Jean Claude Jauniaux

A cis-dominant regulatory mutation linked to the argB-argC gene cluster in Saccharomyces cerevisiae☆

Abstract The argB-argC gene cluster of Saccharomyces cerevisiae , a genetic unit of coordinate expression, is presented as a good tool for investigating the possibility of eucaryotic cells producing more than a single polypeptide directly from one polycistronic messenger RNA. This conclusion is based on the following. The argB-argC gene cluster is transcribed as a single messenger RNA molecule, starting from the argB end (Minet et al. , 1979). New data are presented supporting the idea that two separate proteins, N -acetylglutamate kinase and N -acetylglutamylphosphate reductase, are produced in vivo from the argB-argC gene cluster. Further evidence is provided that the low levels of both enzyme activities in arginine-grown cells are due to repression rather than to inactivation of the enzymes. A regulatory mutation has been isolated which specifically affects the expression of the argB-argC gene cluster. It is tightly linked to the cluster and close to the argB end, the putative origin of transcription. It is cis -dominant in heterozygous diploids. It does not qualitatively affect the affinity constants of N -acetylglutamate kinase and N -acetylglutamylphosphate reductase for their respective main substrates. Additional characteristics are described showing that the mutation most probably affects an operator or attenuator site, or any other regulatory site with analogous properties controlling arginine repression on the argB-argC gene cluster. The data are consistent with several interpretations. 1. (1) The argB-argC gene cluster of S. cerevisiae is translated into a single polypeptide and subsequently processed in vivo into two distinct proteins by a specialized cutting mechanism. 2. (2) The cluster is organized as a bacterial operon. 3. (3) The dicistronic mRNA corresponding to the cluster is processed into two pieces, each of which is read independently.

The Saccharomyces cerevisiae NPR1 gene required for the activity of ammonia-sensitive amino acid permeases encodes a protein kinase homologue

SummaryTheNPR1 gene ofSaccharomyces cerevisiae plays a central role in controlling permease activity; its product is required to promote the activity of at least six distinct transport systems for nitrogenous nutrients under conditions of nitrogen catabolite derepression. We report here the nucleotide sequence of the clonedNPR1 gene. The predicted amino acid sequence indicates thatNPR1 encodes a protein of 86 kDa which appears to be organized into two distinct structural domains. The amino-terminal domain of NPR1 (residues 1 to 440) contains 26% serine residues and several regions strongly enriched for PEST residues suggesting a short half-life for the NPR1 protein. The carboxy-terminal region of NPR1 contains consensus sequences characteristic of the catalytic domains of protein kinases. Therefore, NPR1-dependent positive control of nitrogen transport systems most likely involves protein phosphorylation. Northern analysis indicates that the absence of general amino acid permease (GAP1) activity innpr1 mutants is not due to reduction in transcription or messenger stability. Hence, the NPR1 protein probably acts at the post-transcriptional level. Proteins that may serve as substrates for phosphorylation are discussed.

Cloning and expression of the UGA4 gene coding for the inducible GABA-specific transport protein of Saccharomyces cerevisiae

SummaryTransport of 4-aminobutyric acid (GABA) in Saccharomyces cerevisiae is mediated by three transport systems: the general amino acid permease (GAP1 gene), the proline permease (PUT4 gene), and a specific GABA permease (UGA4 gene) which is induced in the presence of GABA. The UGA4 gene encoding the inducible GABA-specific transporter was cloned and sequenced and its expression analyzed. The predicted amino acid sequence shows that UGA4 encodes a 62 kDa protein having 9–12 putative membrane-spanning regions. The predicted UGA4 protein shares significant sequence similarity with the yeast choline transporter (CTR gene), exhibiting but limited similarity to the previously reported GABA transporters, i.e. the yeast GAP1 and PUT4 permeases and the rat brain GAT-1 transporter. Induction of UGA4 in the presence of GABA is exerted at the level of UGA4 mRNA accumulation, most probably at the level of transcription itself. This induction is conferred by the 5′ flanking region and requires the integrity of two positive regulatory proteins, the inducer-specific factor UGA3 and the pleiotropic factor UGA35/DURL/DAL81. In the absence of the pleiotropic UGA43/DAL80 repressor, UGA4 is constitutively expressed at high level.

Nucleotide sequence of the Saccharomyces cerevisiae PUT4 proline-permease-encoding gene: similarities between CAN1, HIP1 and PUT4 permeases

The complete nucleotide (nt) sequence of the PUT4 gene, whose product is required for high-affinity proline active transport in the yeast Saccharomyces cerevisiae, is presented. The sequence contains a single long open reading frame of 1881 nt, encoding a polypeptide with a calculated Mr of 68,795. The predicted protein is strongly hydrophobic and exhibits six potential glycosylation sites. Its hydropathy profile suggests the presence of twelve membrane-spanning regions flanked by hydrophilic N- and C-terminal domains. The N terminus does not resemble signal sequences found in secreted proteins. These features are characteristic of integral membrane proteins catalyzing translocation of ligands across cellular membranes. Protein sequence comparisons indicate strong resemblance to the arginine and histidine permeases of S. cerevisiae, but no marked sequence similarity to the proline permease of Escherichia coli or to other known prokaryotic or eukaryotic transport proteins. The strong similarity between the three yeast amino acid permeases suggests a common ancestor for the three proteins.

