HMGB proteins involved in TOR signaling as general regulators of cell growth by controlling ribosome biogenesis.

The number of ribosomes and their activity need to be highly regulated because their function is crucial for the cell. Ribosome biogenesis is necessary for cell growth and proliferation in accordance with nutrient availability and other external and intracellular signals. High-mobility group B (HMGB) proteins are conserved from yeasts to human and are decisive in cellular fate. These proteins play critical functions, from the maintenance of chromatin structure, DNA repair, or transcriptional regulation, to facilitation of ribosome biogenesis. They are also involved in cancer and other pathologies. In this review, we summarize evidence of how HMGB proteins contribute to ribosome-biogenesis control, with special emphasis on a common nexus to the target of rapamycin (TOR) pathway, a signaling cascade essential for cell growth and proliferation from yeast to human. Perspectives in this field are also discussed.

Dual function of Ixr1 in transcriptional regulation and recognition of cisplatin-DNA adducts is caused by differential binding through its two HMG-boxes.

Ixr1 is a transcriptional factor involved in the response to hypoxia, which is also related to DNA repair. It binds to DNA through its two in-tandem high mobility group box (HMG-box) domains. Each function depends on recognition of different DNA structures, B-form DNA at specific consensus sequences for transcriptional regulation, or distorted DNA, like cisplatin-DNA adducts, for DNA repair. However, the contribution of the HMG-box domains in the Ixr1 protein to the formation of different protein-DNA complexes is poorly understood. We have biophysically and biochemically characterized these interactions with specific DNA sequences from the promoters regulated by Ixr1, or with cisplatin-DNA adducts. Both HMG-boxes are necessary for transcriptional regulation, and they are not functionally interchangeable. The in-tandem arrangement of their HMG-boxes is necessary for functional folding and causes sequential cooperative binding to specific DNA sequences, with HMG-box A showing a higher contribution to DNA binding and bending than the HMG-box B. Binding of Ixr1 HMG boxes to specific DNA sequences is entropy driven, whereas binding to platinated DNA is enthalpy driven for HMG-box A and entropy driven for HMG-box B. This is the first proof that HMG-box binding to different DNA structures is associated with predictable thermodynamic differences. Based on our study, we present a model to explain the dual function of Ixr1 in the regulation of gene expression and recognition of distorted DNA structures caused by cisplatin treatment.

Comparative transcriptome analysis of yeast strains carrying slt2, rlm1, and pop2 deletions.

The function of the genes SLT2 (encoding the Mpk1 protein), RLM1, and POP2 have previously been related to several stress responses in yeasts. DNA arrays have been used to identify differences among the transcriptomes of a Saccharomyces cerevisiae wild type strain and its derivative Δslt2, Δrlm1, and Δpop2 mutants. Correspondence analyses indicate that the vast majority of genes that show lower expression in Δrlm1 also show lower expression in Δslt2. In contrast, there is little overlap between the results of the transcriptome analyses of the Δpop2 strain and the Δslt2 or Δrlm1 strains. The DNA array data were validated by reverse Northern blotting and chromatin immunoprecipitation (ChIp). ChIp assays demonstrate Rlm1p binding to specific regions of the promoters of two genes that show expression differences between the Δrlm1 mutant and wild type strains. Interestingly, RLM1 deletion decreases the transcription of SLT2, encoding the Mpk1p kinase that phosphorylates Rlm1p, suggesting a feedback control in the signal transduction pathway. Also, deletion of RLM1 causes a decrease in the mRNA level of KDX1, which is paralogous to SLT2. In contrast, deletion of POP2 is accompanied by an increase of both SLT2 and KDX1 levels. We show that SLT2 mRNA increase in the Δpop2 strain is due to a decrease in RNA turnover, consistent with the expected loss of RNA-deadenylase activity in this strain.

Proteomic analysis of the oxidative stress response in Kluyveromyces lactis and effect of glutathione reductase depletion.

Yeasts are unicellular eukaryotes that provide useful models for studying the oxidative stress (OS) response. Most investigations to date have been performed on the fermentative Saccharomyces cerevisiae. The respiratory Kluyveromyces lactis is emerging as an alternative model. Our previous studies showed that glutathione reductase (Glr1) is an interesting point of difference in the OS response between the two yeasts. In the present study, using extensive proteomic analyses, the response to H(2)O(2) and its relationship to Glr1 were investigated in wild-type and glr1-deletion mutant K. lactis strains. We identified 46 proteins that showed modified expression after H(2)O(2) addition and 42 for which the change was Glr1-dependent. As expected, these proteins include a variety of antioxidant enzymes, chaperones, and oxidoreductases related to defense against OS and damage repair. They also include a number of proteins necessary for energy production and carbohydrate and amino acid metabolism. H(2)O(2) addition causes down-regulation of enzymes from the glycolytic pathway and Krebs cycle in wild-type K. lactis, whereas glr1-deletion prevents this effect and actually causes up-regulation of the glycolytic, Krebs cycle, and oxidative pentose phosphate pathways. To our knowledge, this is the first global proteomic analysis performed on K. lactis.

