiration. This protein does not contain a cleavable N-terminal mitochondrial targeting sequence, and it is not understood how the Qcr7 protein is imported into mitochondria and assembled into the complex. To test the role of
the N terminus of the Qcr7 protein in mitochondrial import, assembly of
the complex, and proton translocation, we inactivated the endogenous
QCR7 gene and expressed mutated qcr7 genes
capable of synthesizing proteins truncated by 7, 10, 14, and 20 residues (Qcr7p- The mitochondrial respiratory chain consists of multisubunit
enzyme complexes that are embedded in the inner mitochondrial membrane.
Electron transport through the ubiquinol-cytochrome c
reductase and cytochrome oxidase complexes in S. cerevisiae is coupled to vectorial H+ translocation into the
intermembrane space, resulting in the establishment of a H+
gradient and subsequent membrane potential. The energy from this gradient is then used as the driving force for ion translocation, protein import into mitochondria, and ATP synthesis, which is catalyzed
by the F0F1 ATPase (1, 2).
Ubiquinol-cytochrome c oxidoreductase consists of 10 subunits in yeast (2, 3). There are the three heme-containing subunits cytochrome b, cytochrome c1, and the
Rieske iron-sulfur protein as well as an additional six so-called
supernumerary subunits whose functions remain largely unknown. The
homologous complex in some prokaryotes such as P. dentrificans consists of only the three catalytic subunits (4)
that have been conserved throughout evolution. Thus, it is possible
that the supernumerary subunits do not have any functions in the energy
transducing activity of the bc1 complex.
However, gene inactivation studies of these supernumerary subunits have
shown that all are necessary for the integrity and normal functioning
of complex III (5-7).
Cytochrome b is encoded by the mitochondrial genome, the
other polypeptides being nuclear encoded (8). These nuclear encoded subunits are targeted for import into mitochondria after synthesis on
cytoplasmic ribosomes. Thus there is a requirement for the coordination
of the two separate genetic systems for assembly of the
bc1 complex.
QCR7, the yeast gene encoding the 14.5-kDa polypeptide, has
been cloned and sequenced and was found to be homologous to the 13.4-kDa subunit of the bc1 complex in beef
heart mitochondria (9-11). Studies performed on the
bc1 complex in beef heart suggest that the N
terminus of the 13.4-kDa subunit protrudes into the matrix where it has
been postulated to contribute to the H+ conducting
pathway(s) from the matrix phase to the primary protolytic redox center
(12). Proteolytic cleavage of 7-11 residues from the N terminus of
this subunit was associated with decoupling of redox-linked
H+ pumping (13).
We have now investigated the involvement of the Qcr7p N terminus with
respect to mitochondrial targeting, complex III assembly, and proton
conduction. By CD spectroscopy of synthetic peptides we show that the
secondary structures of the N termini of the yeast and beef heart
proteins strongly resemble one another and are largely L-Amino acids, uracil, adenine, serum
albumin (essentially fatty acid-free), succinate, horse heart
cytochrome c, and Total Protein ReagentTM were obtained
from Sigma. Yeast extract, peptone, tryptone, and yeast nitrogen base
without amino acids were purchased from Difco. Most restriction and
modification enzymes were from Pharmacia Biotech Inc. T7 GEN in
vitro mutagenesis kit was obtained from U. S. Biochemical Corp.
Yeast lytic enzyme, zymolyase 100T, and galactose were purchased from
ICN Pharmaceuticals. Oligodeoxynucleotides were synthesized by the
Department of Clinical Biochemistry at the University of Toronto. Yeast
expression vector pG-3 (14) was a gift from Jacqueline Segall at the
University of Toronto. Alkaline phosphatase substrates
p-nitro blue tetrazolium chloride and
5-bromo-4-chloro-3-indolyl phosphate-toluidine were purchased from
Bio-Rad.
Saccharomyces cerevisiae strain W303-1B (Mat
Plasmid DNA from E. coli (16)
and genomic DNA from yeast (14) were isolated as described.
E. coli strain JMED3
was made competent by growing a culture to A600 < 0.5, pelleting the cells, and resuspending them in 0.12 volumes of
cold 0.1 M CaCl2 with gentle shaking by hand. Cells were left on ice for 30 min, pelleted, and resuspended in 0.02 volumes of cold 0.1 M CaCl2 containing 15%
(v/v) glycerol. After another 30-min incubation on ice, cells were
aliquoted and stored at The original pG-3 expression
plasmid was modified by digestion with SacI to excise a
1700-base pair fragment in the polylinker region and self-ligated to
create the new pG-3 The coding
region of the QCR7 gene was amplified by polymerase chain
reaction from yeast genomic DNA and subcloned into the pCR II vector
(Invitrogen, San Diego). This construct was linearized with
HincII to generate one fragment, disrupted in the middle of
the QCR7 gene. The LEU2 gene, isolated from the
vector pFJ110 by the blunt cutting enzyme PVUII, was subcloned into
this HincII site. The fragment containing the
LEU2 gene flanked by the disrupted qcr7 gene was
excised from the plasmid and transformed into yeast strain W303-1B.
