From the
Subunit 6 of the mitochondrial cytochrome bc
QSR1 was cloned by
rescuing the synthetic lethality of a qsr1-1 mutant.
QSR1 encodes a 25.4-kDa protein which is 65% identical to a
protein encoded by QM, a highly conserved human gene which has
been implicated in tumorigenesis. In mammals QM is
down-regulated during adipocyte, kidney, and heart differentiation, and
in Nicotiana the homolog of QM is also down-regulated
during differentiation. When one chromosomal copy of QSR1 was
deleted in a diploid yeast strain, haploid spores derived therefrom and
carrying the deletion were unable to grow on fermentable or
non-fermentable carbon sources. Although QCR6 allows the
qsr1-1 mutant to grow, it will not substitute for
QSR1, since the deletion of QSR1 is lethal even if
QCR6 is present. These results indicate a novel genetic
relationship between a subunit of the mitochondrial respiratory chain
and an essential gene in yeast which is homologous to a gene implicated
in differentiation in other eukaryotes.
The cytochrome bc
Subunit 6 of
the bc
The fact that the absence of subunit 6
modifies the kinetic properties of the bc
We have cloned
and characterized QSR1, a gene which complements a
qsr1-1 mutant. QSR1 is an essential gene in
yeast and encodes a highly conserved protein which has been implicated
in differentiation in higher eukaryotes.
Saccharomyces cerevisiae strains W303-1A (MATa,
ade2-1, his3-11, 15, ura3-1, leu2-3, 112,
trp1-1, can1-100) and W303-1B (MAT
The strain MES11-13A was crossed to
FY23, after which the diploid strain was sporulated and one haploid
strain, MMY-15D (MAT
Polyclonal antibodies to
the GST-QSR1 fusion protein were raised in rabbits by
Cocallico Biologicals (Reamstown, PA). Preimmune sera were screened to
identify rabbits which did not have antibodies to yeast proteins. When
diluted 1:2000 the immune serum recognized a single protein which
migrated with an apparent molecular mass of 28 kDa when 10 µg of
total yeast proteins were analyzed by Western analysis
(22) .
Yeast proteins which migrated with apparent molecular masses of
25-32.5 kDa were used to affinity purify anti- QSR1 protein antibodies for immunofluoresence microscopy
(23) .
MES12-1D was mutagenized with EMS
to a 60-75% kill ratio, and the cells were then plated at 400
colonies/plate on either YPD plates, to look for mutations that cause
non-sectoring on a fermentable carbon source, or onto D-plates, to
search for mutations that cause non-sectoring only on non-fermentable
carbon sources. Approximately 60,000 yeast colonies were screened.
Strains that sectored on 10% dextrose plates but failed to sector on
non-fermentable carbon source plates were searched for, but none were
found. Thirty-five strains that failed to sector on any carbon source
were saved as non-sectoring mutants. Twenty-eight of these mutant
strains failed to regain sectoring after being crossed to a parental
strain of opposite mating type and were thus considered genomic
ade3-26 revertants or omnipotent suppressors. In order
to examine whether the non-sectoring phenotype was due to a mutation
which enhanced stability of the high copy plasmid, we tested the
remaining seven non-sectoring mutants for growth on plates containing
5-FOA. Two of the seven non-sectoring mutants were able to grow,
indicating they do not require the plasmid carrying QCR6 for
viability, but rather maintained it with high efficiency when incubated
on YPD plates.
After backcrossing and sporulation, non-sectoring
mutants of opposite mating type were crossed. The parents of diploids
which failed to regain sectoring were placed into the same
complementation groups. The mutants in these two complementation groups
were designated qsr1 and qsr2, for quinol-cytochrome
c reductase subunit requiring. Again, the non-sectoring
phenotype of all of these mutants was independent of carbon source. In
addition, both qsr1 and qsr2 grew considerably slower
than the MES12-1D and MMY-15D parental strains. QCR6 Copy Number Requirements of the qsr1-1
Mutant-MES12-1D carries QCR6 on a 2-µm plasmid
and is an extensive overproducer of subunit 6 (see ``Experimental
Procedures''). In order to measure the extent of overproduction
required to rescue the qsr1-1 mutant and to establish
that QCR6, and not some other element on the plasmid, is
covering the lethality of the qsr1-1 mutant, plasmids
pMES34 and pMES36, centromeric and 2 µ-m plasmids, respectively,
both marked with TRP1 and carrying the QCR6 gene
(), were transformed into the qsr1-1 mutant.
The TRP1-marked plasmids carrying QCR6 were exchanged for pMES32 by isolating Trp
The qsr1-1 mutation can also
be covered by a single chromosomal copy of QCR6. The
qsr1-1 mutant was backcrossed with the wild-type strain
W303 to obtain a strain with a wild-type chromosomal QCR6 locus ( i.e. Leu
Although there is no growth advantage from
overexpression of the QCR6 protein when the qsr1-1 mutant is grown on dextrose medium, there is a pronounced effect
of QCR6 copy number when the mutant is grown on
ethanol-glycerol. On the non-fermentable carbon source, the
qsr1-1 mutant grows more slowly than the parental strain
when complemented by a high copy QCR6 plasmid, but the mutant
does not grow on these carbon sources when QCR6 is expressed
from the single chromosomal locus (results not shown). This difference
probably relates to the dual function of QCR6 protein and
sequestration of limited amounts of that protein by other subunits of
the bc
Two of the complementing plasmids contained
genomic fragments which were overlapping for approximately 7 kbp and
which did not contain QCR6. A restriction map of one of these
clones, pMY1, is shown in Fig. 2. Sequence analysis from the
multicloning site into the 5` end of the insert in pMY1 yielded a
stretch of 158 nucleotides which is identical to a sequence within the
open reading frame of RGR1, a previously mapped gene which is
required for glucose repression
(28) . On this basis and
subsequent sequencing of QSR1, we conclude that QSR1 is located on the right arm of chromosome XII, approximately 4 kbp
from RGR1 as shown in Fig. 2. The orientation of
RGR1 and QSR1 relative to the centomere have not been
established.
There are no
obvious conserved sequence motifs in the QSR1 protein.
