Interorganellar Communication

ALTERED NUCLEAR GENE EXPRESSION PROFILES IN A YEAST MITOCHONDRIAL DNA MUTANT*

Ana TravenDagger §, Johnson M. S. Wong§, Deming Xu§, Mary SoptaDagger , and C. James Ingles§||**

From the Dagger  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

Received for publication, July 28, 2000, and in revised form, September 20, 2000



    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.



    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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-), 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

Yeast Strains and Growth Media-- The strain used in this study is W303-1B (MATalpha 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.

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- 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.

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).


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.


                              
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Table I
Genes with induced expression in rho0 cells
The values of -fold change are the average of two independent experiments. Genes indicated in boldface type contain functional or putative STREs.


                              
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Table II
Genes with decreased expression in rho0 cells
The values of -fold change are the average of two independent experiments.



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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.

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.



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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.

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).


                              
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Table III
STRE-containing genes induced in both rho0 cells and at the diauxic transition

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.


                              
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Table IV
Increased resistance of rho0 cells to heat shock treatment
The cells were grown in YPD medium at 23 °C to midlog phase and then transferred to 52 °C for 12 min. 1000 cells were plated on YPD plates, and surviving colonies were counted after 2-3 days of growth at 23 °C.

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).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    ACKNOWLEDGEMENTS

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.


    FOOTNOTES

* 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


    ABBREVIATIONS

The abbreviations used are: PKA, protein kinase A; STRE, stress response element; 4-NQO, 4-nitroquinoline-1-oxide; ORF, open reading frame.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES


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