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From the
Received for publication, July 28, 2000, and in revised form, September 20, 2000
Communication between mitochondria and the
nucleus is important for a variety of cellular processes such as
carbohydrate and nitrogen metabolism, mating and sporulation, and cell
growth and morphogenesis. It has long been known that the functional
state of mitochondria can influence nuclear gene expression. For
example, in yeast cells lacking the mitochondrial genome, the
expression of several nuclear genes, such as CIT2 (citrate
synthase), MRP13 (mitochondrial ribosomal protein), and
DLD3 (D-lactate dehydrogenase) has been
reported to be altered. Here we show by microarray analysis of
the genome-wide transcription profile of Saccharomyces
cerevisiae that yeast petite mutants lacking mitochondrial DNA
induce genes coding for mitochondrial proteins, enzymes of the
glycolytic pathway and of the citric acid cycle, cell wall components,
membrane transporters, and genes normally induced by nutrient
deprivation and a variety of stresses. Consistent with the observed
induction of genes related to cell stress and those encoding membrane
transporters, yeast petite cells showed increased resistance to severe
heat shock and exhibited a pleiotropic drug resistance phenotype. The
observed changes in nuclear gene expression in cells lacking
mitochondrial DNA may have implications for the role of mitochondria in
processes such as carcinogenesis and aging.
Mitochondria are cellular organelles that perform the reactions
necessary for energy production through respiration and contain enzymes
that catalyze key steps in a variety of degradative and biosynthetic
pathways. Mitochondrial respiration occurs on the respiratory chain
situated in the inner mitochondrial membrane. The components of the
respiratory complexes are encoded by both the mitochondrial and nuclear
genomes, and there has to be a precise coordination of gene expression
between these two genomes to allow biosynthesis of functional
mitochondria (1). In addition, communication between the nucleus and
the mitochondria is important for a variety of cellular functions in
yeast including carbohydrate and nitrogen metabolism, mating and
sporulation, cell division, growth and morphogenesis, and perhaps even
for the determination of longevity (1-8). In the yeast
Saccharomyces cerevisiae, the expression of genes required
for mitochondrial biogenesis is controlled mainly by oxygen and a
carbon source (1, 2). When a fermentative carbon source such as glucose
is present, yeast cells obtain energy through the nonmitochondrial
reactions of fermentation, converting glucose to ethanol. Transcription
of nuclear genes encoding mitochondrial proteins is repressed, and
therefore mitochondrial biogenesis is reduced. Exhaustion of glucose
leads to a transient growth arrest called the diauxic transition,
whereupon cells induce transcription of nuclear genes coding for
proteins of the mitochondrial transcriptional and translational
apparatus and for components of the respiratory complexes as they adapt
to respiratory metabolism (9). Glucose represses respiratory activity
and mitochondrial biosynthesis irrespective of whether oxygen is
present. This transcriptional regulation by oxygen and glucose of
nucleus-encoded proteins related to mitochondrial functions is mediated
via regulation of the activity of the Hap1 and Hap2/3/4/5 activator
proteins, respectively (7), which together regulate the expression of
genes encoding components of the electron transport complexes as well
as genes coding for enzymes of the tricarboxylic acid cycle and
the heme, sterol, and fatty acid biosynthetic pathways (2). The
regulation exerted by oxygen is largely dependent on the
transcriptional activator Hap1, which senses oxygen availability
through the cytochrome cofactor heme (10, 11), whose own biosynthesis
in mitochondria is regulated by oxygen. Regulation by carbon source is
dependent on the Hap2-5 complex (12-14), and glucose negatively
regulates the activity of this complex by repressing transcription of
HAP4, the gene encoding its transactivation component
(12).