However, two regions of the protein, from Leu
The QSR1/qsr1
That deletion of QSR1 was responsible for the
lethality was demonstrated by rescuing the lethality by a plasmid born
copy of QSR1. The HFF10 and HFF12 diploids were transformed
with pHFF22, a CEN/ARS, URA3, QSR1 plasmid (),
and the Ura
These
results indicate that the presence of QCR6, which encodes a
regulatory subunit of a mitochondrial respiratory enzyme complex, is
allowing the qsr1-1 mutation to function. One
possibility is that the qsr1-1 mutation alters energy
metabolism of the yeast cells and that QCR6 compensates by
up-regulating the rate of mitochondrial respiration. However, we found
that the qsr1-1 mutant is covered by QCR6 in
rho° cells grown on YPD medium, which lack respiration and
mitochondrial ATP synthesis due to the absence of the mitochondrial
genome (results not shown). Thus, the energy requirements of the
qsr1-1 mutant can be met by fermentation.
Subunit 6 of the cytochrome bc
Since QCR6 encodes
a protein whose only known function is in a respiratory enzyme complex,
we expected to find mutants that required QCR6 to grow only on
non-fermentable carbon sources. For example, yeast strains in which
both QCR6 and CYC1 are deleted are petite, while
strains in which either gene alone is deleted are not
(35) .
Similarly, we have observed that combined deletions of QCR6 and QCR9, the gene for subunit 9 of the bc
We cloned QSR1 from a yeast genomic library by
complementing the qsr1-1 mutant for ability to grow
without QCR6. QSR1 is an essential yeast gene which encodes a
protein which is 65% identical to a protein encoded by a human cDNA
named QM (31) . The human QM cDNA was isolated
by subtractive hybridization between a tumorigenic cell line derived
from Wilms' tumor and a transfected, non-tumorigenic form of the
same cell line. The QM transcript is underexpressed in the
tumorigenic cells.
QM encodes a protein which has been
highly conserved throughout evolution
(32) . A mouse cDNA
encoding a protein homologous to the QM protein has been
cloned by differential hybridization of preadipocytes and adipocytes
(36) . The QM mRNA decreases 80% in primary cultures of
adipocytes compared to preadipocytes and does not decrease when
differentiation is blocked by growth factors. The QM mRNA also
decreases markedly in adult heart and kidney
(31) . In
Nicotiana tabacum QM transcription is turned on when seeds
germinate, highly expressed in growing cell suspensions, but absent in
adult leaves
(37) . There is also a preliminary report that the
QM transcript fluctuates during differentiation of Arabadopsis (38) . Taken together these results suggest that QM is down-regulated during differentiation in a variety of
eukaryotic cells.
A chicken cDNA encoding a protein identical to
QM has been cloned by screening an expressed cDNA library for
proteins which interact with c-Jun
(34) . Jif-1, the
``Jun interacting factor'' from chicken, which is 94%
identical to the protein encoded by human QM, binds Jun and
inhibits DNA binding and transactivation by Jun. From this study it was
suggested that QM is a component of a system that modulates
the activity of transcription factors through protein complex
formation. However, from the steady state mRNA levels, we could find no
evidence of a transcriptional interaction between QSR1 and
QCR6. In addition, we find that the protein encoded by
QSR1 is concentrated in the yeast cytoplasm and appears to be
excluded from the nucleus, although we cannot exclude the possibility
that a subpopulation of QSR1 might traffic to the nucleus
under some specific conditions that we have not tested.
At the
present time, the only mutation known in the QM protein family
is the G194D allele we have characterized in the qsr1-1 allele. The G194D allele of qsr1-1 is semidominant,
as indicated by the retention of the QCR6 plasmid when
heterozygous qsr1-1/QSR1 diploids are plated onto
non-selective medium. This means that the lethality of the
qsr1-1 allele is only partially compensated by either
QSR1 or QCR6. Glycine residues very often mark a turn
or terminate helices in proteins and are considered a punctuation in
peptide sequences. It is possible that replacement of glycine 194 by
aspartic acid would induce a conformational change in the QSR1 protein and modify its function. It has been found that Jif-1 can homodimerize
(34) . If yeast QSR1 protein also
forms an oligomer, the qsr1 (G194D) protein may form a
heterodimer with the QSR1 protein, thus explaining the
semidominant behavior.
Although glycine 194 is totally conserved,
this amino acid is in a region of the protein that is the most
divergent among species
(32) . The repetitive peptides
167-181 and 200-216 which flank glycine 194 in the QSR1 protein are not found in the other proteins of the QM family thus
far identified, which allows for the possibility that this region
reflects a function of the QSR1 protein specific to yeast.
Secondary structure predictions suggest that this motif forms two
Our finding that
QCR6 covers the qsr1-1 mutation raises the
possibility that subunit 6 of the mitochondrial cytochrome
bc
Our results indicate that the QSR1 protein is concentrated
in the cytoplasm. If the QCR6 protein interacts with the
qsr1-1 mutant protein, as suggested below, we would
predict that a population of subunit 6 of the bc
At present we do not understand how
QCR6 covers the qsr1-1 mutation. QCR6 can not substitute for QSR1, since deletion of QSR1 is lethal when QCR6 is present. In some manner, the
presence of QCR6 allows the qsr1-1 mutant to
function. Transcription of QSR1 is not affected by deletion or
over-expression of QCR6. One possibility is that there is an
interaction between the QCR6 and QSR1 proteins. If an
interaction between these two proteins is involved, it is significant
that suppression of the qsr1-1 phenotype by QCR6 appears to be specific. QCR6 was the only gene, other
than QSR1, that we cloned by complementing the
qsr1-1 mutant, and we isolated QCR6 eight times
more frequently than QSR1. A single chromosomal copy of
QCR6 will cover the qsr1-1 mutation, and this
is the only gene for a mitochondrial protein which will do so, since
the qsr1-1 mutant contains functional chromosomal copies
of all of the other genes required for mitochondrial respiration and
oxidative phosphorylation. The observation that overexpression of the
QCR6 protein does not enhance the growth rate of the
qsr1-1 mutant on dextrose medium in comparison to a
chromosomal copy of QCR6 also argues that the speculated
interaction between the qsr1-1 and QCR6 proteins is specific, since a nonspecific interaction would likely
be enhanced by overexpression of either protein.