A functional analysis of Kluyveromyces lactis glutathione reductase.

Glutathione reductase (GLR) null mutants of the yeast Kluyveromyces lactis, a model eukaryotic respiratory cell, were created and phenotypically analysed. We found that cells lacking GLR show decreased resistance to oxidative stress and higher levels of reactive oxygen species and catalase activity than the wild-type strain. However, glutathione redox levels (GSH : GSSG ratio) were similar in the DeltaKlglr1 mutant and wild-type strains. The thioredoxin-thioredoxin reductase system is proposed as an alternative system for maintaining the GSH : GSSG ratio in the DeltaKlglr1 mutant. The involvement of GLR in glucose metabolism in K. lactis is suggested by the improved growth on glucose caused by the DeltaKlglr1 mutation and by the increased GLR activity in the DeltaKlgcr1 strain, KlGcr1 being a transcriptional activator of glycolytic genes. We also studied the subcellular location of GLR in K. lactis, showing that it is present in mitochondria; however, the DeltaKlglr1 mutation does not affect mitochondrial morphology. Genomic DNA integrity and life span are also unaffected by the DeltaKlglr1 mutation, at least under the conditions tested in this study.

The nuclear genes encoding the internal (KlNDI1) and external (KlNDE1) alternative NAD(P)H:ubiquinone oxidoreductases of mitochondria from Kluyveromyces lactis.

Cloning, sequence and functional analyses of the Kluyveromyces lactis genes KlNDI1 and KlNDE1 are reported. These genes encode for proteins with high homology to the mitochondrial internal (Ndi1p) and external (Nde1p) alternative NADH:ubiquinone oxidoreductases from Saccharomyces cerevisiae and complement the respective mutations. Analysis of KlNDI1 transcriptional regulation showed that expression of this gene is lower in 2% glucose than in 0.5% glucose or non-fermentable carbon sources. Beta-galactosidase activity values, shown by lacZ fusions of KlNDI1 promoter deletions, suggested that two Adr1p binding sites mediate this carbon source regulation of KlNDI1. The expression of the KlNDE1 gene in S. cerevisiae mutant strains and measurement of respiration with isolated mitochondria showed that the protein encoded by KlNDE1 oxidizes NADPH, this being an important difference with respect to the conventional yeast S. cerevisiae. Moreover, Northern blot experiments using a phosphoglucose isomerase mutant showed that KlNDE1 gene transcription increases with glucose metabolism through the pentose phosphate pathway.

Characterization of a gene similar to BIK1 in the yeast Kluyveromyces lactis.

In Saccharomyces cerevisiae, Bik1p is a microtubule plus-end-tracking protein that plays several roles in mitosis and ploidy. KlBik1p (from Kluyveromyces lactis) maintains the same structural-domain organization as does S. cerevisiae Bik1p. As part of its characterization, we constructed a stable klbik1 mutant which is sensitive to benomyl only at 14 degrees C and has a higher frequency of crescent-shaped nuclei than S. cerevisiae bik1 mutants. This phenotype is partially rescued by S. cerevisiae BIK1. Other phenotypes associated with bik1 are not present in the K. lactis mutant. By fusion to GFP we were able to show the functionality of the KlBik1p CAP-Gly domain and found that the fusion protein changes its cellular location during the cell cycle.

Isolation and characterization of two nuclear genes encoding glutathione and thioredoxin reductases from the yeast Kluyveromyces lactis.

Response to oxidative stress has been hitherto scarcely studied in the respiratory yeast Kluyveromyces lactis. The genes coding for reductases of glutathione and thioredoxin, KlGLR1 and KlTRR1, respectively, have been cloned and characterized in this work. H(2)O(2) treatment increased transcription and enzyme activity of KlTRR1 but not of KlGLR1, suggesting a different situation from that reported for the fermentative yeast Saccharomyces cerevisiae. A consensus for Yap1p binding is functional in the KlTRR1 promoter.

Cloning genes from a library using a clustering strategy and PCR.

A new polymerase chain reaction (PCR)-based method is described for the isolation of clones of interest from a library when only part of a sequence is available. In actuality, this occurs with many genomes that have been partially sequenced using a random strategy. The method presented here, discriminating clusters by PCR (DCbyPCR), is a nonradioactive and improved alternative to colony hybridization.

Genome-wide analysis of the yeast transcriptome upon heat and cold shock.