In addition to repressing mitochondrial biogenesis and respiratory
activity, glucose also represses genes involved in metabolism of other
sugars as well as the genes coding for enzymes that function in
gluconeogenesis and the glyoxylate cycle (15). Glucose also activates
the cAMP-protein kinase A
(PKA)1 pathway, which in turn
positively controls glycolysis and growth and negatively influences the
accumulation of glycogen and trehalose, stress resistance, and
gluconeogenesis, the physiological responses to nutrient deprivation
(16, 17). Although the proteins phosphorylated by PKA are the metabolic
enzymes themselves, the transcription factors Msn2 and Msn4 are also
subject to PKA-dependent regulation (18, 19). High activity
of PKA represses transcription via Msn2 and Msn4 (18, 19), which in
turn regulate genes containing stress response elements (STREs) in
their promoter regions (20). In addition to nutrient deprivation,
activation of transcription by Msn2 and Msn4 occurs in response to
other kinds of stress (18, 20) and is also important for induction of
gene expression at the diauxic transition (21). The cAMP-PKA pathway
also has a repressing activity on transcriptional activation by Msn2
and Msn4 during the diauxic transition and upon heat shock (21, 22).
Moreover, it has been suggested that low activity of the cAMP-PKA
pathway facilitates mitochondrial biogenesis (23-25).
Yeast cells can exist as petites, defined as cells that lack functional
mitochondria, as a consequence of nuclear mutations, deletions of large
segments (rho Yeast Strains and Growth Media--
The strain used in this
study is W303-1B (MAT Heat Shock Assay--
Cells were grown at 23 °C to midlog
phase and heat-shocked at 52 °C for 12 min. Dilutions of control and
heat-shocked cells were made and plated on YPD plates. The number of
viable cells was determined after 2-3 days at 23 °C. Three
independent experiments were performed, and the average viability of
the cells was determined.
Microarray Analysis--
RNA from wild type (rho+)
and rho0 cells was isolated by the hot phenol method as
described (37) and further purified with a RNeasy kit (Qiagen)
according to the manufacturer's instructions. Cy3- or Cy5-labeled
cDNA was synthesized from 50 µg of total yeast RNA with
SuperScript II RNaseH
Two independent comparisons of gene expression levels were made using
wild type and rho0 cultures. For 95% of the genes, the
expression ratios measured in the duplicate experiments differed by
less than a factor of 2. Reproducibility was also assessed by comparing
gene expression profiles of two independently grown cultures of wild
type cells; no significant changes in expression levels were observed
for the vast majority of the genes.
Northern Analysis--
Northern blot analyses were performed as
described (37). Probes corresponding to 500 base pairs of the indicated
ORFs were generated by polymerase chain reaction and labeled with
32P by random priming essentially as described (37).
Using DNA microarray technology, we compared the whole genome
transcription profile of wild type S. cerevisiae cells with that of cells lacking mitochondrial DNA (rho0). Two
independent experiments involving RNA preparations from wild type and
rho0 cells were performed, and genes that were reproducibly
induced or repressed in both experiments
were identified (Tables I and II). The genes that had reproducibly
altered transcript levels in rho0 cells represented ~4%
of the genome. Of these genes, 86% were induced between 1.5- and
6-fold, while 14% were repressed between 1.4- and 2.3-fold. The
microarray data were independently confirmed by Northern blot analysis
of several genes (Fig. 1), using RNA samples from one of the microarray experiments and from a third, independent set of RNA preparations.
A number of genes that showed relatively small induction or repression
levels and showed variable expression levels in our comparison of the
RNAs of two wild type cultures were omitted from Tables I and II. Five
such genes encoding mitochondrial ribosomal proteins, however, are
listed in Table I, since we observed elevated expression levels for
many components of the mitochondrial ribosome in rho0
cells, and these same genes were shown to be coregulated in the study
of Hughes et al. (38) of multiple mutant strains and
drug-treated cultures.
Not unexpectedly, the differences in gene expression between wild type
and rho0 cells we observed were, for the most part,
relatively small. The yeast strain W303-1, which we have used in this
study, was previously shown by Brown and Trumpower to be the least
responsive of five different strains and showed the smallest
differences in expression levels for nucleus-encoded mitochondrial
proteins under glucose-repressed and -nonrepressed conditions (39).