Transformants were selected for on leucine-deficient medium.
Mutants were
constructed using polymerase chain reaction. For the deletion mutants,
degenerate oligodeoxynucleotides were synthesized missing the bases
corresponding to the N-terminal 2-8, 2-11, 2-15, and 2-21 amino
acids, respectively. Point mutants were generated from the Qcr7p- Mutant genes generated by
polymerase chain reaction were digested with SalI and
KpnI and subcloned into the respective sites of the pG-3 Total yeast RNA was prepared by growing a
25-ml culture overnight, harvesting the cells, and resuspending them in
2 ml of AE buffer (50 mM NaOAc, pH 4.8, 10 mM
EDTA). The suspension was vortexed after the addition of 200 µl of
10% SDS and again after the addition of 450 µl of phenol saturated
with AE buffer. The suspension was incubated at 65 °C for 5 min
during which it was vortexed three times. Then the mixture was washed
with equal volume of phenol/chloroform, and the RNA was precipitated
from the supernatant with 2.5 volumes of ethanol. Northern blotting was
performed as described by Fourney et al. (17).
NADH-cytochrome c reductase and
succinate-cytochrome c reductase activities were assayed in
0.1 M potassium Pi buffer, pH 7.0, containing
94 µM cytochrome c and 1 mM azide.
To start the reaction, either 34.2 µM NADH or 10 mM succinate was added, and reduction of cytochrome
c was monitored at 550 nm. Cytochrome oxidase activities
were assayed in 0.1 M potassium Pi buffer, pH
7.0. Cytochrome c was reduced with 3.2 mM
ascorbate followed by dialysis against 0.1 M potassium
Pi for 2 days, during which the buffer was changed twice.
Reduced cytochrome c (94 µM) was added to
start the reaction and oxidation of cytochrome c was followed at 550 nm. For spectral analyses of the cytochromes, mitochondria were resuspended in 0.1 M potassium
Pi, pH 7.4, 0.25 M sucrose, 0.5% cholic acid
(10). To obtain a spectrum containing cytochromes c and
c1, cytochrome b, and cytochromes
a and a3, a ferricyanide (grains)
oxidized spectrum was deducted from a dithionite (grains) reduced
spectrum. To obtain a spectrum containing cytochrome b only,
dithionite reduced minus ascorbate
(grains)-N,N,N SDS-polyacrylamide gel electrophoresis was
run according to the published procedure (16). Proteins were
transferred for 2 h at 55 V at 4 °C in 1 × running buffer
(3.02 g of Tris, 14.4 g of glycine/liter), 0.1% SDS, and 20%
methanol. Blots were subsequently blocked with 2% gelatin in Blotto
(10 mM Tris-Cl, pH 7.5, 150 mM NaCl, 0.05%
Tween 20) for 1 h and then incubated overnight with primary
antibody in Blotto containing 1% gelatin. Blots were washed 4 × 30 min in Blotto and then incubated for 1-2 h under the above
conditions with a secondary antibody coupled to alkaline phosphatase.
Membranes were washed 4 × 15 min in Blotto, and the proteins were
visualized using p-nitro blue tetrazolium chloride and
5-bromo-4-chloro-3-indolyl phosphate-toluidine as substrates in 0.1 M NaHCO3-1 mM
MgCl2.
Cultures were grown for 2 days in
synthetic medium containing a variety of different carbon sources.
Mitochondria were isolated essentially as outlined (14) with the
substitution of breaking buffer (0.6 M sucrose, 20 mM HEPES-KOH, pH 6.5, 0.1% bovine serum albumin, 1 mM phenylmethylsulfonyl fluoride) for mitochondrial isolation buffer. The final pellet was resuspended in breaking buffer
without phenylmethylsulfonyl fluoride (0.6 M sucrose, 20 mM HEPES-KOH, pH 6.5, 0.1% bovine serum albumin).
Freshly prepared coupled mitochondria
were resuspended in 200 µl of breaking buffer without
phenylmethylsulfonyl fluoride. To start the reaction, 5 mM
potassium Pi, pH 7.4, 1 mM ADP, and 5 mM succinate were freshly added. The mixture was incubated
at 37 °C for 45 min, and the reaction was stopped with 80 mM perchloric acid. Proteins were pelleted, and the
supernatant was assayed for the amount of ATP synthesized by using a
hexokinase/glucose-6-phosphate dehydrogenase coupled assay (18), and
the NADPH generated was measured by an Eppendorf fluorimeter.
Peptides were chosen as follows from the N
terminus of the Qcr7 protein in yeast and the homolog in beef heart and
synthesized by the Alberta Peptide Institute: AGRPAVSASSRWLEG (beef
heart, peptide 1), AGRPAVSASSRWLEGIRKWYYNAAG (beef heart,
peptide 2), PQSFTSIARIGDY (yeast, peptide 3), and
PQSFTSIARIGDYILKSPVLSKL (yeast, peptide 4).
