Chemicals or mutations that target mitochondrial translation can rescue the respiratory deficiency of yeast bcs1 mutants.

Bcs1p is a chaperone that is required for the incorporation of the Rieske subunit within complex III of the mitochondrial respiratory chain. Mutations in the human gene BCS1L (BCS1-like) are the most frequent nuclear mutations resulting in complex III-related pathologies. In yeast, the mimicking of some pathogenic mutations causes a respiratory deficiency. We have screened chemical libraries and found that two antibiotics, pentamidine and clarithromycin, can compensate two bcs1 point mutations in yeast, one of which is the equivalent of a mutation found in a human patient. As both antibiotics target the large mtrRNA of the mitoribosome, we focused our analysis on mitochondrial translation. We found that the absence of non-essential translation factors Rrf1 or Mif3, which act at the recycling/initiation steps, also compensates for the respiratory deficiency of yeast bcs1 mutations. At compensating concentrations, both antibiotics, as well as the absence of Rrf1, cause an imbalanced synthesis of respiratory subunits which impairs the assembly of the respiratory complexes and especially that of complex IV. Finally, we show that pentamidine also decreases the assembly of complex I in nematode mitochondria. It is well known that complexes III and IV exist within the mitochondrial inner membrane as supramolecular complexes III2/IV in yeast or I/III2/IV in higher eukaryotes. Therefore, we propose that the changes in mitochondrial translation caused by the drugs or by the absence of translation factors, can compensate for bcs1 mutations by modifying the equilibrium between illegitimate, and thus inactive, and active supercomplexes.

A new Hansenula polymorpha HAP4 homologue which contains only the N-terminal conserved domain of the protein is fully functional in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, the HAP transcriptional complex is involved in the fermentation-respiration shift. This complex is composed of four subunits. Three subunits are necessary for DNA-binding, whereas the Hap4p subunit, glucose-repressed, contains the transcriptional activation domain. Hap4p is the key regulator of the complex activity in response to carbon sources in S. cerevisiae. To date, no HAP4 homologue has been identified, except in Kluyveromyces lactis. Examination of these two HAP4 sequences led to the identification of two very short conserved peptides also identified in other yeasts. In the yeast Hansenula polymorpha, two possible HAP4 homologues have been found. Their deduced amino acid sequences are similar to the ScHap4p and KlHap4p proteins only in the N-terminal 16-amino-acid basic motif. Since molecular genetic tools exist and complete genome sequence is known for this yeast, we expressed one of these putative HpHap4 proteins in S. cerevisiae and showed that this protein is able to restore the growth defect of the S. cerevisiae hap4-deleted strain. A set of experiments was performed to confirm the functional homology of this new gene with ScHAP4. The discovery of a Hap4-regulatory protein in H. polymorpha with only the N-terminal conserved domain of the S. cerevisiae protein indicates that this domain may play a crucial role during evolution.

Functional dissection of Pdr1p, a regulator of multidrug resistance in Saccharomyces cerevisiae.

Pleiotropic drug resistance in the yeast Saccharomyces cerevisiae results mainly from the overexpression of genes encoding membrane efflux pumps, the so-called ABC and MFS transporters. These pleiotropic drug resistance loci are under the control of the key transcription factors Pdr1p and Pdr3p. We have identified and characterized several new domains of Pdr1p. By testing a series of LexA-PDR1 derivatives for their capacity to activate a GAL1-lacZ reporter gene we have shown that the C-terminal domain of Pdr1p comprising amino acids 879-1036 is involved in transcriptional activation, and that the point mutation pdr1-8 increases its efficiency. Removal of amino acids 1006-1029, which include a polyasparagine stretch, decreases the activation function. Internal deletions within Pdr1p reveal the presence of a large regulatory domain, and a short but strong inhibitory subdomain spanning amino acids 257-316, in which the up-regulating mutations pdr1-2, pdr1-6 and pdr1-7 are located. A mini-Pdr1p consisting of only the DNA-binding and the activation domains strongly up-regulates the expression of the major target genes PDR5, SNQ2 and YOR1, resulting in enhanced multidrug resistance.

Pse1/Kap121-dependent nuclear localization of the major yeast multidrug resistance (MDR) transcription factor Pdr1.