Hughes et al. (38) have also shown that the large group of
coregulated mitochondrial proteins in general exhibit less than 2-fold
alterations in gene expression in different conditions.
Transcription of Nuclear Genes Involved in Mitochondrial Biogenesis
Is Altered in rho0 Cells--
In the absence of
mitochondrial DNA, the expression of a large number of nuclear genes
involved in mitochondrial biogenesis is derepressed (Table I). These
genes include those encoding mitochondrial ribosomal proteins; assembly
factors; components of the respiratory complexes; proteins required for
import, stabilization, and processing of cytoplasmically synthesized
mitochondrial proteins; and known and putative members of the
family of mitochondrial carrier proteins. Although it has been
previously reported that mRNA levels for the mitochondrial
ribosomal proteins Mrp13, Mrp49, and Rml2 are induced in
rho0 cells (27, 40, 41), other genes such as
MRP2, MRP7, MRPL16, and
CYC1 had been reported to not change (26, 42-44). In our study, although all of those particular genes showed increased expression, MRPL33 and MRP20, reported to be
induced in rho0 cells (40, 45), were among those genes we
saw not up-regulated. Some of these differences may be due to the use
of different strain backgrounds.
Although petite cells seem to up-regulate the transcription of many
genes whose products function in mitochondrial biosynthesis, the
transcripts for some subunits of the mitochondrial complexes were
reduced (Table II). They include the COX4 and
QCR2 genes, for which down-regulation in rho0
cells has already been reported (1, 46) and ATP20 and
ATP5, which encode subunits of the ATP synthase complex.
Our results indicate that glucose repression of the transcription of
nuclear genes required for mitochondrial biosynthesis was partially
alleviated. The induction of a number of these genes is probably due to
an increase in the activity of the Hap2/3/4/5 complex. The genes
CYC1, COX5B, and CYB2, known targets
of this transcription factor complex, and MRP2 and
MRPL13 all possess consensus binding sites for the Hap2-5
complex and were all induced in our rho0 strain. In
addition, the genes MRP13, MRP7,
MRP49, and MRPL25, which were also induced in
rho0 cells, all have sequences in their promoter regions
that differ from the consensus binding site for the Hap2-5 complex by
a single nucleotide (40). Transcription of HAP4 was also
induced in rho0 cells, and this may have led to the
increased activity of the complex and up-regulation of some of its
target genes.
The expression of genes encoding chaperones involved in mitochondrial
protein import and processing (SSC1, HSP60,
HSP10, and HSP78) was also induced, and so too
was the transcript for HSF1. Elevated expression of the
transactivator Hsf1 could contribute to the elevated transcription of
these genes; however, the activity of this transcription factor is
known to be regulated posttranslationally (47, 48).
Among the genes required for mitochondrial biogenesis that we found
induced in rho0 cells, many are also induced at the diauxic
transition (9) and during evolutionary adaptation of yeast cells to
growth in limiting glucose conditions (49). A similar set of genes also showed coregulation in the study of Hughes et al. (38) of
300 diverse mutations and chemical treatments.
Transcripts Encoding Tricarboxylic Acid Cycle and Glycolytic
Enzymes Are Elevated in rho0 Cells--
Transcription of
genes coding for enzymes of the tricarboxylic acid cycle is repressed
in cells growing in glucose. When yeast cells lack functional
mitochondria, the expression of CIT1, ACO1, IDH1, and IDH2, under both repressing
(i.e. glucose) and derepressing (i.e. raffinose)
growth conditions, requires Rtg1, Rtg2, and Rtg3 (50), which also
mediate the retrograde response in petite cells (31, 32).