Our discovery that
the qsr1 and qsr2 mutants require QCR6 to
grow on fermentable and non-fermentable carbon sources indicates a
genetic relationship between a protein of the mitochondrial respiratory
chain and two genes or gene products which are essential under
respiring and non-respiring growth conditions. Further genetic and
biochemical studies in yeast should allow us to better understand the
nature of this relationship.
The nucleotide
sequence(s) reported in this paper has been submitted to the
GenBank/EMBL Data Bank with accession number(s) U06952.
We thank Martha Jiminez and Pascale Tron for technical
assistance and Tim Brown and Henry Riley for their helpful comments and
stimulating discussion. We also thank Drs. P. Hieter, R. Rothstein, R.
S. Sikorski, A. Tzagoloff, and F. Winston for yeast strains and
plasmids.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
complex regulates the activity of the bc
complex in Saccharomyces cerevisiae but is not essential
for respiration. To test whether QCR6, the nuclear gene which
encodes subunit 6, might be functionally redundant with any other
gene(s), we screened for mutations in yeast genes which are essential
when the otherwise non-essential QCR6 is deleted from the
yeast chromosome. We obtained such q uinol-cytochrome c reductase subunit-requiring mutants in two complementation
groups, which we named qsr1 and qsr2. The qsr mutants require QCR6 for viability on fermentable and
non-fermentable carbon sources, indicating that QCR6 is
covering lethal mutations in qsr1 and qsr2, even when
the yeast do not require respiration.
complex is an oligomeric
energy transducing enzyme occurring in specialized membranes of
numerous respiratory and photosynthetic organisms
(1) . The
mechanism by which the bc
complex transfers
electrons from ubiquinol to cytochrome c requires three redox
proteins, cytochrome b, an iron-sulfur protein, and a
membranous cytochrome c. While only these three redox subunits
are present in the bc
complexes of numerous
bacteria
(2, 3, 4, 5) , the
bc
complexes of mitochondria contain 7 or 8
additional subunits lacking prosthetic groups
(6, 7) .
The functions of these supernumerary subunits in the mitochondrial
bc
complexes are largely unknown.
complex is an acidic protein which resides
on the electropositive surface of the complex in association with
cytochrome c
(8) and regulates
half-of-the-sites reactivity of the dimeric bc
complex in response to protonmotive force
(9) . Disruption
or deletion of QCR6, the nuclear gene for subunit 6
(10) , does not impair growth of yeast on respiratory substrates
at temperatures up to 35 °C
(9, 11, 12) ,
although it does result in a temperature-sensitive petite phenotype at
37 °C
(13) .
complex
without impairing respiration raises the possibility that deletion of
QCR6 might be covered by another, functionally redundant gene.
To test this possibility, we screened for mutants that require the
otherwise nonessential subunit 6 and identified quinol-cytochrome c reductase subunit requiring mutants in two complementation groups.
Surprisingly, the qsr mutants require QCR6 to grow
under conditions where respiration is not required.
Materials
L-Amino acids, uracil, and
adenine sulfate were obtained from Sigma. Phenol was purchased from
Mallinckrodt. Yeast extract, peptone, tryptone, and yeast nitrogen base
without amino acids were from Difco. Restriction enzymes and T4-DNA
ligase were purchased from New England Biolabs or Life Technologies,
Inc.
,
ade2-1, his3-11, 15, ura3-1, leu2-3, 112,
trp1-1, can1-100) were from Dr. R. Rothstein (Columbia
University). S. cerevisiae strains YPH499 (MATa,
ura3-52, lys2-801 (amber), ade2-101 (ochre),
trp1-
63, his3-
200, leu2-
1) and YPH500 (MAT
,
ura3-52, lys2-801 (amber), ade2-101 (ochre),
trp1-
63, his3-
200, leu2-
1) were from Dr. R. S.
Sikorski (John Hopkins University). W303-1A
W303-1B
and YPH499
YPH500 diploid strains were used to construct
deletion strains as described below. Strain SL210-2A (MAT
his5-2, lys2-1, trp1-1, tyr7-1, ilv1-1,
leu2-1, ade3-26, met8-1) was obtained from the Cold
Spring Harbor Collection (Cold Spring Harbor, NY). Strain FY23
(MATa , ura3-52, trp1
63, leu2
1) was obtained
from Dr. F. Winston (Harvard Medical School). Yeast strains constructed
in this study are described in . Escherichia coli strain DH5-
( psi80d lacZ
M15 ,
endA1 , recA1 , hsdR17 (r
m
) , supE44 ,
thi-1 ,
, gyrA96 ,
relA1 ,
( laczya- argF)U169 , F
(14) was used to amplify plasmids.
Construction of the Plasmid pMES32
The sectoring
plasmid pMES32, shown in Fig. 1, was constructed from pMES20,
which contains a 1.9-kbp SphI fragment encompassing QCR6 (9) in the yeast episomal plasmid YEp352
(15) . A
5.3-kbp BamHI/ SalI fragment containing the ADE3 gene was isolated from pSB32
(16) and cloned into the
BamHI/ SalI sites of pMES20 to create the plasmid
pMES32 ().
Figure 1:
Plasmid sectoring protocol used to
isolate qsr mutants. The parental yeast strain, MES12-1D, has
mutations in ade2, ade3, ura3, trp1, and
qcr6 and carries a yeast episomal plasmid, pMES32, that
contains URA3, ADE3, and QCR6. Yeast
carrying the plasmid are red due to the ade2 mutation, which
causes accumulation of a red pigment. In the absence of selection the
plasmid is spontaneously lost at low frequency, giving rise to red and
white sectored colonies. Mutants that require the plasmid for viability
form solid red, non-sectored colonies.
Mutagenesis of Yeast
Yeast grown to late
exponential phase (10cells/ml) on selective minimal
dextrose media were resuspended in 1 ml of 100 mM NaPi, pH 7.0
at 10
cells/ml. EMS
(
)
was added to a
concentration of 3%, after which the cells were incubated on a rotator
wheel for 1 h at 30 °C. EMS was then inactivated by adding 50 ml of
5% sodium thiosulfate. The cells were collected by centrifugation,
washed once with distilled water, and then spread on selection plates.