For recording CD spectra, peptides were dissolved at 1 mg/ml in either
10 mM NaCl, 10 mM
NaH2PO4·H2O, methanol, or SDS (to a 30-fold molar excess diluted in 10 mM NaCl, 10 mM NaH2PO4·H2O). Spectra were recorded on a Jasco J-720A spectropolarimeter and scanned
two to three times each from 250 to 190 nm at 25 °C. Base-line spectra for each solvent were subtracted from the peptide spectra.
The yeast strain YSM-qcr7 Immunoblotting with an antibody recognizing the core proteins, the
iron-sulfur protein and the 17-, 14-, and 11-kDa subunits, showed that
no Qcr7 protein is present in mitochondrial membranes isolated from
YSM-qcr7
Link
et al. (11) have previously suggested that the N terminus of
the 13.4-kDa subunit of complex III from beef heart mitochondria forms
an amphiphilic CD spectra were obtained for all four peptides in aqueous buffer,
methanol, and SDS micelles. All peptides formed only a limited amount
of secondary structure in aqueous buffer; however, as shown in Fig.
2, peptides 2 (long beef heart peptide,
top panel, middle) and 4 (long yeast
peptide, bottom panel, middle) have considerable
Many nuclear encoded mitochondrial proteins possess
targeting sequences, 15-70 amino acids long, which are usually located at the N terminus. Some proteins such as cytochromes
c1 and b2 even have
bipartite signal sequences that are cleaved in two steps (19, 20).
Targeting sequences have no obvious homology but are generally rich in
hydrophobic and hydroxylated amino acids, have a net positive charge,
and are able to form amphipathic The Qcr7 protein does not contain a cleavable N-terminal mitochondrial
targeting sequence. However, given the
Growth studies
were performed at 30 °C on two types of solid media to determine
whether any of the mutants are respiration-deficient. Fig.
4 (top) shows that when grown on synthetic
deficient medium with ethanol/glycerol containing 0.1% glucose, the
yeast Qcr7p-
Having established
that the majority of mutant proteins are present in the mitochondria,
it was of interest to find out whether they assemble into a functional
enzyme complex. Mitochondrial membranes from the yeast YSM-qcr7
Strains carrying Qcr7p- When examining the levels of the other
subunits in the complex (Fig. 1), it can be seen that core protein 1 is
present at comparable levels in all the mutant strains and the wild
type. Intermediate iron-sulfur and cytochrome c1
proteins are present in high amounts in the wild type and in somewhat
lower amounts in the strain expressing the Qcr7p- To determine more precisely what the function of the N terminus may be,
we constructed two point mutants in the context of a
Because truncation of the N-terminal seven
amino acids does not impair electron transport, we were interested in
determining whether this segment contributes to the proton conducting
pathways from the matrix phase to the primary protolytic redox center. Studies have been performed on the homologous subunit in beef heart
where cleavage of 7-11 residues from the N terminus was correlated
with decoupling of redox-linked proton pumping (13). To establish a
proton gradient, electron transport has to be intact; hence, of the
strains tested, only Qcr7p- The
growth of the yeast strain expressing the Qcr7p-
Previous studies of the QCR7 gene have shown that this
subunit is an essential component of ubiquinol-cytochrome c
oxidoreductase (10) and that the C terminus may be involved in the
assembly of a functional enzyme complex (25). In the current work, we have investigated the role of the N terminus of the Qcr7 protein with
respect to proton translocation, because previous studies have
postulated that this subunit faces the matrix (13, 26) and is involved
in the uptake of protons from the matrix (13). We were also interested
in determining the importance of this region with respect to assembly
of the bc1 complex and mitochondrial targeting,
because the Qcr7 protein does not contain a cleavable N-terminal signal
sequence but displays features characteristic such sequences. We have
approached the above issues by expressing a number of point and
deletion mutants in the strain YSM-qcr7 We have confirmed earlier findings of Schoppink et al. (10)
that inactivating the QCR7 gene gives rise to a
respiration-deficient strain. Complementation of this strain with the
gene encoding Qcr7p- When complementing YSM-qcr7 The phenotype of the strains containing the Qcr7p- To determine more precisely the function of the N terminus of the
Qcr7p, we mutated the two charged residues Arg-10 and Asp-13 in the
context of a Although the N-terminal 23 amino acids spontaneously assume a largely
All the mutants with the exception of the strain expressing the
Qcr7p- In summary, the N terminus of the Qcr7p of the
bc1 complex may have some characteristic
features of mitochondrial targeting sequences and may indeed facilitate
import. Nevertheless, this region is not essential for the localization
of the Qcr7 protein into mitochondria. This notion is reinforced by
Western blot analyses of cytosolic protein fractions, which do not show
an accumulation of nonimported Qcr7 proteins (result not shown).
Because the steady-state levels of Qcr7p- We thank Joses Jones for help while he was a
summer student. We also thank Dr. Frank Merante, Dr. Sandeep Raha, and
the members of the Jim Friesen laboratory at the Hospital for Sick
Children for many useful discussions.