We found in rho0 cells grown under repressing conditions
that the mRNA levels for CIT1, ACO1,
IDH1, and IDH2 were elevated (Table I). These
same genes have been reported not to change in the absence of
mitochondrial DNA (50); however, that study, in contrast to our
experiment, used growth conditions that derepress transcription of
these genes. Our findings are also supported by the recent report that
expression of ACO1, IDH1, and IDH2 is elevated in cells harboring mutations in nuclear genes that cause respiratory deficiency (38). Since it has been demonstrated that
CIT1, ACO1, IDH1, and IDH2
expression in rho0 cells is dependent on the functions of
Rtg1, Rtg2, and Rtg3 (50), the induction of their expression is, by
definition, a retrograde response.
We also observed elevated mRNA levels for two genes encoding the
mitochondrial and cytosolic malate dehydrogenase (MDH1 and MDH2). MDH1 and MDH2 contain STREs in
their upstream regulatory regions, and since we saw induction of STRE
containing genes in petites (see below), it is likely that their
induction is mediated by these elements.
In addition to these genes coding for enzymes of the tricarboxylic acid
cycle, genes coding for glycolytic enzymes were also induced (Table I).
This induction may in part account for the observed stimulation of
glycolysis in petite strains (51). This alteration of metabolism is not
surprising, since in respiration-deficient mutants energy production is
absolutely dependent on glycolysis.
rho0 Cells Induce Transcription of Genes Conferring a
Pleiotropic Drug Resistance Phenotype--
The pleiotropic drug
resistance phenomenon in the yeast S. cerevisiae is mediated
by ABC transporters whose expression is regulated by the transcription
factors Pdr1 and Pdr3 (52-54). Recently, a genome microarray analysis
has been done in yeast strains overexpressing Pdr1 or Pdr3, and several
known and putative new targets of these factors have been identified
(55). Among these, PDR5, YGR035C, YOR049C, LPG20, YOR152C, and
HXK1 were also induced in rho0 cells (Table I).
Up-regulation of PDR5, YGR035C, and
YOR049C in respiration-deficient cells has also been
reported recently (38).
We tested the growth of rho0 versus wild type
cells on three different drugs, namely 4-NQO, cycloheximide, and
ketoconazole, which are known to be substrates for different members of
the ABC transporter family. As shown in Fig.
2, our petite strain was clearly more
resistant to each of these drugs. Cycloheximide and ketoconazole are
known to be substrates of the Pdr5 transporter (56-58), whose
transcription is induced in rho0 cells. 4-NQO is normally a
substrate for the Snq-2 transporter (57), whose expression did not
appear to be induced in our study. It is quite possible, however, that
increased expression of an as yet uncharacterized member of the
transporter gene family, such as YOR049C, could mediate the
resistance of petite strains to 4-NQO.
Among the genes listed in Table I, PDR5, YGR035C,
YOR049C, and LPG20 have pleiotropic drug response
elements in their promoter regions, pointing to their regulation by
Pdr1 and/or Pdr3. The transcripts encoding these two transcription
factors were not elevated in rho0 cells, but they could be
regulated posttranslationally. It has been shown that Pdr1 could also
have target genes that code for proteins related to mitochondrial
functions, since cells with a point mutation of PDR1,
pdr1-2, are partially respiration-deficient (59).
These observations suggest that Pdr1-dependent
transcription may be induced in petite cells as the cell attempts to
compensate for mitochondrial defects.
The Expression of Genes Regulated by the cAMP-PKA Pathway and/or
Msn2 and Msn4 Is Elevated in rho0 Cells--
The
expression of genes whose transcription is negatively regulated by the
cAMP-PKA pathway in wild type cells grown on glucose, such as
TPS1, TPS2, TPS3, TSL1,
GSY2, HXK1, HOR2, GPD2,
UBI4, GLK1, ARA1, MDH1,
MDH2, and YOR173W (Table I), was elevated in rho0 cells, suggesting that the activity of this pathway
may be low. In this group of genes, all but GPD2 are
dependent on the STRE-binding transcription factors Msn2 and Msn4 for
their induction. Other genes, such as HSP78,
YML128C, and YNL200C, are listed in Table I in
the same grouping because they are regulated by Msn2 and Msn4, although
a direct repressing effect of the cAMP-PKA pathway on their expression
has not been demonstrated. Nevertheless, these same genes are induced
upon nutrient limitation or during the diauxic transition, two
conditions for which a negative effect of the cAMP-PKA pathway on gene
expression has been shown (19, 21, 60, 61).