This method of mutagenesis gave a 30-50% survival rate when
compared to yeast treated without EMS
(17, 18) .
Screening for Sectoring Mutants
The sectoring
strain MES12-1D (MATa, his5-2, ura3-1,
leu2-3, 112, trp1-1, ade2, ade3-26, qcr61::LEU2, pMES32 [ADE3, URA3, QCR6]) was constructed by
crossing MES8 (MATa, ade2-1, his3-11, 15, ura3-1,
leu2-3, 112, trp1-1, can1-100, qcr6
1::LEU2)
to SL210-2A (MAT
, his5-2, lys2-1,
trp1-1, tyr7-1, ilv1-1, leu2-1, ade3-26,
met8-1). The diploid was sporulated and haploid cells that
were ade2
, ade3
,
ura3
, Leu2
were saved. Two such
haploid strains, MES11-13A (MAT
, ade2, leu2-3,
112, ade3-26, ura3-1, trp1-1, met8-1,
lys1-1, ilv1-1, his3-11, 15, qcr6
1::LEU2)
and MES11-14A (MATa, ade2, ade3-26, ura3-1,
trp1-1, his5-2, leu2-3, 112, qcr6
1::LEU2),
were transformed with the plasmid pMES32 (see below). These were then
crossed, and the diploid was sporulated. One haploid strain from this
cross, MES12-1D, was selected for mutagenesis due to its ability
to produce a dark red color when maintaining the plasmid pMES32.
MES12-1D also grew well on non-fermentable carbon sources and did
not agglutinate. To test for high copy maintenance of the plasmid in
the strain MES12-1D, genomic DNA was purified and Southern
analysis was performed. The strain contained at least 35 copies of the
1.9-kbp SphI fragment encompassing QCR6 and a single
3.7-kbp SphI fragment remaining from the LEU2 marked,
deleted chromosomal copy of QCR6 (18) . Hence the only
source of QCR6 in this strain is from the plasmid, pMES32, and
this plasmid is maintained at a substantially high copy number. The
strain MES12-1D was also subjected to Northern analysis for
QCR6 transcripts, and as shown below, the QCR6 transcript is present in substantially greater amounts in the
strain expressing pMES32.
, ade2-1, ade3-26,
leu2-3, 112, trp1-1, ura3-52, lys1-1,
qcr6
1::LEU2), was then used for backcrossing and
complementation analysis. MES12-1D was mutagenized as described
above and plated on D-plates (1% yeast extract, 2% peptone, 4% ethanol,
3% glycerol, 0.1% dextrose) at a density of 400 colonies/plate. Plates
were incubated at 30 °C for 1 week and then at room temperature for
4-5 days. A total of 60,000 colonies were screened. Colonies that
remained solid red were collected. These potential mutants were
streaked onto minimal dextrose plates to recover, and then restreaked
on D-plates to retest the non-sectoring phenotype. Non-sectoring
mutants were crossed to MMY-15D to test recessiveness of the mutation.
Complementation Grouping
Five non-sectoring
strains from the screening described above were crossed to MMY-15D, and
15 or more tetrads from each cross were analyzed for segregation of the
non-sectoring phenotype. Three of the strains always segregated the
non-sectoring phenotype in 2:2 fashion, and these non-sectoring mutants
also grew more slowly than the parental strain at 15 °C on both
fermentable and non-fermentable carbon sources. Mutant spores were then
crossed to each other for assignment of complementation groups. Two
complementation groups, qsr1 and qsr2, were
determined by complementation analysis.
Cloning of QSR1
QSR1 was cloned by a
two-step plasmid shuffling procedure in which the requirement of the
qsr1-1 mutant for the QCR6 plasmid was
complemented from a yeast genomic library. The library contains
8-10 kbp inserts cloned into pRS200, a single copy plasmid
carrying TRP1 as a prototrophic marker. The library in pRS200
and the pRS200 plasmid without inserts were provided by Dr. P. Hieter
(Johns Hopkins University). The qsr1-1 mutant was
transformed with the library and approximately 13,000 Trptransformants were selected on plates containing minimal medium.
The Trp
transformants were then replica plated onto
plates containing 5-FOA to select for cells carrying a library plasmid
that allowed the mutant to live without the URA3 plasmid
carrying QCR6. Transformants able to grow on 5-FOA-containing
plates were cultured in YPD (1% yeast extract, 2% peptone, 2%
dextrose), and the complementing plasmid DNA was isolated and subjected
to restriction analysis.
Sequencing of the QSR1 Gene
Restriction fragments
from the plasmid pMY7 (see below) were subcloned into pRS200, and the
DNA was sequenced on both strands by chain termination using
double-stranded DNA templates. M13 universal and reverse primers for
sequencing were obtained from the Molecular Genetics Center, Dartmouth
College. Nucleic acid and protein data bases were searched by the
FASTA-FASTP algorithms using the Genetics Computer Group Sequence
Analysis software package
(19) on a Digital VAX117-80
computer at Dartmouth College Computing Center. MacVector Software
(IBI) was used for DNA and protein analysis. Sequencing of the qsr1-1 Mutation-The genomic
qsr1-1 locus was amplified by PCR, using
oligonucleotides P1(GTAGCGGTTATTTCCGTGGGGTGC) and P2
(GGATATATCACTACCCAACATGC) as primers. After purification, the PCR
product was directly sequenced using the CircumVent kit (New England
Biolabs) and oligonucleotides synthesized from various internal
sequences.
