The asymmetric localization of mRNA plays an important role in coordinating posttranscriptional events in eukaryotic cells. We investigated the peripheral mitochondrial localization of nuclear-encoded mRNAs (MLR) in various conditions in which the mRNA binding protein context and the translation efficiency were altered. We identified Puf3p, a Pumilio family RNA-binding protein, as the first trans-acting factor controlling the MLR phenomenon. This allowed the characterization of two classes of genes whose mRNAs are translated to the vicinity of mitochondria. Class I mRNAs (256 genes) have a Puf3p binding motif in their 3'UTR region and many of them have their MLR properties deeply affected by PUF3 deletion. Conversely, mutations in the Puf3p binding motif alter the mitochondrial localization of BCS1 mRNA. Class II mRNAs (224 genes) have no Puf3p binding site and their asymmetric localization is not affected by the absence of PUF3. In agreement with a co-translational import process, we observed that the presence of puromycin loosens the interactions between most of the MLR-mRNAs and mitochondria. Unexpectedly, cycloheximide, supposed to solidify translational complexes, turned out to destabilize a class of mRNA-mitochondria interactions. Classes I and II mRNAs, which are therefore transported to the mitochondria through different pathways, correlated with different functional modules. Indeed, Class I genes code principally for the assembly factors of respiratory chain complexes and the mitochondrial translation machinery (ribosomes and translation regulators). Class II genes encode proteins of the respiratory chain or proteins involved in metabolic pathways. Thus, MLR, which is intimately linked to translation control, and the activity of mRNA-binding proteins like Puf3p, may provide the conditions for a fine spatiotemporal control of mitochondrial protein import and mitochondrial protein complex assembly. This work therefore provides new openings for the global study of mitochondria biogenesis.

Gene expression plays a central role in the orchestration of cellular processes. The use of inducible promoters to change the expression level of a gene from its physiological level has significantly contributed to the understanding of the functioning of regulatory networks. However, from a quantitative point of view, their use is limited to short-term, population-scale studies to average out cell-to-cell variability and gene expression noise and limit the nonpredictable effects of internal feedback loops that may antagonize the inducer action. Here, we show that, by implementing an external feedback loop, one can tightly control the expression of a gene over many cell generations with quantitative accuracy. To reach this goal, we developed a platform for real-time, closed-loop control of gene expression in yeast that integrates microscopy for monitoring gene expression at the cell level, microfluidics to manipulate the cells' environment, and original software for automated imaging, quantification, and model predictive control. By using an endogenous osmostress responsive promoter and playing with the osmolarity of the cells environment, we show that long-term control can, indeed, be achieved for both time-constant and time-varying target profiles at the population and even the single-cell levels. Importantly, we provide evidence that real-time control can dynamically limit the effects of gene expression stochasticity. We anticipate that our method will be useful to quantitatively probe the dynamic properties of cellular processes and drive complex, synthetically engineered networks.model based control | computational biology | high osmolarity glycerol pathway | quantitative systems biology U nderstanding the information processing abilities of biological systems is a central problem for systems and synthetic biology (1-6). The properties of a living system are often inferred from the observation of its response to static perturbations. Timevarying perturbations have the potential to be much more informative regarding the dynamics of cellular functions (7-12). Currently, it is not possible to precisely perturb protein levels in an analogous manner, even though this perturbation would be instrumental in our understanding of gene regulatory networks. Indeed, despite the development of novel regulatory systems, including various RNA-based solutions (13), transcriptional control by means of inducible promoters is still the preferred method for manipulating protein levels (14, 15). Unfortunately, inducible promoters have several generic limitations. First, there is a significant delay between gene expression activation and effective protein synthesis. Second, many cellular processes can interfere with gene expression through internal feedback loops whose effects are hard to predict. Third, the process of gene expression shows significant levels of noise (16)(17)(18). Given these limitations, novel experimental strategies are required to gain quantitative, real-time control of gene expression in vivo.Here, we see the...

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.

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.The diverse responses of eukaryotic cells to variations in the external environment are essential in shaping their identities. In this respect, one of the most important and general phenomena is the multidrug resistance process in which a cell can respond to a wide range of unrelated compounds. This process seems to apply to the vast majority of eukaryotic cells and has considerable fundamental and applied implications in both medicine and agriculture. The known key molecules in this resistance phenomenon are the membrane proteins belonging to either ABC (ATP-binding-cassette) or MFS (major facilitators superfamily) transport family. In the yeast Saccharomyces cerevisiae, genes of the ABC family include STE6, a homolog of the mammalian MDR1 gene (21, 22) whose product is involved in the secretion of the mating pheromone a, and the genes PDR5 (4), also known as STS1 (6), and SNQ2 (34), which are involved in the resistance to many different drugs. Many other ABC or MFS homologs are being discovered in the framework of the European project of systematic sequencing of the yeast genome (3), demonstrating the importance and complexity of this phenomenon.The regulatory elements controlling the expression of these transporter-encoding genes are poorly understood. Two networks of regulators are known to be involved in the drug resistance phenomenon in S. cerevisiae: (i) Yap1p, a leucine zipper factor that acts on the gene YCF1, an ABC homolog of the human cystic fibrosis transmembrane conductance regulator factor (25, 38), and Yap2p, a homologous factor conferring cadmium resistance (9, 41), and (ii) Pdr1p and Pdr3p, which belong to the C6 zinc cluster family of t...