Among the genes induced in rho0 cells, many possess STREs
in their promoter regions. The genes marked in boldface type in Table I
are known either to be regulated through their STREs or to contain a
putative STRE identified by a computer search (62, 63). Since the
stress response element appears extremely frequently in the genome of
S. cerevisiae and most functional STREs occur in clusters,
the search criteria used by these authors had excluded genes that had
just one STRE element or did not have clustered STREs. Some
STRE-regulated genes such as GAC1 (64), however, contain
only a single STRE. Many genes that were induced in rho0
cells contain single or unclustered STRE elements (e.g.
XKS1, FBP26, YBR262C,
YDR516C, YHR087W, and others). Regardless of the nature of the STRE(s) they contain, the genes listed in Table III, in addition to their induction in
rho0 cells, were also shown to be induced at least 2-fold
at the diauxic transition (9, 21), a condition that activates
STRE-dependent transcription and requires the
transactivators Msn2 and Msn4 for induction of a large number of genes
(21).
STRE-dependent induction may also contribute to induction
of genes related to mitochondrial biogenesis, for the mitochondrial proteins encoded by CYC7, COX5B,
HSP78, and MSF1 also contain STREs.
rho0 Cells Are More Resistant to Severe Heat
Shock--
Low activity of the cAMP-PKA pathway is associated with a
variety of cellular phenotypes including enhanced resistance to heat
shock and other types of cellular stress (16). Enhanced resistance to
heat shock is a consequence of elevated expression of heat shock
proteins, elevated accumulation of trehalose, and perhaps elevated
expression of metabolic enzymes for which a direct physiological role
in heat shock response has been suggested (22). In agreement with lower
activity of the cAMP-PKA pathway, rho0 cells were more
resistant to severe heat shock than wild type cells (Table
IV). An elevated mRNA levels of
several genes, such as several of the HSPs, UBI4,
the genes for the components of the trehalose synthase complex, and
other genes coding for metabolic enzymes (e.g.
HXK1 or GLK1) may account for this phenotype.
Another possible contribution to the increased heat shock resistance of
rho0 cells is the induced expression of MPK1,
PST1, SED1, CWP1, PIR1, PIR3, and HSP150 (Table I); these genes are
regulated at the transcriptional level by the protein kinase
C-dependent cell wall integrity signaling pathway (65),
whose induction in response to heat shock is important for
thermotolerance in yeast (66).
By analyzing the whole genome transcription profile of S. cerevisiae, we have shown that in the absence of mitochondrial DNA the expression of a large number of genes is altered. Petite cells induce transcription of genes involved in mitochondrial biogenesis which are normally repressed by glucose and genes regulated by the
cAMP-PKA pathway and by the transcription factors Msn2 and Msn4. Also
induced is the expression of genes coding for cell surface components,
membrane transporters, and enzymes of the tricarboxylic acid cycle and
of glycolysis.
The transcription profile of rho0 cells is in part similar
to that of cells undergoing the diauxic transition (9) or adaptive evolution in glucose limiting conditions (49) in that genes required
for mitochondrial biogenesis and those coding for the enzymes of the
tricarboxylic acid cycle are induced. The expression of genes coding
for glycolytic enzymes is not repressed, however, but induced in
rho0 cells, since glycolysis in respiration-deficient
strains is the only means to generate energy.