Pdr1 and Pdr3 are two very similar transcription factors that mainly control membrane biogenesis by adjusting the production of different membrane proteins, such as different ABC or major facilitator superfamily (MFS) transporters. We observed that the pse1-1 mutation in the importin/beta-karyopherin Pse1/Kap121 specifically induced the cytoplasmic localization of Pdr1, but not that of Pdr3. Interactions between Pse1 and Pdr1 could be observed in vivo, and a short peptide of 44 amino acids from Pdr1 was shown to contain the information necessary and sufficient for Pse1-dependent nuclear import. This Pdr1-NLS sequence, absent in Pdr3, although rich in serine and tyrosine, is different from the Pse1-dependent nuclear localization signal (NLS) of Pho4. Furthermore, we showed that Pse1/Kap121 is likely to be the sole import receptor for the regulator Pdr1. Together, these new observations underscore the diversity of cellular processes that address to the nucleus two very similar transcription factors involved in the control of the same phenotype, thus securing their function in the cell.

Isolation and molecular characterization of the carboxy-terminal pdr3 mutants in Saccharomyces cerevisiae.

Multidrug resistance in Saccharomyces cerevisiae mainly results from the overexpression of genes coding for the membrane efflux pumps, the major facilitators and the ABC binding cassette transporters, under the control of key transcription regulators encoded by the PDR1 and PDR3 genes. Pdr3p transcriptional activator contains a weak activation domain near the N-terminal zinc finger, a central regulatory domain, and a strong activation domain near the carboxyl terminus. Here we report the results of the mutational analysis of the C-terminal region of Pdr3p. After in vitro mutagenesis of the PDR3 gene six single amino acid substitutions were identified and resulted in resistance to cycloheximide, sulfomethuron methyl, 4-nitroquinoline oxide, fluconazole, mucidin, chloramphenicol and oligomycin. All the C-terminal pdr3 mutant alleles also conferred multidrug resistance in the presence of the wild-type PDR3 gene. The pdr3 mutations resulted in overexpression of both the PDR3 and PDR5 genes as revealed by transactivation experiments involving the PDR3-lacZ and PDR5-lacZ fusion genes and Western blot analyses using antibodies against Pdr5p. Most of the C-terminal pdr3 mutations were found in two sequence stretches exhibiting a high degree of amino acid identity with Pdr1p indicating that they might play a significant role in protein-protein interactions during the initiation of transcription of genes involved in multidrug resistance.

Clustered amino acid substitutions in the yeast transcription regulator Pdr3p increase pleiotropic drug resistance and identify a new central regulatory domain.

In the yeast Saccharomyces cerevisiae mutations in the genes encoding the transcription factors Pdr1p and Pdr3p are known to be associated with pleiotropic drug resistance mediated by the overexpression of the efflux pumps Pdr5p, Snq2p, and Yor1p. Mutagenesis of PDR3 was used to induce multidrug resistance phenotypes and independent pdr3 mutants were isolated and characterized. DNA sequence analysis revealed seven different pdr3 alleles with mutations in the N-terminal region of PDR3. The pdr3 mutants were semidominant and conferred different drug resistance patterns on host strains deleted either for PDR3 or for PDR3 and PDR1. Transactivation experiments proved that the mutated forms of Pdr3p induced increased activation of the PDR3, PDR5, and SNQ2 promoters. The amino acid changes encoded by five pdr3 mutant alleles were found to occur in a short protein segment (amino acids 252-280), thus revealing a regulatory domain. This region may play an important role in protein-DNA or protein-protein interactions during activation by Pdr3p. Moreover, this hot spot for gain-of-function mutations overlaps two structural motifs, MI and MII, recently proposed to be conserved in the large family of Zn2Cys6 transcription factors.

Multiple-drug-resistance phenomenon in the yeast Saccharomyces cerevisiae: involvement of two hexose transporters.