DNA arrays were used to measure changes in transcript levels as yeast cells responded to temperature shocks. The number of genes upregulated by temperature shifts from 30 to 37 or 45 was correlated with the severity of the stress. Pre-adaptation of cells, by growth at 37 previous to the 45 shift, caused a decrease in the number of genes related to this response. Heat shock also caused downregulation of a set of genes related to metabolism, cell growth and division, transcription, ribosomal proteins, protein synthesis and destination. Probably all of these responses combine to slow down cell growth and division during heat shock, thus saving energy for cell rescue. The presence of putative binding sites for Xbp1p in the promoters of these genes suggests a hypothetical role for this transcriptional repressor, although other mechanisms may be considered. The response to cold shock (4) affected a small number of genes, but the vast majority of those genes induced by exposure to 4 were also induced during heat shock; these genes share in their promoters cis-regulatory elements previously related to other stress responses.

Genome-wide analysis of yeast transcription upon calcium shortage.

Several regulatory circuits related to important functions, like membrane excitation, immunoresponse, replication, control of the cell cycle and differentiation, among others, cause an increase in intracellular calcium level that finally has a consequence upon transcription of specific genes. The sequencing of the whole genome of eukaryotic cells enables genome-wide analysis of gene expression under many conditions not yet assessed by conventional methods. Using the array technology, the effect of calcium shortage in yeast cells was studied. Correspondence analysis of data showed that there is a response in transcription that is correlated to calcium shortage. The distribution of up-regulated-genes in functional categories suggests a regulatory connection between the cell-cycle progression and the energetic metabolic requirements for growth and division. In silico analysis of promoters reveals the frequent appearance of the Mlu I cell cycle box (MCB) cis element that binds the transcriptional regulatory factor Mcm1.

The KlCYC1 gene, a downstream region for two differentially regulated transcripts.

KlCYC1 encodes for cytochrome c in the yeast Kluyveromyces lactis and is transcribed in two mRNAs with different 3'-processing points. This is an uncommon transcription mechanism in yeast mRNAs. The 3' sequence encompassing the whole region that is needed to produce both mRNAs is analysed. We have determined identical processing points in K.lactis and in Saccharomyces cerevisiae cells transformed with KlCYC1; positions 698 and 1092 (with respect to the TAA) are the major polyadenylation points. This shows that the cis-elements present in the KlCYC1 3'-untranslated region (3'-UTR) direct a processing mechanism that has been conserved in yeast. In K. lactis there is a high predominance of the shorter transcript (1.14 kb) only at the initial logarithmic growth phase. Interestingly, this growth phase-dependent regulation of 3'-UTR processing is lost when the gene is expressed in S. cerevisiae.

New secretory strategies for Kluyveromyces lactis beta-galactosidase.

We examined several strategies for the secretion of Kluyveromyces lactis beta-galactosidase into the culture medium, in order to facilitate the downstream processing and purification of this intracellular enzyme of great industrial interest. We constructed plasmids by fusing the LAC4 gene or engineered variants to the secretion signal of the K.lactis killer toxin or to the secretion signal of the Saccharomyces cerevisiae alpha-factor. With these plasmids we transformed strains of the yeasts K.lactis and S.cerevisiae, respectively and tested beta-galactosidase extracellular activity in different culture media. We achieved partial secretion of beta-galactosidase in the culture medium since the high molecular weight and oligomeric nature of the enzyme, among other factors, preclude full secretion. The percentage of secretion was improved by directed mutagenesis of the N-terminus of the protein. We developed several deletion mutants which helped us to propose structure-function relationships by comparison with the available data on the homologous Escherichia coli beta-galactosidase. The influence of the culture conditions on heterologous beta-galactosidase secretion was also studied.

Transcript analysis of 1003 novel yeast genes using high-throughput northern hybridizations.

The expression of 1008 open reading frames (ORFs) from the yeast Saccharomyces cerevisiae has been examined under eight different physiological conditions, using classical northern analysis. These northern data have been compared with publicly available data from a microarray analysis of the diauxic transition in S.cerevisiae. The results demonstrate the importance of comparing biologically equivalent situations and of the standardization of data normalization procedures. We have also used our northern data to identify co-regulated gene clusters and define the putative target sites of transcriptional activators responsible for their control. Clusters containing genes of known function identify target sites of known activators. In contrast, clusters comprised solely of genes of unknown function usually define novel putative target sites. Finally, we have examined possible global controls on gene expression. It was discovered that ORFs that are highly expressed following a nutritional upshift tend to employ favoured codons, whereas those overexpressed in starvation conditions do not. These results are interpreted in terms of a model in which competition between mRNA molecules for translational capacity selects for codons translated by abundant tRNAs.