Molecular cloning, DNA structure, and RNA analysis of the arginase gene in Saccharomyces cerevisiae. A study of cis-dominant regulatory mutations

The Saccharomyces cerevisiae gene cargA + or CAR1, encoding arginase has been cloned by recovering function in transformed yeast cells. It was used to analyse RNA and chromosomal DNA from six strains bearing cis‐dominant regulatory mutations leading to constitutive arginase synthesis. The DNA from the four cargA + O‐ strains in which constitutive arginase synthesis was independent of the mating‐type functions showed no detectable differences with the wild‐ typye . The cargA + O‐ mutations were, therefore, small alterations, possibly single base substitutions. On the other hand, the cargA + Oh‐1 and cargA + Oh‐2 mutations, leading to a constitutive and mating‐type dependent arginase synthesis, were identified as insertions. Their size and restriction pattern strongly suggested that they were induced by the Ty1 yeast transposable element. This was confirmed by cloning and analysis of the cargA + Oh‐1 mutant gene. The concentration of arginase RNA was significantly increased in the mutants, indicating that the regulation of arginase synthesis was exerted, at least in part, at the level of RNA synthesis or stability. In the cargA + Oh‐2 strain the Ty1 element was located at a distance of approximately 600 base pairs from the insertion present in the cargA + Oh‐1 strain. This result suggests either a surprisingly large arginase regulatory region or an indirect influence of the Ty1 element on gene expression over long distances.

Inhibition of ornithine carbamoyltransferase by arginase among yeasts: correlation with energy production, subcellular localization and enzyme synthesis

Summary: Thirty-two yeast species belonging to twelve genera were examined for the occurrence of inhibition of ornithine carbamoyltransferase by arginase (epiarginasic regulation) and related properties. Obligate aerobes were devoid of this regulation. Among fermenting species, Schizosaccharomyces and budding genera had different properties: all Schizosaccharomyces species were devoid of this regulation whereas all species of budding yeasts showing a weak or absent Pasteur effect had this regulation. Strains showing a strong Pasteur effect and taxonomically related to Saccharomyces (Kluyveromyces) had the regulation, whereas species classified in genera that include species which are obligate aerobes did not. We confirm that the absence of epiarginasic regulation is correlated with a mitochondrial localization of ornithine carbamoyltransferase but not with the function of the mitochondria in Schizosaccharomyces japonicus. In this case, compartmentation could serve to channel anabolic and catabolic functions. Although, in general, the arginase of ‘negative epiarginasic’ species had no affinity for cytosolic ornithine carbamoyltransferase, one exception was found (Hansenula anomala). Regulation by repression of ornithine carbamoyltransferase and induction of arginase by arginine was the general rule. These types of regulation of enzyme synthesis were only absent in yeasts in which compartmentation is present and could serve as the basis for anabolism—catabolism exclusion.

Organization and expression of a two-gene cluster in the arginine biosynthesis of Saccharomyces cerevisiae

SummaryIn Saccharomyces cerevisiae, argB and argC define two adjacent and complementing loci, with mutants defective in two consecutive steps of arginine biosynthesis: N-acetylglutamate kinase (AG-kinase) and N-acetylglutamyl-phosphate reductase (AGPreductase). These enzymic activities are readily separated by ammonium sulfate fractionation or Sephadex G-200 chromatography. This suggests that each activity is carried in vivo by a different protein. The synthesis of the two enzymes is coordinately regulated, with an 85-fold difference in specific activities between fully repressed and fully derepressed cells. Missense mutations in the argB locus are defective in AGkinase only. Nonsense mutations in the argB locus are defective in both activities. Missense and nonsense mutations in the argC locus are defective in AGPreductase, with a few alleles also showing a reduced level of AGkinase. These data are best explained by assuming that argB and argC are two genes transcribed as a single messenger from argB to argC. This messenger produces in vivo two distinct proteins corresponding to the argB and argC gene products, either because translation can be initiated at the beginning of both genes, or because a large polypeptide is specifically cut in vivo to yield the gene products of argB and argC.

Schizosaccharomyces pombe Pmf1p is structurally and functionally related to Mmf1p of Saccharomyces cerevisiae

A novel family of small proteins, termed p14.5 or YERO57c/YJGFc, has been identified. Independent studies indicate that p14.5 family members are multifunctional proteins involved in several pathways, e.g. regulation of translation or activation of the protease µ‐calpain. We have previously shown that Mmf1p, a p14.5 of the budding yeast Saccharomyces cerevisiae, is localized in the mitochondria and influences mitochondrial DNA stability. In addition, we have demonstrated that Mmf1p is functionally related to p14.5 of mammalian cells. To explore further the evolutionary conservation of the mitochondrial function(s) of the p14.5s we have extended our study to the fission yeast, Schizosaccharomyces pombe. In this organism two p14.5 homologous proteins are present: Pmf1p (pombe mitochondrial factor 1) and Hpm1p (homologous Pmf1p factor 1). We have generated a specific Pmf1p antibody, which recognizes a single band of approximately 15 kDa in total cellular extracts. Cellular fractionation experiments indicate that Pmf1p localizes in the mitochondria as well as in the cytoplasm. We also show that Pmf1p shares several properties of S. cerevisiae Mmf1p. Indeed, Pmf1p restores the wild‐type phenotype when expressed in Δmmf1 S. cerevisiae cells. Deletion of the leader sequence of Pmf1p abrogates its ability to localize in mitochondria and to functionally replace Mmf1p. Thus, these data together with our previous study show that the mitochondrial function(s) of the p14.5 family members are highly conserved in eukaryotic cells. Copyright