Marjolaine Crabeel

A New Yeast Metabolon Involving at Least the Two First Enzymes of Arginine Biosynthesis ACETYLGLUTAMATE SYNTHASE ACTIVITY REQUIRES COMPLEX FORMATION WITH ACETYLGLUTAMATE KINASE

Open reading frame YJL071W ofSaccharomyces cerevisiae was shown to be ARG2and identified as the structural gene for acetylglutamate synthase, first step in arginine biosynthesis. The three Ascomycete acetylglutamate synthases characterized to date appear homologous, but unlike the other enzymes of the yeast arginine biosynthesis pathway, they showed no significant similarity to their prokaryotic equivalents. The measured synthase activity did not increase with the number ofARG2 gene copies unless the number of ARG5,6gene copies was increased similarly. ARG5,6 encodes a precursor that is maturated in the mitochondria into acetylglutamate kinase and acetylglutamyl-phosphate reductase, catalyzing the second and third steps in the pathway. The results imply that the synthase must interact stoichiometrically in vivo with the kinase, the reductase, or both to be active. Results obtained with synthetic ARG5 and ARG6 genes suggested that both the kinase and the reductase could be needed. This situation, which has completely escaped notice in yeast until now, is reminiscent of the observation in Neurospora crassa that nonsense arg-6 kinase/reductase mutants lack synthase activity (Hinde, R. W., Jacobson, J. A., Weiss, R. L., and Davis, R. H. (1986) J. Biol. Chem. 261, 5848–5852). In immunoprecipitation experiments, hemagglutinin-tagged synthase coprecipitated with a protein proven by microsequencing to be the kinase. Western blot analyses showed that the synthase has reduced stability in the absence of the kinase/reductase. Our data demonstrate the existence of a new yeast arginine metabolon involving at least the first two, and possibly the first three, enzymes of the pathway. Hypotheses regarding the biological significance of this interaction are discussed.

The ARG11 Gene of Saccharomyces cerevisiae Encodes a Mitochondrial Integral Membrane Protein Required for Arginine Biosynthesis

Prototype strain MG409 (arg11-1) is a severe arginine bradytroph with greatly reduced ornithine and arginine pools, although all known enzymes required for arginine biosynthesis are functional. To identify the function required for normal arginine production impaired in MG409, we have cloned, sequenced, and performed a first molecular characterization of ARG11. We show that the ARG11 open reading frame encodes a putative 292-residue protein with a predicted molecular mass of 31.5 kDa. Sequence similarities, a tripartite organization, and six potential hydrophobic transmembrane spans suggest that Arg11p belongs to the mitochondrial integral inner membrane carrier family. We have used immuno-Western blotting and hemagglutinin epitope-tagged derivatives of Arg11p, Arg8p (a mitochondrial matrix marker), and Arg3p (a cytosolic marker) to demonstrate that Arg11p is confined to the mitochondria and behaves like an integral membrane protein. A deletion created in ARG11 causes the same arginine-leaky behavior as the original arg11-1 mutation, which yields a premature stop codon at residue 266. Arg11p thus appears to fulfill a partially redundant function requiring its 27 carboxyl-terminal amino acids. As a working hypothesis, we propose that Arg11p participates in the export of matrix-made ornithine into the cytosol.

Escherichia coli and Saccharomyces cerevisiae acetylornithine aminotransferases: evolutionary relationship with ornithine aminotransferases

Genes argD and ARG8, encoding the acetylornithine aminotransferase (ACOAT) subunit in Escherichia coli and Saccharomyces cerevisiae, respectively, have been cloned and sequenced. The deduced amino acid sequences show substantial similarity. Moreover, they resemble ornithine aminotransferase (OAT) sequences (i.e., those from yeast, rat and man); the observed similarities are statistically significant, indicating that the enzymes are homologous. However, in contrast to OATs, which appear to be substrate (i.e., ornithine)-specific, S. cerevisiae ACOAT transaminates ornithine about as efficiently as E. coli does. The evolutionary relationship between ACOATs and OATs is discussed in terms of substrate ambiguity.

Cloning and endonuclease restriction analysis of argF and of the control region of the argECBH bipolar operon in Escherichia coli.

A 1.8 kb DNA fragment, liberated by endonuclease HindIII, contains the control region of the argECBH bipolar operon near one end and the weak secondary promoter of argH at the other extremity; it has been cloned in plasmid pBR322. The same plasmid vector has been used to clone the argF gene liberated from the chromosome by endonuclease BamHI. Restriction patterns for the two hybrid plasmids have been determined, using enzymes AluI, BglI, EcoRI, HaeIII, HincII, HindIII, HpaI and II, PstI and SalI. Two AluI sites situated on either side of and close to a HincII target delineate two short fragments covering the whole of the argECBH control region. The argF control elements are located in a region accessible to further dissection by BamHI, EcoRI, PstI and HindIII. Carriers of the argF plasmid produce extremely high amounts of ornithine carbamoyltransferase, a feature useful for purification of this enzyme.

Arginine-specific repression in Saccharomyces cerevisiae: kinetic data on ARG1 and ARG3 mRNA transcription and stability support a transcriptional control mechanism.