Genetics and
Biochemistry,
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
7, Qcr7p-
10, Qcr7p-
14, and Qcr7p-
20,
respectively) from the N terminus. In addition, we studied two mutants
containing Qcr7 proteins with point mutations in addition to a
7
truncation, Qcr7p-
7(D13V) and Qcr7p-
7(R10K). All the mutant
proteins with the exception of Qcr7p-
10 were present in the
mitochondria at 30 °C, although most at lower steady-state levels
than the Qcr7p from the strain overexpressing wild type
QCR7. The absence of the Qcr7p-
10 may be the result of
an unstable protein or a decrease in the efficiency of mitochondrial
import due to its compromised amphipathic
-helix and the presence of
a negative charge exposed at the N terminus. Cytochrome c
reductase activities and the amounts of ATP synthesized were comparable
with the wild type in the strain expressing Qcr7p-
7. The strain
expressing Qcr7p-
7(R10K) had an identical phenotype to the one
containing the Qcr7p-
7, whereas strains expressing the Qcr7p-
10,
Qcr7p-
14, Qcr7p-
20, and Qcr7p-
7(D13V) were all
respiration-deficient. Examination of the steady-state levels of
complex III subunits showed that core protein 2, cytochrome c1, the iron-sulfur protein, and the 11-kDa
subunit are reduced in respiration-deficient mutant strains. Results
from deletion analyses indicate that the N-terminal 20 residues (after
Met-1) of the Qcr7 protein are not essential for import into
mitochondria and that the N-terminal seven residues (after Met-1) are
not involved in proton translocation. The results of this work show,
however, that the N terminus of the Qcr7 protein is essential for the
biosynthesis of ubiquinol-cytochrome c reductase.
-helical when
inserted into the membrane-mimetic environment of SDS micelles. By
deletion analysis we found that the N-terminal seven amino acids are
not required for conducting protons and that the N-terminal 20 residues
are not required for import into mitochondria. However, the N terminus
does contain information required for the assembly of
ubiquinol-cytochrome c oxidoreductase.
Materials
, ade 2-1, his 3-11, 15, ura 3-1, leu 2-3, 112, trp 1-1,
can 1-100) was used as the parent strain. Rho
strain (Mat a, ura 1 [rho
][C321R E514°]) was obtained
from ATCC 42209 (donated by L. A. Grivell). Transformation of yeast was
carried out by the Me2SO-enhanced whole cell yeast
transformation method (15). Transformants were selected on minimal
medium containing 0.87% (w/v) yeast nitrogen base, 2% (w/v) glucose,
2% agar and supplemented with the appropriate amino acid mix (0.87 mg/liter). Escherichia coli strain JMED3 was used for
propagation of recombinant DNA constructs in LB broth containing 100 µg/ml ampicillin.
70 °C. Yeast were made competent for
transformation as described in Ref. 15.
vector. Recombinant plasmids were constructed by
digesting pG-3
with SalI and KpnI in two steps
followed by ligation to an accordingly restricted QCR7 gene
generated by polymerase chain reaction mutagenesis. Ligations were
carried out overnight at 12 °C.
7
by using oligonucleotides containing the missense mutations R10K and
D13V, respectively.
vector. DNA was sequenced by the dideoxy chain termination method using
the T7 Sequencing kit from Pharmacia. The sequencing primers used were
in the central portion, 5
-GGGAGTCTTCTTAAAGCGG-3
, and in the 3
downstream region of the coding sequence,
5
-CCGTCGACCGGGTTGTGTGTTCGTGGTG-3
.
,N
-tetramethyl-p-phenylenediamine (0.2 mM) reduced samples were run. Spectra were recorded on
a DW-2a Aminco spectrophotometer from 520-620 nm.
Characterization of Strain YSM-qcr7 Containing the qcr7 Gene
Disruption
, which contains the
chromosomal qcr7
:LEU2 disruption, lacks a
mRNA transcript for the qcr7 gene (results not shown).
The absence of mRNA indicates that a fusion transcript containing
the LEU2 gene within the disrupted qcr7 coding
region is not synthesized, nor is a stable transcript made corresponding to the 5
end of the qcr7 gene. A message of
approximately 450 bases can be seen in YSM-qcr7
when complemented
with QCR7 on the expression vector pG-3
(not shown).
(Fig. 1, lane 5). A band,
however, can be seen in Fig. 1 (lane 6) corresponding to the
Qcr7p from the strain YSM-qcr7
complemented with the wild type
QCR7 gene. This indicates that the Qcr7p is being produced
from the expression plasmid and imported into mitochondria.
Fig. 1.