The increased expression of genes coding for proteins involved in
mitochondrial biogenesis and function in rho0 cells is
possibly a combined effect of increased activity of the Hap2-5 complex
and reduced activity of the cAMP-PKA pathway. It has been suggested
that the activity of the Hap2-5 complex is reduced in rho0
cells during growth on raffinose (derepressing conditions), as judged
by lower expression of Hap2-5-regulated genes encoding enzymes of the
tricarboxylic acid cycle (50). We now show that under repressing
conditions (i.e. growth on glucose) the expression of the
target genes of the Hap2-5 complex coding for the mitochondrial proteins Cyc1, Cox5b, and Cyb2 is increased. Glucose regulates the
activity of the complex by repressing transcription of HAP4, and remarkably the expression of HAP4 is also elevated in
rho0 cells. These observations suggest that, in repressed
rho0 cells, the activity of the Hap2-5 complex is
increased. Our observations provide further evidence for the
involvement of mitochondrial functions in regulating the activity of
this complex, although how this is achieved is currently not clear.
The expression of genes known to be negatively regulated by the
cAMP-PKA pathway in wild type cells is induced in rho0
cells. Among those genes, many possess functional STREs in their promoter regions, suggesting that the activity of the transactivators Msn2 and Msn4, which function through STREs, is elevated. Evidence that
petite cells may have lower activity of the cAMP-PKA pathway and thus
an activated stress response is further corroborated by our observation
that cells lacking mitochondrial DNA were more resistant to severe heat
shock (Table IV). Our study, however, did not reveal increased mRNA
levels for the STRE-regulated genes such as HSP104,
SSA3, HSP26, CTT1, and
DDR2. The absence of an increased basal mRNA level for
heat shock proteins such as Hsp104 in rho0 cells, which are
more heat shock-resistant than wild type cells, was surprising in light
of previous work demonstrating a central role of this protein in
thermotolerance in yeast (67).
The reason for the lack of induction of some genes regulated through
their STREs is not clear. However, of the 29 S. cerevisiae genes that are known to be regulated through their STRE (62), not all
demonstrated increased transcription during the diauxic transition (9),
a condition that activates STRE-dependent gene expression
(21). It has been suggested that the promoter context of each
particular STRE may influence responsiveness (62).
Although we did not observe induced transcription of genes coding for
cytosolic chaperones (e.g. HSP104 and SSA3) in
petite cells, the genes coding for mitochondrial chaperones
(SSC1, HSP60, HSP10, HSP78)
were induced. The induction of mitochondrial chaperones in
rho0 cells probably reflects an attempt to maintain
efficient import of cytoplasmically synthesized proteins in the
impaired organelle. Interestingly, a similar differential induction of
chaperones following mitochondrial DNA depletion occurs in mammalian
cells (68).
Mitochondria have also been implicated in the processes of
carcinogenesis and aging. In neoplastic mammalian cells, multiple rearrangements of mtDNA occur, and those events may contribute to the
development and maintenance of the tumorigenic phenotype (69).
Remarkably, yeast cells treated with a variety of known carcinogens
lose mitochondrial DNA and show cell surface changes, such as
alteration in lectin agglutinability, cellular electrokinetic properties, and glycoprotein composition, that are analogous to changes
observed in neoplastic cells (70). Tumor cells also acquire multidrug
resistance, and Wilkie et al. (70) had speculated that
modulation of nuclear gene expression following mitochondrial DNA
depletion could be at the basis of these phenotypes. Our studies support such a hypothesis; yeast cells lacking mitochondrial DNA acquire a multidrug-resistant phenotype perhaps as a result of increased expression of genes coding for membrane transporters (e.g. PDR5 and YOR049C) and show increased
expression of genes coding for a number of cell surface proteins
(e.g. Hsp150, Sed1, and Cwp1) that may account for the
changes in cell surface characteristics reported by Wilkie et
al. (70). Interestingly, up-regulation of the P-glycoprotein and a
consequent multidrug-resistant phenotype has been reported for rat
hepatoma rho0 cells (71).