Construction of QSR1 Deletion Strains
A 3.0-kbp
EcoRV fragment, beginning approximately 670 bp 3` of QSR1 and extending into the polylinker of pRS200, was deleted from pMY3
to form pHFF15. An 825-bp SpeI- EcoRI fragment
carrying the first 636 bp of the QSR1 reading frame and 189 bp
of sequence 5` of the reading frame was removed from pHFF15 and
replaced with a 1.8-kbp XbaI- EcoRI fragment carrying
the yeast HIS3 gene
(20) . From the resulting deletion
plasmid (pHFF19), a 3.3-kbp XbaI- EcoRV fragment was
released which carried the HIS3 gene flanked by genomic DNA
from the QSR1 region. This fragment was transformed into a
his3diploid yeast strain (W303-1A
W303-1B or YPH499
YPH500), and His
transformants were selected. The QSR1/qsr1
1::HIS3 diploid strain derived from W303-1A
W303-1B
was named HFF10; that derived from YPH499
YPH500 was named
HFF12. Insertion of HIS3 at the QSR1 locus was
verified by Southern analysis of genomic DNA.
Complementation of the QSR1 Deletion Strain
HFF10
was transformed with pHFF22, a CEN/ARS vector containing a
3.4-kbp KpnI- ClaI QSR1 fragment and a
URA3 gene as a selectable marker, and uracil prototrophs were
selected. After sporulation on acetate medium, asci were dissected by
micromanipulation, and spores were allowed to germinate on YPD.
His, Ura
spores were selected and
assayed for viability on YPD and on SD medium (0.67% yeast nitrogen
base, 2% dextrose, 1% yeast extract, 2% peptone, 4% ethanol, 3%
glycerol, 0.1% dextrose) containing 5-FOA.
Northern Analysis
Total RNA was isolated from
cells in logarithmic phase growing on the carbon source indicated in
the figure legends, separated by electrophoresis on 1% agarose, and
transferred to nitrocellulose. The blots were probed with a
P
-labeled 1.2 kbp SpeI- ClaI
fragment of QSR1 excised from pMY7. Actin mRNA was probed as a
control for RNA loading, using a 1.6-kbp
EcoRI- HindIII fragment of the actin gene
( ACT1).
Preparation of Antibodies
A plasmid for expressing
a GST-QSR1 fusion protein was constructed by ligating the
0.5-kbp EcoRI fragment from the QSR1 open reading
frame into the EcoRI site of pGEX-1T to create the
plasmid pHFF17, which was amplified in DH5-
. E. coli cultures were grown to mid-log phase on LB (Luria
broth)-ampicillin medium, then induced with 1 mM IPTG for 3 h.
The insoluble GST-QSR1 fusion protein was purified from
inclusion bodies by SDS-polyacrylamide gel electrophoresis and
recovered by electroelution
(21) .
Microscopy
Cells were prepared for microscopy as
described by Pringle and co-workers
(23) . W303-1B cells
were grown in YPD to an optical density of 0.4 at a wavelength of 600
nm. Formaldehyde was added to the YPD to a final concentration of 3.7%,
and the cells were incubated at room temperature for 1 h. The cells
were pelleted and washed three times in solution A (1.2 M
sorbitol, 50 mM potassium P, pH 7.5). Spheroplasts
were prepared by treating the cells with 15 µg/ml yeast lytic
enzyme (ICN Biochemicals) in 0.3%
-mercaptoethanol for 30 min at
37 °C. Cells were mounted on a 1% polyethylenimine coated slide and
fixed in place with 1.8% formaldehyde. Slide wells were washed twice
with TBS (150 mM NaCl, 20 mM Tris, pH 7.5) containing
0.1% BSA and once with TBS containing 0.1% BSA and 0.1% Nonidet P-40
(Calbiochem). Cells were incubated with affinity purified anti-QSR1
protein antibody diluted 1:25 in TBS with 0.1% BSA and incubated for 2
h. The cells were washed as before incubation with the primary antibody
except that the first wash contained 1 µg/ml
4`-6-diamidino-2-phenylindole. Cells were then incubated with a
fluorescein-conjugated goat anti-rabbit IgG secondary antibody for 1 h
and washed again. The cells were then mounted and the coverslip sealed
with clear nail polish. Cells were viewed at
1200 magnification
with a Nikon Optiphot microscope with the appropriate optics or
fluorescence filter and photographed at
375 magnification with
Kodak T-Max 400 film.
Isolation of QSR Mutants
The screen for mutants
exhibiting synthetic lethality with the QCR6 deletion is based
on a plasmid loss assay as depicted in Fig. 1. We transformed a
2-µm plasmid carrying URA3 as a selective marker, ADE3 as a screening marker, and QCR6 into a ura3,
ade2, ade3 yeast strain lacking QCR6. In the
absence of any selection, this plasmid is lost at a rate of about
10%/generation, and the resulting cells grow as white, plasmid-free
sectors in red colonies (Fig. 1). Any mutant cells that require
QCR6 will retain the ADE3-containing plasmid, and the
colony will be red. This type of sectoring screen was originally
developed to monitor mitotic stability of minichromosomes
(24, 25) and has also been used to look for haploid lethal mutations
(23) and mutations exhibiting synthetic lethality with the
cyclin genes
(27) .
transformants, which were then replicated onto plates containing
5-FOA to select for loss of the plasmid pMES32, which carries a
URA3 gene in addition to QCR6. The qsr1-1 mutant was able to grow on 5-FOA plates after transformation with
either the centromeric or the 2-µm plasmid carrying QCR6 (results not shown). This establishes that qsr1-1 can be complemented by either a single copy or high copy plasmid
carrying QCR6.
) and which had lost the
plasmid carrying QCR6 ( i.e. Ura
).
Two Leu
, Ura
spores from the first
backcross were used to form diploids with W303, and these were again
sporulated. The slow growth phenotype segregated in a 2:2 manner
through both backcrosses. After the second backcross, the two
Leu
,Ura
spores, designated
MMY3-3B and MMY3-3C, exhibited the slow growth phenotype of
the original qsr1-1 mutant on YPD medium, indicating
that a chromosomal QCR6 can cover the mutation in the absence
of the plasmid carrying QCR6 (see Fig. 3, below). The qsr1-1 Mutant Is Semidominant-During the backcrossing of
the qsr mutants, we observed that when the
qsr1-1, QCR6 haploid is mated to a parental
strain carrying a wild-type QSR1 allele (MMY-15D), the
resulting diploid fails to regain a normal sectoring phenotype. The
diploid colonies are mostly red with very small white sectors appearing
after a long period of incubation on YPD (results not shown).
