Yeast nitrogen catabolite repression is sustained by signals distinct from glutamine and glutamate reservoirs.

Nitrogen catabolite repression (NCR) is a wide transcriptional regulation program enabling baker's yeast to downregulate genes involved in the utilization of poor nitrogen sources when preferred ones are available. Nowadays, glutamine and glutamate, the major nitrogen donors for biosyntheses, are assumed to be key metabolic signals regulating NCR. NCR is controlled by the conserved TORC1 complex, which integrates nitrogen signals among others to regulate cell growth. However, accumulating evidence indicate that the TORC1-mediated control of NCR is only partial, arguing for the existence of supplementary regulatory processes to be discovered. In this work, we developed a genetic screen to search for new players involved in NCR signaling. Our data reveal that the NADP-glutamate dehydrogenase activity of Gdh1 negatively regulates NCR-sensitive gene transcription. By determining the total, cytoplasmic and vacuolar pools of amino acids, we show that there is no positive correlation between glutamine/glutamate reservoirs and the extent of NCR. While our data indicate that glutamine could serve as initial trigger of NCR, they show that it is not a sufficient signal to sustain repression and point to the existence of yet unknown signals. Providing additional evidence uncoupling TORC1 activity and NCR, our work revisits the dogmas underlying NCR regulation.

Proteomics as the tool to search for lung disease markers in bronchoalveolar lavage.

Most lung disorders are known to be associated to considerable modifications of surfactant composition. Numerous of these abnormalities have been exploited in the past to diagnose lung diseases, allowing proper treatment and follow-up. Diagnosis was then based on phospholipid content, surface tension and cytological features of the epithelial lining fluid (ELF), sampled by bronchoalveolar lavage (BAL) during fiberoscopic bronchoscopy. Today, it appears that the protein content of ELF displays a remarkably high complexity, not only due to the wide variety of the proteins it contains but also because of the great diversity of their cellular origins. The significance of the use of proteome analysis of BAL fluid for the search for new lung disease marker proteins and for their simultaneous display and analysis in patients suffering from lung disorders has been examined.

Differences in regulation of yeast gluconeogenesis revealed by Cat8p-independent activation of PCK1 and FBP1 genes in Kluyveromyces lactis.

The yeast Kluyveromyces lactis is can utilise a wide range of non-fermentable carbon compounds as sole sources of carbon and energy, and differs from Saccharomyces cerevisiae in being able to carry out oxidative and fermentative metabolism simultaneously. In S. cerevisiae, growth on all non-fermentable carbon sources requires Cat8p, a transcriptional activator that controls the expression of gluconeogenic and glyoxylate cycle genes via CSREs (Carbon Source Responsive Elements). The down-regulation of Cat8p by fermentable carbon sources is the primary factor responsible for the tight repression of gluconeogenesis by glucose in S. cerevisiae. To analyse the regulation of gluconeogenesis in K. lactis, we have cloned and characterised the K. lactis homologue of CAT8 (KlCAT8). The gene was isolated by multicopy suppression of a fog2/klsnf1 mutation, indicating a similar epistatic relationship between KlSNF1 and KlCAT8 as in the case of the S. cerevisiae homologues. KlCAT8 encodes a protein of 1445 amino acids that is 40% identical to ScCat8p. The most highly conserved block is the putative Zn(II)2Cys6 DNA-binding domain, but additional conserved regions shared with members of the zinc-cluster family from Aspergillus define a subfamily of Cat8p-related proteins. KlCAT8 complements the growth defect of a Sccat8 mutant on non-fermentable carbon sources. In K. lactis, deletion of KlCAT8 severely impairs growth on ethanol, acetate and lactate, but not on glycerol. Derepression of enzymes of the glyoxylate cycle--malate synthase and particularly isocitrate lyase--was impaired in a Klcat8 mutant, whereas Northern analysis revealed that derepression of KlFBP1 and KlPCK1 does not require KlCat8p. Taken together, our results indicate that in K. lactis gluconeogenesis is not co-regulated with the glyoxylate cycle, and only the latter is controlled by KlCat8p.

Kluyveromyces marxianus exhibits an ancestral Saccharomyces cerevisiae genome organization downstream of ADH2.

In Saccharomyces cerevisiae, the alcohol dehydrogenase genes ADH1 and ADH5 are part of a duplicated block of genome, thought to originate from a genome-wide duplication posterior to the divergence from the Kluyveromyces lineage. We report here the characterization of Kluyveromyces marxianus ADH2 and the five genes found in its immediate downstream region, MRPS9, YOL087C, RPB5, RIB7 and SPP381. The order of these six genes reflects the structure of the ancestral S. cerevisiae genome before the duplication that formed the blocks including ADH1 on chromosome XV and ADH5 on chromosome II, indicating these ADH genes share a direct ancestor. On the one hand, the two genes found immediately downstream of KmADH2 are located, for the first, downstream ADH5 and, for the second, downstream ADH1 in S. cerevisiae. On the other hand, the order of the paralogs included in the blocks of ADH1 and ADH5 in S. cerevisiae suggests that two of them have been inverted within one block after its formation, and that inversion is confirmed by the gene order observed in K. marxianus.

Glucose repression of the Kluyveromyces lactis invertase gene KlINV1 does not require Mig1p.

Kluyveromyces lactis, a budding yeast related to Saccharomyces cerevisiae, can grow on a wider variety of substrates and shows less sensitivity to glucose repression than does Saccharomyces cerevisiae. Many genes that are subject to glucose repression in S. cerevisiae are repressed only weakly or not at all in K. lactis. The molecular basis for this difference is largely unknown. To compare the mechanisms that regulate glucose repression in K. lactis and S. cerevisiae, we decided to clone and analyse an invertase gene from K. lactis. The SUC2 gene, which encodes invertase in S. cerevisiae, is strongly regulated by glucose and serves as a model system for studies on glucose repression. The invertase gene of K. lactis, KlINV1, was isolated by colony hybridization using a conserved region within the inulinase gene of K. marxianus as a probe. Two independent clones obtained were shown to contain the same ORF of 1827 bp. The deduced amino acid sequence is 59% similar to that of the K. marxianus inulinase and shows 49% similarity to ScSuc2p. Gene disruption experiments and low-stringency Southern analysis indicate that KlINV1 is a unique gene in K. lactis. Northern analysis revealed that the transcription of KlINV1 is strongly repressed in the presence of glucose, but, in contrast to the case in S. cerevisiae, repression is independent of KlMig1p.

The MIG1 repressor from Kluyveromyces lactis: cloning, sequencing and functional analysis in Saccharomyces cerevisiae.

Sequence comparisons between Saccharomyces cerevisiae ScMig1 and Aspergillus nidulans CREA proteins allowed us to design two sets of degenerate primers from the conserved zinc finger loops. PCR amplification on Kluyveromyces marxianus and K. lactis genomic DNA yielded single products with sequences closely related to each other and to the corresponding regions of ScMig1 and CREA. The KIMIG1 gene of K. lactis was cloned from a genomic library using the K. marxianus PCR fragment as probe. KIMIG1 encodes a 474-amino acid protein 55% similar to ScMig1. Besides their highly conserved zinc fingers, the two proteins display short conserved motifs of possible significance in glucose repression. Heterologous complementation of a mig1 mutant of S. cerevisiae by the K. lactis gene demonstrates that the function of the Mig1 protein is conserved in these two distantly related yeasts.