Studies in yeast have also shed light on the aging process (72, 73). In
this respect, the increased life span of petite cells has been
attributed to the activation of the Rtg-dependent retrograde response in cells with dysfunctional mitochondria (8). It is
also known that increased stress resistance positively correlates with
increased life span in many organisms (74-76). However, as reported by
Kirchman et al. (8), petites derived from W303-1 cells, the
strain also used in this study, did not show increased life span. It
was argued by Kirchman et al. (8) that the W303-1 strain
might have a constitutively active retrograde response that cannot be
further activated by the presence of dysfunctional mitochondria.
However, we did observe an elevated expression of tricarboxylic acid
cycle genes that are regulated by Rtg proteins in petite cells (50),
and thus rho0 W303-1 cells seem to induce a retrograde
response. We also demonstrated that petite cells were more resistant to
heat, and this phenotype has been correlated to increased life span in
different organisms (75, 76). These findings open the possibility that,
in addition to stress resistance and the induction of retrograde
response, some other critical factor, not present in the W303-1 strain
background, is required for the prolonged life span of yeast cells
having impaired mitochondrial functions.
We have demonstrated that the absence of mitochondrial DNA influences
expression of nuclear genes involved in multiple cellular pathways. A
major task now is to elucidate the mechanism of mitochondria to nucleus
signaling. Although it has been demonstrated recently that the
functional state of mitochondria influences the subcellular localization of Rtg1 and Rtg3 (34), the actual signals sent by the
dysfunctional mitochondrion to regulate nuclear gene expression remain
unknown. It has been proposed that the intracellular level of glutamate
could be a key signal in the Rtg-dependent pathways (34,
50). In addition to such metabolic signals, a relocalization of
proteins, such as the transcription factor Abf2, between the mitochondria and the nucleus may occur (2). We are currently exploring
the possibility that additional mitochondrial proteins may move between
the two organelles and thus influence mitochondria to nucleus signaling.
We thank the Microarray Center at the Ontario
Cancer Institute for providing the chips and additional assistance. We
also thank Dr. Andrew Goryachev and Michael Shales for assistance with data analysis. We are grateful to Dr. Helena Friesen and Dr. Thomas Fox
for critically reading the manuscript.
*
This work was supported by a grant from the Medical Research
Council (MRC) of Canada (to C. J. I.) and from the Croatian Ministry of Science and Technology (to M. S.). The Microarray Centre at the
Ontario Cancer Institute is supported by funding from the MRC of
Canada, the National Research Council of Canada, and the National
Science and Engineering Research Council of Canada.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.
¶
Recipient of a graduate scholarship from the Croatian Ministry
of Science and Technology.
**
To whom correspondence should be addressed: Banting and Best Dept.
of Medical Research, University of Toronto, 112 College St., Toronto,
Ontario M5G 1L6, Canada. Tel.: 416-978-7400; Fax: 416-978-8528; E-mail:
cj.ingles@utoronto.ca.
Published, JBC Papers in Press, October 27, 2000, DOI 10.1074/jbc.M006807200
The abbreviations used are:
PKA, protein kinase
A;
STRE, stress response element;
4-NQO, 4-nitroquinoline-1-oxide;
ORF, open reading frame.
Interorganellar Communication
ALTERED NUCLEAR GENE EXPRESSION PROFILES IN A YEAST
MITOCHONDRIAL DNA MUTANT*
§¶,, and
**
Department of Molecular Genetics, Institute
Rudjer Boskovic, Bijenicka 54, 10000 Zagreb, Croatia and
§ Banting and Best Department of Medical Research and
Department of Molecular and Medical Genetics, University of
Toronto, Ontario M5G 1L6, Canada
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
), or total absence of mitochondrial DNA
(rho0). Petite strains have been used as models to study
the influence of mitochondrial functions on nuclear gene expression. In
petite cells, altered expression has been observed for several nuclear transcripts, including spacer regions of the rDNA repeat; the genes
encoding Cit2 (peroxisomal citrate synthase), Dld3 (cytosolic D-lactate dehydrogenase), and Mrp13 (mitochondrial
ribosomal protein); and the COX5A and COX6
genes, which encode subunits of the cytochrome c oxidase
complex (26-30). One mode of mitochondria to nucleus signaling,
retrograde regulation (31), is mediated by the transcription factors
Rtg1 and Rtg3 and a cytoplasmic protein Rtg2 (31-34) and leads to
induction of nuclear gene expression as a result of altered mitochondrial functions. By this stress-responsive mechanism, cellular
metabolic and biosynthetic activities are adjusted to compensate for
dysfunctional mitochondria (29, 35). To gain a global perspective
on the phenomenon of mitochondria to nucleus signaling, we used
a microarray technology to compare the whole genome transcription
profile of wild type yeast to that of a rho0 petite strain.