In the yeast Saccharomyces cerevisiae, multidrug resistance to unrelated chemicals can result from overexpression of ATP-binding cassette (ABC) transporters such as Pdr5p, Snq2p, and Yor1p. Expression of these genes is under the control of two homologous zinc finger-containing transcription regulators, Pdr1p and Pdr3p. Here, we describe the isolation, by an in vivo screen, of two new Pdr1p-Pdr3p target genes: HXT11 and HXT9. HXT11 and HXT9, encoding nearly identical proteins, have a high degree of identity to monosaccharide transporters of the major facilitator superfamily (MFS). In this study, we show that the HXT11 product, which allows glucose uptake in a glucose permease mutant (rag1) strain of Kluyveromyces lactis, is also involved in the pleiotropic drug resistance process. Loss of HXT11 and/or HXT9 confers cycloheximide, sulfomethuron methyl, and 4-NQO (4-nitroquinoline-N-oxide) resistance. Conversely, HXT11 overexpression increases sensitivity to these drugs in the wild-type strain, an effect which is more pronounced in a strain having both PDR1 and PDR3 deleted. These data show that the two putative hexose transporters Hxt11p and Hxt9p are transcriptionally regulated by the transcription factors Pdr1p and Pdr3p, which are known to regulate the production of ABC transporters required for drug resistance in yeast. We thus demonstrate the existence of genetic interactions between genes coding for two classes of transporters (ABC and MFS) to control the multidrug resistance process.

The yeast ATP binding cassette (ABC) protein genes PDR10 and PDR15 are novel targets for the Pdr1 and Pdr3 transcriptional regulators.

The yeast transcription factors Pdr1 and Pdr3 control pleiotropic drug resistance (PDR) development, since they regulate expression of ATP-binding cassette (ABC) drug efflux pumps through binding to cis-acting sites known as PDREs (PDR responsive elements). In this report, we show by Northern blotting, gel shift mobility assays and DNase I footprinting that transcription of the ABC genes PDR10 and PDR15 is also controlled by Pdr1 and Pdr3. In addition, in vitro band shift assays demonstrate that a GST-Pdr1 fusion protein can bind to the PDREs of PDR10 and PDR15. DNase I footprinting allowed the identification of the precise PDRE binding motifs, indicating the presence of a novel slightly degenerate PDRE motif in the PDR15 promoter. Finally, PDR10 and PDR15 mRNA levels vary dramatically in abundance in isogenic yeast strains carrying either deltapdr1, deltapdr3 and deltapdr1 deltapdr3 deletions or pdr1-3 and pdr3-2 gain-of-function mutations, demonstrating that both PDR10 and PDR15 are new members of the yeast PDR network.

The ATP-binding cassette multidrug transporter Snq2 of Saccharomyces cerevisiae: a novel target for the transcription factors Pdr1 and Pdr3.

Pleiotropic drug resistance (PDR) in the yeast Saccharomyces cerevisiae can arise from overexpression of ATP-binding cassette (ABC) efflux pumps such as Pdr5 and Snq2. Mutations in the transcription factor genes PDR1 and PDR3 are also associated with PDR. We show here that a pdr1-3 mutant exhibits a PDR phenotype, including elevated resistance to the mutagen 4-nitroquinoline-N-oxide, a known substrate for Snq2 but not for Pdr5. Northern analysis and immunoblotting demonstrated that the SNQ2 gene is 10-fold overexpressed in a pdr1-3 gain-of-function mutant strain, whereas Snq2 expression is severely reduced in a delta pdr1 deletion strain, and almost abolished in a delta pdr1 delta pdr3 double disruptant when compared to the PDR1 strain. However, expression of the Ste6 a-factor pheromone transporter, another yeast ABC transporter not associated with PDR, is unaffected in pdr1-3 mutant cells and in strains carrying delta pdr1, delta pdr3, or delta pdr1 delta pdr3 deletions. Finally, DNA footprint analysis revealed that the SNQ2 promoter contains three binding sites for Pdr3. Our results identify SNQ2 as a novel target for both Pdr1 and Pdr3, and demonstrate that the PDR phenotype of a pdr1-3 mutant strain results from overexpression of more than one ABC drug-efflux pump.

Positive autoregulation of the yeast transcription factor Pdr3p, which is involved in control of drug resistance.