Composite of Western blot analyses of
mitochondrial proteins from YSM-qcr7 strains overexpressing wild
type and N-terminally truncated proteins Qcr7p-
7, Qcr7p-
10,
Qcr7p-
14, and Qcr7p-
20. Mitochondrial proteins (100 µg)
were dissolved in SDS-polyacrylamide gel electrophoresis buffer
containing dithiothreitol and heated for 3 min at 95 °C. Samples
were run on 16% polyacrylamide gels and then transferred to
nitrocellulose membranes. The blot containing core proteins 1 and 2 and
the blot containing the 17-, the 14- (indicated by arrows),
and the 11-kDa subunits were probed with antibodies detecting all those
subunits as well as the iron-sulfur protein. The two blots containing
cytochrome c1 and the iron-sulfur protein,
respectively, were probed with monoclonal antibodies raised against
each of these subunits. Lane 1, protein from YSM-qcr7
overexpressing Qcr7p-
14; lane 2, protein from YSM-qcr7
overexpressing Qcr7p-
20; lane 3, protein from YSM-qcr7
overexpressing Qcr7p-
10; lane 4, protein from YSM-qcr7
overexpressing Qcr7p-
7; lane 5, protein from YSM-qcr7
;
lane 6, protein from YSM-qcr7
overexpressing wild type
Qcr7 protein. cyt c1, cytochrome
c1; ISP, iron-sulfur protein;
i, intermediate processed forms of ISP or cytochrome c1.
[View Larger Version of this Image (46K GIF file)]
-helix. To confirm this helicity and to compare the
yeast N terminus of the Qcr7 protein with its beef heart homolog, two
peptides corresponding to amino acids 2-16 and 2-26, respectively,
for the bovine sequence and 2-14 and 2-24, respectively, for the
yeast sequence were synthesized (see "Experimental Procedures").
-helix content in methanol. In SDS micelles, the secondary structure
of peptides 2 and 4 are similarly
-helical
(Fig. 2). Spectra and helical content of peptide 4 (bottom panels) strongly resemble that of peptide
1 (short beef heart peptide, spectra not shown), an expected
result, because these two regions can be shown to be homologous upon
comparison of the yeast Qcr7 protein with the beef heart 13.4-kDa
protein (11).
Fig. 2.
Circular dichroism spectra. Peptides
corresponding to the N-terminal 25 amino acids of the beef heart
13.4-kDa subunit and the N-terminal 23 amino acids of the yeast
14.5-kDa subunit were assayed for secondary structure by CD. Peptides
were dissolved at a concentration of 1 mg/ml in buffer (10 mM NaCl, 10 mM
NaH2PO4·H2O), methanol, or SDS to
a 30-fold molar excess (diluted in above buffer). Spectra were recorded
in duplicates at 25 °C on a Jasco J-720A spectropolarimeter.
Top panels, beef heart peptide 2; bottom panels, yeast peptide 4.
[View Larger Version of this Image (23K GIF file)]
-helices when in contact with the
lipid bilayer (21-23).
-helical nature of the wild
type Qcr7p N terminus and its potential for forming an amphiphilic
helix (Fig. 3), we decided to investigate whether this
region contains any information for the mitochondrial localization of
the protein. Accordingly, we constructed four deletion mutants in which
residues 2-8, 2-11, 2-15, and 2-21 (Qcr7p-
7, Qcr7p-
10, Qcr7p-
14, and Qcr7p-
20, respectively) were deleted from the N
terminus (Fig. 3). In each case the initial Met (residue 1) was
retained; however, a number of residues typical for import sequences
were deleted. When expressing the Qcr7p-
7, the Qcr7p-
14, and the
Qcr7p-
20 in the strain YSM-qcr7
at 30 °C, the respective, truncated Qcr7 proteins are synthesized and transported into
mitochondria, as can be seen from the Western analysis in Fig. 1
(lanes 1, 2, and 4). However, it is
evident that the amount of protein present in the mitochondria is lower
in yeast transformed with the deletion mutants than in yeast
transformed with the wild type gene. It is also obvious that in yeast
transformed with the gene encoding Qcr7p-
10, the protein is not
present in the mitochondria (Fig. 1, lane 3). This protein
may be unstable and degraded in the cytoplasm or in the
mitochondria.
Fig. 3.
Helical wheel plots and Qcr7 amino acid
sequence. The N-terminal 18 residues (starting from residue 2) are
plotted (top). WT, wild type. Qcr7 protein was
truncated by residues 2-8 from the N terminus (7). A helical wheel
projection of the wild type shows that all the charged and most of the
polar residues are located on one face of the helix, whereas the
majority of the hydrophobic residues are located on the opposing face.
A helical wheel projection of the Qcr7p-
7 shows that charged
residues are located on one face of the helix, whereas hydrophobic and
polar residues are interspersed throughout.
[View Larger Version of this Image (27K GIF file)]
7 is comparable in size with the wild type, whereas
YSM-qcr7
as well as yeast strains containing Qcr7p-
10, -
14,
and -
20 are pet
mutants, indicative of a
respiratory chain defect. Similarly, when grown on synthetic deficient
medium containing glucose as the sole carbon source (Fig. 4,
bottom), the deficient deletion mutants remain white. The
wild type strain has a red phenotype due to the ade2
mutation, which causes a pigment to accumulate (24). In respiration
incompetent mutants, this pigment is not formed, and the cells retain a
white phenotype. Growth characteristics of the strains containing the
point mutants (not shown) were in agreement with the results from the
enzyme activities. The strain expressing Qcr7p-
7(R10K) was
comparable with the wild type, whereas that containing Qcr7p-
7(D13V)
resembled the profile of the respiration-deficient deletion
mutants.