This approach permitted a broader assessment of the dependence of
various cellular processes on mitochondrial functions than described previously.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
ho can1-100 ade2-1 trp-1
leu2-3, 112 his3-11, 15 ura3-1). The rho0
derivative was induced by ethidium bromide as described (36) and
verified by staining with 4',6-diamino-2-phenylindole. For RNA
isolation, cells were grown in YPD (1% yeast extract, 2% glucose, 2%
peptone) at 30 °C to midlog phase. For the drug resistance experiments, cells were grown in YPD to saturation, and serial dilutions were spotted onto YPD plates containing
4-nitroquinoline-1-oxide (4-NQO), cycloheximide, and ketoconazole at
the following concentrations: 4-NQO (0.15 and 0.3 µg/ml),
cycloheximide (0.05 and 0.5 µg/ml), ketoconazole (1 and 2 µg/ml).
Only experiments with the higher concentration of drugs are shown in
Fig. 2.
reverse transcriptase (Life
Technologies, Inc.) in the presence of Cy3- or Cy5-dCTP (Amersham
Pharmacia Biotech) using the T20VN (where V represents A,
G, or C and N is A, G, C, or T) primer. Yeast genome chips containing
6218 S. cerevisiae ORFs (Toronto Microarray Consortium) were
hybridized with the mixture of Cy3- and Cy5-labeled cDNA as
follows: chip A Cy3-cDNA (rho+) and Cy5-cDNA
(rho+); chip B Cy3-cDNA (rho+) and
Cy5-cDNA (rho0); chip C Cy3-cDNA (rho0)
and Cy5-cDNA (rho+); chip D Cy3-cDNA
(rho0) and Cy5-cDNA (rho0). The chips were
scanned on a GSI scanner, and the signals were quantified with the
QuantArray software (GSI). For each ORF, the ratios of
rho0/rho+ signals collected from chips B and C
were normalized by dividing the ratio of these
rho0/rho+ signals with the factor for the same
ORF obtained from chips A and D. After normalization, 16 values were
obtained for each ORF, and the coefficient of variation was calculated.
Only those genes for which the ratios had a coefficient of variation
less than 0.35 were further analyzed. The values of gene expression obtained by this procedure were confirmed by using a somewhat different
normalization procedure being developed by Dr. A. Goryachev of the
Bioinformatics group, Research Information Systems, Princess Margaret
Hospital, Toronto.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
Genes with induced expression in rho0 cells
Genes with decreased expression in rho0 cells
View larger version (42K):
[in a new window]
Fig. 1.
Confirmation of microarray data by Northern
analysis. 10 µg of total RNA from a microarray experiment
(A) and from an independent preparation of RNA
(B) were hybridized with probes specific for the indicated
genes. 18 S rRNA is shown as the loading control.
View larger version (47K):
[in a new window]
Fig. 2.
rho0 cells exhibit a pleiotropic
drug-resistant phenotype. Yeast cells were grown to saturation,
and serial dilutions were made and spotted onto YPD plates supplemented
with or without 4-NQO, ketoconazole, and cycloheximide at the indicated
concentrations.
STRE-containing genes induced in both rho0 cells and at the
diauxic transition
Increased resistance of rho0 cells to heat shock treatment
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
ACKNOWLEDGEMENTS
FOOTNOTES
ABBREVIATIONS
REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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