Simultaneous resistance to an array of drugs with different cytotoxic activities is a property of Saccharomyces cerevisiae, in which the protein Pdr3p has recently been shown to play a role as a transcriptional regulator. We provide evidence that the yeast PDR3 gene, which encodes a zinc finger transcription factor implicated in certain drug resistance phenomena, is under positive autoregulation by Pdr3p. DNase I footprinting analyses using bacterially expressed Pdr3p showed specific recognition by this protein of at least two upstream activating sequences in the PDR3 promoter. The use of lacZ reporter constructs, a mutational analysis of the upstream activating sequences, as well as band shift experiments enabled the identification of two 5'TC CGCGGA3' sequence motifs in the PDR3 gene as consensus elements for the binding of Pdr3p. Several similar sequence motifs can be found in the promoter of PDR5, a gene encoding an ATP-dependent drug pump whose Pdr3p-induced overexpression is responsible for drug resistance phenomena. Recently one of these sequence elements was shown to be the target of Pdr3p to elevate the level of PDR5 transcription. Finally, we provide evidence in the absence of PDR1 for a PDR3-controlled transcriptional induction of the drug pump by cycloheximide and propose a model for the mechanism governing the transcriptional autoregulation of Pdr3p.

PDR3, a new yeast regulatory gene, is homologous to PDR1 and controls the multidrug resistance phenomenon.

The Saccharomyces cerevisiae PDR3 gene, located near the centromere of chromosome II, has been completely sequenced and characterised. Mutations pdr3-1 and pdr3-2, which confer resistance to several antibiotics can be complemented by a wild-type allele of the PDR3 gene. The sequence of the wild-type PDR3 gene revealed the presence of a long open reading frame capable of encoding a 976-amino acid protein. The protein contains a single Zn(II)2Cys6 binuclear-type zinc finger homologous to the DNA-binding motifs of other transcriptional activators from lower eukaryotes. Evidence that the PDR3 protein is a transcriptional activator was provided by demonstrating that DNA-bound LexA-PDR3 fusion proteins stimulate expression of a nearby promoter containing LexA binding sites. The use of LexA-PDR3 fusions revealed that the protein contains two activation domains, one localised near the N-terminal, cysteine-rich domain and the other localised at the C-terminus. The salient feature of the PDR3 protein is its similarity to the protein coded by PDR1, a gene responsible for pleiotropic drug resistance. The two proteins show 36% amino acid identity over their entire length and their zinc finger DNA-binding domains are highly conserved. The fact that the absence of both PDR1 and PDR3 (simultaneous disruption of the two genes) enhances multidrug sensitivity strongly suggests that the two transcriptional factors have closely related functions.

Connections between RNA splicing and DNA intron mobility in yeast mitochondria: RNA maturase and DNA endonuclease switching experiments.

The intron-encoded proteins bI4 RNA maturase and aI4 DNA endonuclease can be faithfully expressed in yeast cytoplasm from engineered forms of their mitochondrial coding sequences. In this work we studied the relationships between these two activities associated with two homologous intron-encoded proteins: the bI4 RNA maturase encoded in the fourth intron of the cytochrome b gene and the aI4 DNA endonuclease (I-SceII) encoded in the fourth intron of the gene coding for the subunit I of cytochrome oxidase. Taking advantage of both the high recombinogenic properties of yeast and the similarities between the two genes, we constructed in vivo a family of hybrid genes carrying parts of both RNA maturase and DNA endonuclease coding sequences. The presence of a sequence coding for a mitochondrial targeting peptide upstream from these hybrid genes allowed us to study the properties of their translation products within the mitochondria in vivo. We thus could analyze the ability of the recombinant proteins to complement RNA maturase deficiencies in different strains. Many combinations of the two parental intronic sequences were found in the recombinants. Their structural and functional analysis revealed the following features. (i) The N-terminal half of the bI4 RNA maturase could be replaced in total by its equivalent from the aI4 DNA endonuclease without affecting the RNA maturase activity. In contrast, replacing the C-terminal half of the bI4 RNA maturase with its equivalent from the aI4 DNA endonuclease led to a very weak RNA maturase activity, indicating that this region is more differentiated and linked to the maturase activity. (ii) None of the hybrid proteins carrying an RNA maturase activity kept the DNA endonuclease activity, suggesting that the latter requires the integrity of the aI4 protein. These observations are interesting because the aI4 DNA endonuclease is known to promote the propagation, at the DNA level, of the aI4 intron, whereas the bI4 RNA maturase, which is required for the splicing of its coding intron, also controls the splicing process of the aI4 intron. We propose a scenario for the evolution of these intronic proteins that relies on a switch from DNA endonuclease to RNA maturase activity.

A new specific DNA endonuclease activity in yeast mitochondria.