Apparently the sectoring plasmid containing QCR6 is conferring
an advantage to the qsr1-1/QSR1 diploid, even though the
qsr1-1 mutation is covered by a wild-type chromosomal
copy of QSR1. The qsr1-1 mutation is also only
partially covered in the haploid strain by QSR1 on a CEN
plasmid, as indicated by the fact that the QSR1-complemented
strain grows with a doubling time of 3.2 h on YPD at 30 °C,
compared to 1.6 h for that of the parental strain, MMY-15D (results not
shown). This semidominant behavior of the qsr1-1 mutation suggests that it is a missense mutation, resulting in an
altered protein which is non-functional unless covered by QCR6 or QSR1.
Figure 3:
Complementation of qsr1-1.
W303-1A, the parental strain, and MMY3-3B, a
qsr1-1 mutant which has a chromosomal copy of
QCR6, will grow on medium containing 5-FOA. The
qsr1-1 mutant has QCR6 deleted from its
chromosomal locus and depends on a URA3, QCR6 plasmid for
viability. The qsr1-1 mutant will not grow on medium
containing 5-FOA, which selects for loss of the URA3 plasmid.
pMY3, a subclone which carries QSR1 on a TRP1 plasmid
(see Fig. 2), will allow the qsr1-1 mutant to live
without the URA3, QCR6 plasmid.
The qsr1-1 mutation is also
covered by QCR6, and the doubling time of the cells on YPD
medium is the same as when the mutant is covered by QSR1,
whether QCR6 is expressed from its chromosomal locus or from a
high copy plasmid (results not shown). If the QCR6-encoded
protein stabilizes the qsr1-1 mutant protein by a direct
interaction, the latter observation suggests that this interaction is
specific, since no growth advantage derives from overexpression of the
QCR6 protein.
complex, which are expressed on
ethanol-glycerol, as discussed below.
Complementation Cloning of QSR1
QSR1 was
cloned by complementing the ability of the qsr1-1 mutant
to grow without the QCR6 plasmid by a two-step, plasmid
shuffle procedure as described under ``Experimental
Procedures.'' From 13,000 Trptransformants of
the qsr1-1 mutant, 19 colonies were able to grow on
medium containing 5-FOA. Restriction analysis and hybridization of
these 19 colonies with a labeled DNA fragment containing QCR6 indicated that 17 of the complementing plasmids contained genomic
fragments encompassing QCR6. This cloning of QCR6 confirmed the validity of the cloning protocol. However, since our
cloning strategy employed a centromeric plasmid library, it leaves open
the possibility that other, high copy suppressors of qsr1-1 might exist.
Figure 2:
Restriction map of qsr1-1
complementing clone. The original complementing clone, pMY1, was
digested with restriction enzymes to deduce a restriction map.
Subclones pMY2 to pMY7, containing segments of the original
complementing DNA indicated by the thick lines, were tested
for complementation of the qsr1-1 mutation as shown in
Fig. 3. The + or sign to the right indicates whether or
not the subclone complemented the mutation. The black box designates the QSR1 open reading frame. The 1-kbp size
marker refers to distances on the subclones in plasmids pMY1 through
pMY7. The 1.2-kbp SpeI- ClaI insert in pMY7 is
expanded at the bottom of the figure. The abbreviations are:
A, AccI; B, BamHI; BII,
BglII; C, ClaI; E, EcoRI;
H, HindIII; K, KpnI; P,
PstI; R, EcoR5, S, SpeI;
Sau, Sau3A; Sa, SalI; X,
XbaI.
Restriction fragments of pMY1 were subcloned into
pRS200 to generate the plasmids pMY2-pMY7 (Fig. 2), which were
assayed for complementation of qsr1-1 on plates
containing 5-FOA as shown in Fig. 3. The shortest fragment that
complemented the qsr1-1 mutation was the 1.2-kbp
SpeI- ClaI fragment in the subclone pMY7. Although the
SpeI- ClaI fragment allowed the mutant to grow without
the QCR6-containing pMES32 plasmid, this strain grew more
slowly than a strain complemented with the longer pMY3 fragment
(Fig. 2). As discussed below, this difference is probably due to
the elimination of a constitutive promoter element in the DNA 5` of the
SpeI site.
QSR1 Encodes for the Yeast Counterpart of a Mammalian
Protein Implicated in Differentiation
The 1.2-kbp
SpeI- ClaI DNA fragment and approximately 200 base
pairs upstream of the SpeI site were sequenced by chain
termination. The SpeI- ClaI fragment includes a 663-bp
open reading frame, capable of encoding a protein consisting of 221
amino acids, as shown in Fig. 4 a. The DNA sequence
upstream of the QSR1 reading frame contains two putative TATA
boxes at 75 and
109, a putative MIG1 binding
element
(29) at
285, and two poly(dT) tracks at
210 and
240. The poly(dT) tracks are indicative of a
constitutive promoter in yeast
(30) , and these are upstream of
the SpeI restriction site, which accounts for the observation
that the qsr1-1 mutant complemented by the
SpeI- ClaI fragment grows more slowly than the mutant
complemented by pMY3 as noted above.
Figure 4:
Nucleotide sequence of QSR1 and homology
between QSR1 and the human QM protein. a, the deduced amino
acid sequence is represented by single-letter code in bold
face under the nucleotide sequence, which is numbered to the
left starting with the A of the initial methionine codon. The
amino acid sequence is also numbered to the left, starting
with Met. The asterisk refers to the position of
the qsr1-1 mutation G
D.
Restriction sites are underlined and marked. b,
alignment of the amino acid sequences of the human QM and
yeast QSR1 proteins. Amino acids in the yeast QSR1 sequence which are identical to those in the human QM sequence are indicated by a dash (- - - -), and those
which are conservative changes from the human QM sequence are
indicated by lower case letters in the yeast QSR1 sequence.
The segment of DNA within the
1.2-kbp SpeI- ClaI fragment gives rise to a single
mRNA, approximately 850 base pairs in length, as shown in Fig. 5.
The amount of QSR1 mRNA is essentially identical in cells
grown on dextrose, galactose, or ethanol-glycerol, indicating that
expression of this gene is not subject to catabolite repression nor
induced by non-fermentable carbon sources.