Fig. 4.
Growth on ethanol/glycerol containing 0.1%
glucose (top) or synthetic deficient medium containing
glucose (bottom). Top, when grown primarily on
the nonfermentable carbon sources ethanol and glycerol, the mutant
strain containing Qcr7p-7 is comparable in size with the wild type
(WT). Mutant strains expressing Qcr7 proteins truncated by
10, 14, or 20 residues (
10,
14, and
20, respectively) are
pet
mutants, indicative of a respiratory chain
defect. Bottom, when grown on synthetic deficient medium
containing glucose as the sole carbon source, respiration-competent
strains turn red due to the ade2 mutation that
causes a pigment to accumulate. Strains expressing Qcr7 proteins
truncated by 10, 14, and 20 residues remain white, further
indicating a respiration defect.
[View Larger Version of this Image (84K GIF file)]
were
devoid of NADH-cytochrome c reductase (Fig.
5) and succinate-cytochrome c reductase
activities. Cytochrome c oxidase activity was diminished by
about 35% when compared with the wild type (not shown).
Fig. 5.
NADH-cytochrome c reductase
activities of mitochondrial membranes from the qcr7
disruption mutant and yeast expressing wild type and mutant Qcr7
proteins. Mitochondrial membranes from yeast were prepared by the
lytic method and assayed at 37 °C for NADH-cytochrome c
reductase activities in 0.1 M potassium Pi
buffer, pH 7.0, containing 94 µM cytochrome c
and 1 mM azide. To start the reaction, 34.2 µM NADH was added as substrate. The strains expressing
wild type (WT) Qcr7p, Qcr7p-7, and Qcr7p-
7(R10K) have
comparable levels of NADH-cytochrome c reductase activities. Strains containing the mutant proteins Qcr7p-
10, Qcr7p-
14,
Qcr7p-
20, and Qcr7p-
7(D13V) are all devoid of complex III-linked
activities.
[View Larger Version of this Image (29K GIF file)]
7 and the Qcr7p-
7(R10K) were assayed for
respiratory chain complex activities and found to have wild type
activities for NADH-cytochrome c reductase (Fig. 5) as well as succinate-cytochrome c reductase and cytochrome
c oxidase (not shown). Expression of qcr7 genes
encoding Qcr7p-
7(D13V), Qcr7p-
14, and Qcr7p-
20, which produce
stable but truncated protein products that are located into
mitochondria, did not restore NADH-cytochrome c reductase
(Fig. 5) or succinate-cytochrome c reductase activities. Cytochrome c oxidase activities were lowered in these
strains by 35% similarly as in YSM-qcr7
.
7. Strains carrying
Qcr7p-
14 and Qcr7p-
20, however, only contain trace amounts of
these intermediates, and they are not detectable in the strain with
Qcr7p-
10 and in YSM-qcr7
. Mature iron-sulfur and cytochrome
c1 proteins are present in all strains, although
in varying amounts. Of the strains examined in Fig. 1, yeast expressing
wild type and
7 Qcr7 proteins contain comparable levels, whereas the
remaining mutants have lower amounts. A similar pattern is seen for the
11-kDa subunit, with the highest levels in the mutants expressing wild
type and Qcr7p-
7, and the lowest levels in the strain expressing the
Qcr7p-
10 and the YSM-qcr7
.
7 deletion. The
mutations targeted two charged residues, Arg-10 (changed to Lys) and
Asp-13 (mutated to Val). From examining the subunit composition of the
strains containing these mutant proteins (Fig. 6), it is
evident that the mutant Qcr7 proteins are all located in the
mitochondria. Interestingly, in the strain expressing Qcr7p-
7(D13V)
the level of 14-kDa subunit is comparable with the wild type, whereas
in the strain with Qcr7p-
7(R10K) the level is similar to that of the
yeast containing the Qcr7p-
7, Qcr7p-
14, and Qcr7p-
20. All the
other subunits are present at comparable amounts in the yeast
containing Qcr7p-
7(R10K) as in the yeast overexpressing the wild
type QCR7 gene. As for the strain expressing the
Qcr7p-
7(D13V), its subunit composition is similar to that of the
14 and
20 deletion mutants; unchanged amounts of core protein 1, however, lower amounts of mature cytochrome c1
and the iron-sulfur protein. Furthermore, the 11-kDa subunit as well as
intermediate cytochrome c1 and iron-sulfur
proteins are nearly undetectable in this strain. From the spectral
analyses it is evident that the strain carrying the conservative
substitution R10K displays near wild type levels of cytochrome
b, whereas the strain expressing proteins with the D13V
mutation contains low and undetectable amounts of cytochrome
b, respectively (spectra not shown). Taken together with the
results from the enzyme activities, the varying levels of complex III
subunits implicate the Qcr7p N terminus in assembly.
Fig. 6.