Two group I intron-encoded proteins from the yeast mitochondrial genome have already been shown to have a specific DNA endonuclease activity. This activity mediates intron insertion by cleaving the DNA sequence corresponding to the splice junction of an intronless strain. We have discovered in mitochondrial extracts from the yeast strain 777-3A a new DNA endonuclease activity which cleaves the fused exon A3-exon A4 junction sequence of the CO XI gene.

In vivo and in vitro analyses of an intron-encoded DNA endonuclease from yeast mitochondria. Recognition site by site-directed mutagenesis.

The pal 4 nuclease (termed I-Sce II) is encoded in the group I al 4 intron of the COX I gene of Saccharomyces cerevisiae. It introduces a specific double-strand break at the junction of the two exons A4-A5 and thus mediates the insertion of the intron into an intronless strain. To define the sequence recognized by pal 4 we introduced 35 single mutations in its target sequence and examined their cleavage properties either in vivo in E. coli (when different forms of the pal 4 proteins were artificially produced) or in vitro with mitochondrial extracts of a mutant yeast strain blocked in the splicing of the al 4 intron. We also detected the pal 4 DNA endonuclease activity in extracts of the wild type strain. The results suggest that 6 to 9 noncontiguous bases in the 17 base-pair region examined are necessary for pal 4 nuclease to bind and cleave its recognition site. We observed that the pal 4 nuclease specificity can be significantly different with the different forms of the protein thus explaining why only some forms are highly toxic in E. coli. This study shows that pal 4 recognition site is a complex phenomenon and this might have evolutionary implications on the transfer properties of the intron.

Site-specific DNA endonuclease and RNA maturase activities of two homologous intron-encoded proteins from yeast mitochondria.

Two introns of the mitochondrial genome 777-3A of S. cerevisiae, bl4 in cob and al4 in coxl genes, contain ORFs that can be translated into two homologous proteins. We changed the UGA, AUA, and CUN codons of these ORFs to the universal genetic code, in order to study the functions of their translated products in E. coli and in yeast, by retargeting the nuclear encoded protein into mitochondria. The p27bl4 protein has been shown to be required for the splicing of both introns bl4 and al4. The homologous p28al4 protein is highly toxic to E. coli. It can specifically cleave double-stranded DNA at a sequence representing the junction of the two fused flanking exons. We present evidence that this system is a good model for studying the role of mitochondrial intron-encoded proteins in the rearrangement of genetic information at both the RNA (RNA splicing-bl4 maturase) and DNA levels (intron transposition-al4 transposase).

A mitochondrial RNA maturase gene transferred to the yeast nucleus can control mitochondrial mRNA splicing.

bI4 maturase, encoded by the fourth intron of the yeast mitochondrial cytochrome b gene, controls the splicing of both the fourth intron of the cytochrome b gene and the fourth intron of the gene encoding subunit I of cytochrome oxidase. By fusing the encoding presequence of subunit 9 of the Neurospora ATPase to a restriction fragment containing the bI4 maturase coding sequence, we have constructed a hybrid gene that can be translated on yeast cytosolic ribosomes. The resulting protein is imported into mitochondria, which was revealed by its ability to restore to respiratory competence a yeast mutant defective in the bI4 maturase. Moreover, a protein reacting with antimaturase antibodies was detected in the mitochondria of the transformed cells; this imported maturase functioned similarly to the endogenous maturase.

Solubilization and insertion into reverse micelles of the major myelin transmembrane proteolipid.

The Folch-Pi proteolipid has been isolated from bovine white matter and characterized with respect to phospholipid and glycolipid composition. The protein-lipid complex has been solubilized in aqueous reverse micelles of di(2-ethylhexyl) sodium sulfosuccinate and isooctane. Solubilization of this otherwise water-insoluble proteolipid requires small amounts of water, the percent of solubility being maximum for a low molar ratio of water to surfactant (Wo = 5.6). Unlike hydrophilic proteins, the extent of incorporation into the micellar system is negligible at 50 mM surfactant and reaches 90Vo only at 300 mM. However, the conformation of the proteolipid in reverse micelles as studied by fluorescence emission spectroscopy and circular dichroism was not affected by variations of the surfactant concentration. These results are consistent with the peculiar properties of the aqueous environment of the proteolipid within the reverse micelles and may reflect the membrane-like character of these bio-assemblies.