A specific repression mechanism regulates arginine biosynthesis in Saccharomyces cerevisiae. The involvement of regulatory proteins displaying DNA-binding features and the location of an operator region between the TATA box and the transcription start of the structural gene ARG3 suggest that this mechanism operates at the level of transcription. A posttranscriptional mechanism has, however, been proposed to account for the conspicuous lack of proportionality between ARG3 mRNA steady-state levels (as determined by Northern [RNA] assays; F. Messenguy and E. Dubois, Mol. Gen. Genet. 189:148-156, 1983) and the cognate enzyme activities. In this work, we have analyzed the time course of the incorporation of radioactive precursors into ARG1 and ARG3 mRNAs and the kinetics of their decay under different regulatory statuses. The results (expressed in terms of relative mRNA levels, relative transcription rates, and mRNA half-lives) give the picture expected from a purely transcriptional control. A similar analysis of expression of the gene CPA1, for which a translational regulation by arginine has been clearly demonstrated (M. Werner, A. Feller, F. Messenguy, and A. Piérard, Cell 49:805-813, 1987), indicates that this gene is also partly regulated at the transcriptional level by the ARGR repressor system. Moreover, the half-life of CPA1 mRNA is reduced twofold in the presence of excess arginine; we suggest that this could be inherent in the mechanism of translational regulation of CPA1.

General amino acid control and specific arginine repression in Saccharomyces cerevisiae: physical study of the bifunctional regulatory region of the ARG3 gene.

To characterize further the regulatory mechanism modulating the expression of the Saccharomyces cerevisiae ARG3 gene, i.e., the specific repression by arginine and the general amino acid control, we analyzed by deletion the region upstream of that gene, determined the nucleotide sequence of operator-constitutive-like mutations affecting the specific regulation, and examined the behavior of an ARG3-galK fusion engineered at the initiating codon of ARG3. Similarly to what was observed in previous studies on the HIS3 and HIS4 genes, our data show that the general regulation acts as a positive control and that a sequence containing the nucleotide TGACTC, between positions -364 and -282 upstream of the transcription start, functions as a regulatory target site. This sequence contains the most proximal of the two TGACTC boxes identified in front of ARG3. While the general control appears to modulate transcription efficiency, the specific repression by arginine displays a posttranscriptional component (F. Messenguy and E. Dubois, Mol. Gen. Genet. 189:148-156, 1983). Our deletion and gene fusion analyses confirm that the specific and general controls operate independently of each other and assign the site responsible for arginine-specific repression to between positions -170 and +22. In keeping with this assignment, the two operator-constitutive-like mutations were localized at positions -80 and -46, respectively, and thus in a region which is not transcribed. We discuss a hypothesis accounting for the involvement of untranscribed DNA in a posttranscriptional control.

Control-mechanisms acting at the transcriptional and post-transcriptional levels are involved in the synthesis of the arginine pathway carbamoylphosphate synthase of yeast.

In Saccharomyces cerevisiae, the synthesis of the arginine pathway enzyme carbamoylphosphate synthase (CPSase A) is subject to two control mechanisms. One mechanism, the general control of amino acid biosynthesis, influences the expression of both CPA1 and CPA2 genes, the structural genes for the two subunits of the enzyme. The second mechanism, the specific control of arginine biosynthesis, only affects the expression of CPA1. To study these mechanisms in more detail, we have cloned the CPA1 and CPA2 genes and used their DNA to measure the CPA1 and CPA2 mRNA content of cells grown under various conditions. A close coordination was observed in the variation of the levels of CPA1 and CPA2 mRNAs and polypeptide products under conditions where the general control of amino acid biosynthesis operates. In contrast, little correlation was found between the levels of CPA1 mRNA and the corresponding protein for conditions affecting repression by arginine: the total amplitude of variation was 6‐fold higher for the CPA1 protein than for the CPA1 messenger transcript. Such findings are consistent with the conclusion that the general control operates at the transcriptional level and that the specific arginine control acts primarily at a post‐transcriptional level.

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.

Crystallization of ornithine acetyltransferase from yeast by counter-diffusion and preliminary X-ray study

A study is presented on the crystallization of ornithine acetyltransferase from yeast, which catalyzes the fifth step in microbial arginine synthesis. The use of the counter-diffusion technique removes the disorder present in one dimension in crystals grown by either the batch or hanging-drop techniques. This makes the difference between useless crystals and crystals that allow successful determination of the structure of the protein. The crystals belong to space group P4, with unit-cell parameters a = b = 66.98, c = 427.09 A, and a data set was collected to 2.76 A.

Positive and negative regulation of CAR1 expression in Saccharomyces cerevisiae

SummaryWe localized the chromosomal targets of several of the regulatory controls of expression of theCAR1 gene. Fusion tolacZ of several fragments of the 5′ non-coding region showed that induction ofCAR1 by arginine is positively regulated by the products of theARGR genes. The target lies upstream of another site where repression by the CARGRI molecule occurs. The latter control is not specific to arginine catabolism since it also affectsCYC-1 and indeed does not appear to involve arginine. The primary target of the two other regulatory allelesCARGRII andCARGRIII is not situated in the 5′ non-coding region. Deletion analysis supports the fusion data and confirms the order of the regulatory regions: 5′—nitrogen catabolite repression—activation by arginine—CARGRI-mediated repression—CAR1.