Figure 5:
Northern analysis showing a single
transcript derived from the QSR1 locus. Total RNA was isolated from
cells growing exponentially on 10% dextrose ( D), 2% galactose
( G), or 4% ethanol, 3% glycerol ( E/G). The blot was
probed with a P
-labeled 1.1-kbp
SpeI- ClaI fragment excised from pMY7. Numbers to the right indicate the mobility and size of standards
in kilobases.
A search of sequence data
bases revealed that the DNA sequence of the QSR1 open reading
frame is homologous to a human cDNA named QM. The QM cDNA was isolated by subtractive hybridization between a
tumorigenic cell line derived from Wilms' tumor and a
transfected, non-tumorigenic form of the same cell line
(31) .
The amino acid sequences of the proteins encoded by QSR1 and
QM are compared in Fig. 4 b. The calculated
molecular masses of the yeast and human proteins are 25.4 and 24.5 kDa,
and their calculated pI values are 10.1 and 10.5, respectively. The
amino acid sequences of the yeast and human proteins are 63-65%
identical over their entire length and 74% identical over the first 180
amino acids. This unusually high sequence identity suggests that
QSR1 is the yeast counterpart of QM.
to
Tyr
and from Leu
to Tyr
are
more than 50% identical in sequence, indicating that this region is
internally duplicated. In addition, searches of the data bases with
different peptides from the QSR1 sequence revealed a low
homology between the carboxyl terminus of QSR1 and regions of
several proteins containing calcium-binding motifs. The qsr1-1 Allele Contains a Missense Mutation-Approximately
1000 bp encompassing the qsr1-1 allele were sequenced
from genomic DNA extracted from the mutant. The qsr1-1 allele contains a G to A mutation at nucleotide 581, which changes
a codon in the COOH terminus of the open reading frame from GGT to GAT,
resulting in the replacement of a glycine by an aspartic acid
(Fig. 4 a). Although the COOH terminus of the protein is
not highly conserved, glycine 194 is conserved in all species where
QSR1 has been identified
(32) . The occurrence of a
missense mutation in qsr1-1 is consistent with the
semidominant phenotype noted above.
QSR1 Is an Essential Gene in Yeast
To evaluate the
function of the QSR1 protein, we constructed diploid yeast
strains in which one chromosomal copy of QSR1 was replaced
with the auxotrophic marker, HIS3, as shown in Fig. 6.
Since QSR1 has some, as yet uncharacterized, relationship to a
subunit of a mitochondrial respiratory enzyme complex we constructed
the deletion in two diploid strains, HFF10 (W303-1A
W303-1B) and HFF12 (YPH499
YPH500), which differ in their
catabolite repression response,
(
)
to ascertain
whether any phenotype resulting from the QSR1 deletion may
depend on the genetic background of the yeast strain.
Figure 6:
QSR1 is an essential gene. a,
Southern blot hybridization. Genomic DNA isolated from the wild-type
diploid ( W303D) or from the QSR1/qsr11::HIS3 diploid ( HFF10) was digested with EcoRI or
ClaI, separated by agarose gel electrophoresis, and
transferred to nitrocellulose. The blots were probed either with a
1.2-kbp XbaI- XbaI fragment located 5` of the QSR1 ORF ( probe 1) or with a 1.8-kbp fragment containing the
HIS3 gene ( probe 2). b, meiotic segregation
of the qsr1
1::HIS3 allele. The QSR1/qsr1
1::HIS3 diploid ( HFF10) was sporulated, and asci were dissected
on YPD medium. The abbreviations for restriction enzymes are the same
as in Fig. 2.
Integration of
the marker and replacement of the QSR1 locus was verified by
Southern analysis as shown in Fig. 6 a. Integration of
the HIS3 fragment changed a 3.0-kbp EcoRI fragment
overlapping and extending 5` of the QSR1 locus to a 4.5-kbp
fragment (see Fig. 2), both of which are detected in HFF10, the
diploid deletion strain derived from W303. When this same DNA was
digested with ClaI and probed with the HIS3 gene, the
probe detected the endogenous HIS3 DNA as a 3-kbp fragment
(Fig. 6 a), and the integrated HIS3 as an
approximately 6-kbp fragment encomposing the QSR1 locus and
extending into the 5`-terminus of RGR1 (see Fig. 2). The
lack of any other hybridizing fragments also indicates that QSR1 is a single copy gene. Identical results were obtained with HFF12,
a diploid derived from YPH499 YPH500 (results not shown).
1::HIS3 diploid strains were sporulated,
and 20 tetrads were dissected for each strain. In every tetrad from
both strains, only two spores were viable on YPD
(Fig. 6 b), and none of the viable spores were
prototrophic for histidine. Cosegregation of the HIS3 marker
with lethality establishes that QSR1 is an essential gene in
yeast.
prototrophs were sporulated and dissected.
Among the progeny, His
, Ura
prototrophs were found at a frequency compatible with segregation
of a centromeric plasmid, indicating that the URA3 marked
plasmid carrying QSR1 covers the otherwise lethal deletion of
QSR1. Rescue of the qsr1-1 Mutation by QCR6 Does Not Involve
Transcriptional Regulation-It has been reported that
Jif-1, the chicken equivalent of QSR1, interacts with
Jun and may be a negative regulator of transcription
(34) . We
thus tested whether the relationship between QSR1 and QCR6 might involve regulation of transcription. As shown in
Fig. 7
, the amount of QSR1 mRNA detected by Northern
analysis is identical in strain W303 and the qsr1-1 mutant covered by QCR6 on a high copy plasmid, and also
identical in a qsr1-1, rho
strain covered by
the plasmid. The amount of QSR1 mRNA is also identical in the
qsr1-1 mutant covered by QCR6 on a high copy
plasmid, the parental strain MMY-15D, in which QCR6 is
deleted, and the wild-type strain W303. In addition, when normalized to
an internal actin mRNA standard, the amount of QSR1 mRNA is
identical in MMY3-3A, a QSR1,QCR6 haploid and
MMY3-3B, a qsr1-1,QCR6 haploid, which were derived
from the same heterozygous diploid strain. Also, the qsr1-1 mutation does not affect the expression of QCR6. The
amount of QCR6 transcript originating from the single
chromosomal locus is identical in MMY3-3A and MMY3-3B
(Fig. 7).