The variety of environmental stresses is probably the major challenge imposed on transcription activators and the transcriptional machinery. To precisely describe the very early genomic response developed by yeast to accommodate a chemical stress, we performed time course analyses of the modifications of the yeast gene expression program which immediately follows the addition of the antimitotic drug benomyl. Similar analyses were conducted with different isogenic yeast strains in which genes coding for relevant transcription factors were deleted and coupled with efficient bioinformatics tools. Yap1 and Pdr1, two well-known key mediators of stress tolerance, appeared to be responsible for the very rapid establishment of a transient transcriptional response encompassing 119 genes. Yap1, which plays a predominant role in this response, binds, in vivo, promoters of genes which are not automatically up-regulated. We proposed that Yap1 nuclear localization and DNA binding are necessary but not sufficient to elicit the specificity of the chemical stress response.Cellular organisms develop a myriad of strategies to maintain specific internal conditions constantly challenged by the varying drug environment. The complexity of the yeast cell system for detecting and responding to environmental variations is only beginning to come to light. It has been reported previously (13) that a large set of yeast genes (about 900) showed a similar drastic response to a large variety of environmental changes including temperature shocks, hydrogen peroxide, menadione, diamide, dithiothreitol, hyper-or hypoosmotic shock, amino acid starvation, nitrogen source depletion, and progression into stationary phase. Since these pioneering studies were reported, many observations of the global effects of a large variety of drugs on gene expression have been made. In most of these studies, a binary comparison (i.e., control versus stress-exposed cells) was carried out, whereas in some cases, time course experiments over rather long periods (several hours) were conducted. Although much valuable information has been collected in these studies, the heterogeneity in the protocols followed precludes a simple comparison between the different drug responses. In particular, it is extremely difficult to identify the different regulatory networks and to establish their chronological relationships. Time series experiments soon appeared and were much more informative than simple binary experiments. Such approaches were a particularly valuable source of information in the case of cell cycle analyses (24, 27); however, they were less suitable to describe the chronology of transcriptional events in the case of environ-

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.Transmembrane solute transport is ensured in all eukaryotic cells by a set of proteins embedded in the plasma and the internal membranes. Most transport proteins characterized to date catalyze the uptake of solutes across the plasma membrane. Other plasma membrane transporters mediate extrusion of intracellular compounds into the medium, while others, located in intracellular membranes, catalyze efflux from or within the mitochondria, vacuole, peroxisomes, or secretion organelles. These membrane proteins are generally classified in three main categories: channels, facilitators (also named transporters, permeases, or carriers), and pumps (ATPases). Among them, one protein family of particular biological importance is the nonproton ATPase family encoding ATP-binding cassette (ABC) transporters, which appear to be conserved in all living organisms ranging from bacteria to humans (1, 13, 32). Alterations of certain ABC transporters can cause human genetic disorders such as cystic fibrosis (36), Zellweger syndrome (19), X-linked adrenoleukodystrophy (30), and the multidrug-resistance (MDR) phenotype shown by tumor cells which acquire resistance to a variety of chemotherapeutic agents. MDR phenotype is frequently linked to the increased expression, sometimes by gene amplification, of an integral membrane protein, a member of the ABC transporter family. This protein, called P-glycoprotein, functions as an ATP-dependent efflux pump for drugs (18).In the yeast Saccharomyces cerevisiae, a phenotype resembling the mammalian MDR exists and i...

We analysed the genome-wide regulatory properties of an artificial transcription activator in which the DNA-binding domain of the yeast transcription factor, Pdr1, was fused to the activation domain of Gal4 (Pdr1*GAD). This Pdr1*GAD chimera was put under the control of the inducible GAL1 promoter. DNA microarray analyses showed that all the target genes upregulated by the well-studied native gain-of-function Pdr1-3 mutant were similarly activated by the chimerical factor Pdr1*GAD upon galactose induction. Additionally, this kinetic approach led us not only to confirm previously published targets, but also to define a hierarchy among members of the Pdr1 regulon. Our observations prove, for the first time at the complete genome level, that the DNA-binding domain of Pdr1 is sufficient to guide its specificity. We propose that this approach could be useful for the study of new transcription factors identified in silico from sequenced organisms. Complete data are available at www.biologie.ens.fr/yeast-publi.html.

SummaryFlavohemoglobins are the main detoxifiers of nitric oxide (NO) in bacteria and fungi and are induced in response to nitrosative stress. In fungi, the flavohemoglobin encoding gene YHB1 is positively regulated by transcription factors which are activated upon NO exposure. In this study, we show that in the model yeast Saccharomyces cerevisiae and in the human pathogen Candida glabrata, the transcription factor Yap7 constitutively represses YHB1 by binding its promoter. Consequently, YAP7 deletion conferred high NO resistance to the cells. Coimmunoprecipitation experiments and mutant analyses indicated that Yap7 represses YHB1 by recruiting the transcriptional repressor Tup1. In S. cerevisiae, YHB1 repression also involves interaction of Yap7 with the Hap2/3/5 complex through a conserved Hap4-like-bZIP domain, but this interaction has been lost in C. glabrata. The evolutionary origin of this regulation was investigated by functional analyses of Yap7 and of its paralogue Yap5 in different yeast species. These analyses indicated that the negative regulation of YHB1 by Yap7 arose by neofunctionalization after the whole genome duplication which led to the C. glabrata and S. cerevisiae extant species. This work describes a new aspect of the regulation of fungal nitric oxidase and provides detailed insights into its functioning and evolution.