Haem regulation of the mitochondrial import of the Kluyveromyces lactis 5-aminolaevulinate synthase: an organelle approach.

The enzyme 5-aminolaevulinate acid synthase (ALAS) catalyses the first reaction in the haem biosynthetic pathway. In eukaryotes this protein is translated by cytosolic ribosomes and then targeted to the mitochondria. We present evidence that in the yeast Kluyveromyces lactis haem exerts a feedback control upon the import of the ALAS into mitochondria. The ALAS from K. lactis (KlALAS) contains two haem regulatory motifs (HRM) in the mitochondrial targeting signal. Mutagenesis experiments reveal the involvement of these HRM in the response of the KlALAS to haem.

Heme-mediated transcriptional control in Kluyveromyces lactis.

Heme is of great importance in oxygen-dependent biological functions, since it serves as a prosthetic group for many proteins related to oxygen-binding, oxidative damage prevention and electron transport. It also regulates gene expression through the action of specific transcriptional regulatory factors. In this paper, we present an analysis of heme-dependent transcriptional regulation of several respiration-related genes in an aerobic respiratory yeast, Kluyveromyces lactis. We also report that the KlHEM13 gene, encoding the heme biosynthetic enzyme coproporphyrinogen oxidase, is under heme and oxygen transcriptional regulation, thereby controlling the synthesis of the effector, heme. KlHEM13 is induced during hypoxia, which represents the first report of a transcriptionally regulated gene with this behaviour in K. lactis.

Transcript analysis of 203 novel genes from Saccharomyces cerevisiae in hap1 and rox1 mutant backgrounds.

Hap1 and Rox1 are transcriptional regulators that bind regulatory sites in the promoters of oxygen-regulated genes in Saccharomyces cerevisiae. Hap1 is a heme-responsive activator of genes induced in aerobic conditions and Rox1 is a repressor of hypoxic genes in aerobic conditions. We have studied transcriptional regulation of a pool of 203 open reading frames (ORFs) from chromosomes IV, VII, and XIV in wild-type, hap1, and rox1 mutant genetic backgrounds in an attempt to extend the family of oxygen and heme regulated genes. Only three ORFs are significantly repressed by Rox1 but they cannot be considered as typical hypoxic genes because they are not overexpressed during hypoxia.

Kluyveromyces lactis HIS4 transcriptional regulation: similarities and differences to Saccharomyces cerevisiae HIS4 gene.

Sequence analysis of the Kluyveromyces lactis HIS4 (KlHIS4) gene promoter reveals relevant differences in comparison to the Saccharomyces cerevisiae HIS4 homologous gene. Among them are the absence of a Rap1 binding site and the presence of only three putative Gcn4 binding consensus sites instead of the five described in the S. cerevisiae promoter. Since these factors are implicated in the general control, we investigated the transcriptional regulation of the KlHIS4 gene under conditions of amino acid starvation and discovered that the mechanisms previously described for S. cerevisiae HIS4 regulation and related to general control are not functional in K. lactis. The expression analysis of the KlHIS4 gene under phosphate starvation or high adenine supply shows that factors, such as Bas1 or Bas2, involved in the basal control may also operate in a different way in K. lactis. Interestingly, and also in contrast to the HIS4 regulation in S. cerevisiae, we found domains for Nit2-like and yeast-Ap1-like binding sequences. Northern analyses showed transcriptional activation under ammonia starvation and oxidative stress.

Transcript analysis of 250 novel yeast genes from chromosome XIV.

The European Functional Analysis Network (EUROFAN) is systematically analysing the function of novel Saccharomyces cerevisiae genes revealed by genome sequencing. As part of this effort our consortium has performed a detailed transcript analysis for 250 novel ORFs on chromosome XIV. All transcripts were quantified by Northern analysis under three quasi-steady-state conditions (exponential growth on rich fermentative, rich non-fermentative, and minimal fermentative media) and eight transient conditions (glucose derepression, glucose upshift, stationary phase, nitrogen starvation, osmo-stress, heat-shock, and two control conditions). Transcripts were detected for 82% of the 250 ORFs, and only one ORF did not yield a transcript of the expected length (YNL285w). Transcripts ranged from low (62%), moderate (16%) to high abundance (2%) relative to the ACT1 mRNA. The levels of 73% of the 206 chromosome XIV transcripts detected fluctuated in response to the transient states tested. However, only a small number responded strongly to the transients: eight ORFs were induced upon glucose upshift; five were repressed by glucose; six were induced in response to nitrogen starvation; three were induced in stationary phase; five were induced by osmo-stress; four were induced by heat-shock. These data provide useful clues about the general function of these ORFs and add to our understanding of gene regulation on a genome-wide basis.