Figure 7:
QCR6 does not affect expression of QSR1.
Total RNA was isolated from cells growing exponentially on YPD medium.
The blot was probed simultaneously with a
P
-labeled 1.1-kbp SpeI -ClaI
fragment of QSR1 and a 1.6-kbp EcoRI- HindIII
fragment of ACT1. The blot was then stripped and reprobed
simultaneously with a 1.9-kbp SphI fragment of QCR6 and the EcoRI- HindIII fragment of ACT1. Arrows indicate the mobility of the QSR1,
ACT1, and QCR6 transcripts. The figure also shows the
high level of QCR6 expression from pMES32 in the
qsr1-1 mutant, which is diminished in the rho°
mutant. The QCR6 transcript derived from pMES32 is slightly
longer than that derived from the chromosomal locus. This appears to be
due to the use of a cryptic promoter, which is preferentially used when
QCR6 is expressed from this plasmid, since trace amounts of a
similar length transcript can be seen in the mRNA from W303,
MMY3-1A, and MMY3-1B, but are absent in the QCR6 deletion strains qsr1-1[pMY3] and MMY-15D.
On the basis of these results, we conclude that
there is no transcriptional interaction between QCR6 and
QSR1 as reflected in the steady state levels of mRNA.
Additionally, if the QSR1 protein is involved in
transcriptional regulation, it should be located in the nucleus.
However, as shown below, this protein appears to be concentrated in the
cytoplasm and excluded from the nucleus. Rescue of the qsr1-1 Mutation by QCR6 Does Not Depend on
Mitochondrial Function- QCR6 can not substitute for
QSR1, since deletion of QSR1 is lethal in cells
containing a chromosomal copy of QCR6. In addition the
deletion of QSR1 is not rescued by QCR6 carried on a
high copy plasmid. When the QSR1/qsr11::HIS3 diploid
strain was transformed with pMES32 () and sporulated, only
two spores from each tetrad were viable on YPD. None of the viable
spores were His
, although 11 out of the 18 spores were
Ura
, indicating that the QCR6 containing
plasmid had segregated randomly but had not allowed any of the
qsr1
1::HIS3 spores to live (results not shown).
The QSR1 Protein is Located in the Cytoplasm
When
exponentially growing yeast cells are stained with antibody to QSR1 protein the antibody is concentrated in the cytoplasm and appears
to be excluded from the nuclear region, as shown in Fig. 8. An
identical staining pattern for QSR1 protein was observed in
the qsr1-1 mutant covered by QCR6 (results not
shown). The QSR1 antigen consistently exhibits a granular or
punctate staining pattern, which suggests that the protein may be
associated with a larger protein complex or structural element,
although the antigen does not colocalize with mitochondrial DNA
(Fig. 8).
Figure 8:
The
protein encoded by QSR1 is concentrated in the cytoplasm. The
photograph on the left is of strain W303-1B yeast cells
harvested during exponential growth and viewed by differential
interference contrast (Nomarski) optics. The center photograph
is the same field of cells stained for nuclear and mitochondrial DNA
with 4`-6-diamidino-2-phenylindole. The right photograph is
the same field of cells showing the localization of anti-QSR1p antibody
by indirect immunofluoresence after staining with a fluorescein-labeled
secondary antibody.
complex
is one of the supernumerary subunits found in the bc
complexes of mitochondria but absent from the bc
complexes of bacteria. Although this subunit regulates the
activity of the cytochrome bc
complex, it is not
essential for respiration, since deletion of QCR6 does not
prevent growth of yeast on non-fermentable carbon sources. We suspected
that the QCR6 deletion might be covered by another gene and
thus screened for mutants in which QCR6 is essential. We
obtained five such quinol-cytochrome c reductase
subunit- requiring mutants, belonging to two complementation
groups, qsr1 and qsr2.
complex, results in a synthetic petite phenotype.
(
)
That no such synthetic petite mutants were found suggests
that the mutagenesis protocol we employed was probably not saturating.
-helices linked by a short coil segment, and data base searches
with this region of QSR1 revealed a low homology with several
calcium-binding proteins, which typically contain helix-coil-helix
motifs
(39) . When the QSR1 protein has been purified
it will be possible to test whether it binds calcium, and if so,
whether the G194D mutation alters such binding.
complex may have dual functions. In this regard
it is notable that haploid yeast which carry the qsr1-1 allele and a single chromosomal copy of QCR6 are unable
to grow on non-fermentable carbon sources, although QCR6 expressed from the multicopy pMES32 will allow the yeast to grow
slowly on these carbon sources. This is consistent with the idea that
the QCR6 encoded protein (subunit 6) can perform two
functions, but the limited amount of QCR6 protein encoded from
the chromosomal locus is sequestered into the cytochrome
bc
complex when synthesis of the respiratory
enzyme is induced, and not available to cover the qsr1-1 mutation. Such dual function is not unprecedented. For example,
core protein 1 of the Neurospora crassa cytochrome
bc
complex also participates in proteolytic
processing of imported mitochondrial precursor proteins
(40) .
complex is located outside of the mitochondria in the mutant. In
this regard it should be noted that subunit 6 has a 25-amino-terminal
presequence, which is absent from the mature subunit in the
bc
complex. However, whereas presequences which
target proteins to mitochondria are enriched in basic amino acids and
generally lack acidic amino acids, this presequence contains a single
basic amino acid (lysine) and is enriched in acidic amino acids
(10, 18) . Whether subunit 6 can be targeted to both
cytoplasmic and mitochondrial locations and the possible role of the
unusual presequence in such targeting can be tested when antibodies to
subunit 6 become available.
Table:
Yeast strains constructed in this study
Table:
Plasmids used in this study
-D-thiogalactopyranoside;
QSR, quinol-cytochrome c reductase subunit requiring;
QCR, quinol-cytochrome c reductase; bp, base pairs;
kbp, kilobase pairs.
Complex, Ph.D. Thesis, Dartmouth College
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.