Composite of Western blot analyses of
mitochondrial proteins from YSM-qcr7 strains overexpressing wild
type Qcr7p, Qcr7p-
7(D13V), and Qcr7p-
7(R10K). Mitochondrial
proteins (100 µg) isolated from the above strains were dissolved in
SDS-polyacrylamide gel electrophoresis buffer containing dithiothreitol
and heated for 3 min at 95 °C. Samples were run on 16%
polyacrylamide gels and then transferred to nitrocellulose membranes.
The blot containing core proteins 1 and 2 and the blot containing the
11- and 14-kDa subunits were probed with an antibody detecting all
those subunits as well as the ISP. The blots containing cytochrome
c1 and the iron-sulfur protein were probed with
monoclonal antibodies raised against each of these subunits. Lane
1, protein from YSM-qcr7
overexpressing wild type; lane
2, protein from YSM-qcr7
overexpressing Qcr7p-
7(R10K);
lane 3, protein from YSM-qcr7
overexpressing Qcr7p-
7(D13V). WT, wild type; cyt c1,
cytochrome c1; ISP, iron-sulfur protein;
i, intermediate processed forms of cytochrome c1 and ISP.
[View Larger Version of this Image (27K GIF file)]
7 produced ATP in the assay system. With
succinate as substrate, the integrity of the proton gradient that is
established by complexes III and III+IV was measured. When ferricyanide
was used as electron acceptor and cytochrome oxidase was inhibited by
azide, the ATP produced was solely due to the action of complex III. In
both cases, the yield of ATP in the mutant with Qcr7p-
7 was
comparable with the wild type (not shown). This indicates that in
yeast, unlike in beef heart, the N-terminal seven amino acids are
unlikely to be involved in proton translocation.
7 Is Temperature-sensitive
7 displays a
different profile at 37 °C when compared with the phenotype at
30 °C. At 37 °C this strain is a pet
mutant with undetectable NADH-cytochrome c reductase
activity. In this case the immunoblotting (Fig. 7)
showed that the
7 protein was not present in the mitochondria
(lane 1) in contrast to the wild type (lane 2).
This result suggests that the Qcr7p-
7 is not sufficiently stable at
37 °C to be imported into mitochondria and therefore degraded in the
cytoplasm or, alternatively, that it is imported and rapidly degraded
in the mitochondria.
Fig. 7.
Western blot analysis performed with
mitochondrial membranes prepared from YSM-qcr7 (grown at 37 °C)
overexpressing Qcr7p-
7 and the wild type Qcr7 protein.
Mitochondrial proteins (100 µg, from cells grown at 37 °C) were
dissolved in SDS-polyacrylamide gel electrophoresis buffer and heated
for 3 min at 95 °C. Samples were run on a 16% polyacrylamide gel
and then transferred to a nitrocellulose membrane. The blot was probed
with a polyclonal antibody that recognizes core proteins 1 and 2, the
iron-sulfur protein, and the 14- and 11-kDa subunits. Lane
1, protein from the strain YSM-qcr7
overexpressing Qcr7p-
7;
lane 2, protein from strain YSM-qcr7
overexpressing the
wild type Qcr7 protein.
[View Larger Version of this Image (40K GIF file)]
in which we inactivated the
chromosomal QCR7 gene and through investigation of the
secondary structure(s) of selected N-terminal peptides by CD
spectroscopy and comparison with their beef heart homologs.
7 restored cytochrome c reductase
activities to wild type levels at 30 °C (Fig. 5) and resulted in a
strain that was no longer respiration-deficient (Fig. 4). This result
indicated that electron transport had been restored, and we proceeded
to examine whether these N-terminal amino acids play a role in proton
transfer by assaying the amount of ATP synthesized in coupled
mitochondria. Efficiency of ATP synthesis in the mutant containing
Qcr7p-
7, however, was observed to be equivalent to the wild type,
and it was concluded that the involvement of the N-terminal seven amino acids in proton translocation and ATP synthesis is unlikely to be
critical.
with the gene encoding Qcr7p-
10, a
substantially different profile is seen. The Qcr7p-
10 is not present
in the mitochondria (Fig. 1), and as a result, complex III is inactive
and the mutant strain is respiration-deficient. Truncation of 10 amino
acids from the N terminus of the Qcr7p results in the exposure of the
negative charge of Asp-13 at the front end (now residue 2 of mature
Qcr7p) of the protein. It is conceivable that the exposure of this
negative charge is detrimental to the import process. Mitochondrial
signal sequences rarely contain negatively charged residues; however,
depending on their location and orientation they may not always be as
disruptive. In addition, it is not clear whether this or any of the
other truncated proteins can form amphiphilic
-helices because the
charged residues are more widely interspersed with hydrophobic residues
and thus result in sequences uncharacteristic for amphiphilic helices.
Alternatively, this result may simply imply that the protein is
unstable and degraded in the cytoplasm or the mitochondria.
14 and Qcr7p-
20
is significantly different from mutants with Qcr7p-
7 and
Qcr7p-
10. Strains expressing these truncated proteins form stable
products that are imported into mitochondria (Fig. 1), albeit to a
lesser degree than the Qcr7p from the wild type strain in which
QCR7 is overexpressed. Significantly, NADH-cytochrome c reductase activities are absent, and these mutants display
a pet
phenotype (Fig. 4), despite the fact
that their truncated proteins are present in the mitochondria at levels
comparable with those of the respiration-competent mutant containing
Qcr7p-
7. Examination of the steady-state levels of the 11-kDa
subunit, as well as the iron-sulfur protein and cytochrome
c1, shows that these subunits are significantly
reduced in strains with Qcr7p-
14 and Qcr7p-
20 when compared with
the wild type strain.
7 deletion. The strain complemented with the
Qcr7p-
7(R10K), which contains a conservative substitution, has a
phenotype comparable with that carrying the Qcr7p-
7 and the wild
type at 30 °C. On the other hand, the strain expressing Qcr7p-
7(D13V) is completely devoid of NADH-cytochrome c
reductase activities and displays a pet
phenotype. Furthermore, when examining the levels of complex III
subunits in this mutant, it can be seen (Fig. 6) that the levels of
cytochrome c1, iron-sulfur protein, and 11-kDa
subunit are reduced, with the level of 11-kDa subunit being lower than in mutants with Qcr7p-
14 and Qcr7p-
20. This stands in contrast to
the level of Qcr7p, which is higher in this mutant than in the others
and compares with the wild type (Fig. 6). Cytochrome b
spectra of strains with the Qcr7p-
7(R10K) and Qcr7p-
7(D13V) correlate with the results from the NADH-cytochrome c
reductase activities; the R10K mutation resulted in a strain with
nearly wild type levels of cytochrome b, whereas cytochrome
b was not detectable in the strain with
Qcr7p-
7(D13V).
-helical conformation (Fig. 2) when inserted into the membrane-mimetic environment of SDS micelles and there is a potential for the wild type N terminus to form an amphiphilic
-helix (Fig. 3),
in strains expressing Qcr7p-
7 and Qcr7p-
7(R10K) the complex functions just as in the wild type at 30 °C. This finding suggests that the N-terminal seven amino acids are not required for the functioning of the complex. However, in strains complemented with the
Qcr7p-
14 and Qcr7p-
20, as well as with Qcr7p-
7(D13V), the truncated Qcr7 protein products are imported into mitochondria at
30 °C to approximately the same level as Qcr7p-
7, but the bc1 complex is not functional.
7(D13V) contain lower than wild type levels of Qcr7p in the
mitochondria. This fact may point toward a function in mitochondrial
import for the N terminus, especially because Qcr7p-
7(D13V) is
present at wild type levels. One could argue that the elimination of
the negative charge, which is an uncommon feature for signal sequences,
causes the N terminus to assume a more typical character for transit
sequences. This may in turn compensate for the truncation of the seven
residues and restore mitochondrial protein levels back to wild type
levels as seen in the mutant with Qcr7p-
7(D13V). If the Qcr7 protein
were to follow the same import pathway as proteins that have a
cleavable N-terminal signal sequence, then deletion of the N terminus
could conceivably cause a number of problems that result in decreased
import. Truncated proteins may not interact with cytosolic chaperones
as efficiently, or alternatively they may not bind as tightly to the
outer mitochondrial membrane receptors. The absence of the Qcr7p-
7
at 37 °C could thus be due to a higher degradation rate at that
temperature, resulting from impaired binding to cytosolic chaperones,
for example. Roise et al. (27) have suggested that part of
the mechanism of import is the perturbation of the phospholipid bilayer
by the surface active amphiphilic helix of the presequence. This
ability of the presequence to cause a local defect in the membranes
would then create a route for the rest of the protein to follow. It is
thus conceivable that the deletion mutants in our study cannot enter the membrane as efficiently, because the amphiphilic character (and
inherent lytic properties) has been decreased by shortening the
presequence. Hence, this again may reduce the efficiency of import.
14, Qcr7p-
20, and
Qcr7p-
7(D13V) are comparable with or higher than the level of
Qcr7p-
7, the pet
character of the deficient
mutants cannot be attributed to the decreased levels of Qcr7p. We
therefore conclude that the respiratory chain defect of these mutants
is the result of the lowered levels of complex III subunits. This
implicates the N terminus of the Qcr7p in assembly of the
bc1 complex. More precisely, this region may
bind cytochrome b and/or the 11-kDa subunit. Hence,
substitution of critical residues in the N terminus of the Qcr7 protein
may prevent the formation of the subcomplex consisting of cytochrome b and the 11-kDa and the 14-kDa subunits. This would then
lead to the observed lowered steady-state levels of complex III
subunits with the concurrent loss of complex III-linked enzyme
activities.
*
This work was supported in part by grants from the Medical
Research Council of Canada (to B. H. R. and C. M. D.) and National Institutes of Health Grant GM-20379 (to B. L. T.).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.
To whom correspondence should be addressed: Hospital for Sick
Children, 555 University Ave., Toronto, ON M5G 1X8, Canada. Tel.:
416-813-5989; Fax: 416-813-4931; E-mail:
bhr{at}resunix.ri.sickkids.on.ca.
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.