Isabel L. Calderón

Saccharomyces cerevisiae Aqr1 Is an Internal-Membrane Transporter Involved in Excretion of Amino Acids

ABSTRACT Excretion of amino acids by yeast cells was reported long ago but has not been characterized in molecular terms. It is typically favored by overproduction of the amino acid and/or impairment of its uptake. Here we describe the construction of a yeast strain excreting threonine and homoserine. Using this excretor strain, we then applied a reverse-genetics approach and found that the transporter encoded by the YNL065w/AQR1 gene, a protein thought to mediate H+ antiport, is involved in homoserine and threonine excretion. Furthermore, overexpression of AQR1 led to increased excretion of several amino acids (alanine, aspartate, and glutamate) known to be relatively abundant in the cytosol. Transcription of the AQR1 gene is induced severalfold by a number of amino acids and appears to be under the negative control of Gcn4. An Aqr1-green fluorescent protein fusion protein is located in multiple internal membrane structures and appears to cycle continuously between these compartments and the plasma membrane. The Aqr1 sequence is significantly similar to the vesicular amine transporters of secretory vesicles of neuronal cells. We propose that Aqr1 catalyzes transport of excess amino acids into vesicles, which then release them in the extracellular space by exocytosis.

Isolation and characterization of yeast DNA repair genes : II. Isolation of plasmids that complement the mutations rad50-1, rad51-1, rad54-3, and rad55-3.

SummaryPlasmids that complement the yeast mutations rad50-1, rad51-1, rad54-3 and rad55-3 were obtained by transforming strains that carried a leu2 marker and the particular rad mutation, with YEp13 plasmids containing near random yeast DNA inserts. Integration of these plasmids or of fragments of these plasmids was accomplished. Genetic studies using the integrants established the presence of the genes RAD51, RAD54 and RAD55 in the respective plasmids. However, a BamHI subclone of the rad50-1 complementing plasmid failed to integrate at the RAD50 locus, indicating that no homology exists between this fragment and the RAD50 gene.A BamHI fragment from the RAD54 plasmid was shown to be internal to the RAD54 gene: its integration within a wild type copy of RAD54 causes the cell to become Rad−; its excision is X-ray inducible and restores the Rad+ phenotype. Since cells bearing a disrupted copy of RAD54 are able to survive, we conclude that this gene is not essential.

Inhibition by different amino acids of the aspartate kinase and the homoserine kinase of the yeast Saccharomyces cerevisiae.

In this paper, we describe a simple method to measure the yeast homoserine kinase and asparate kinase activities, independently but in the same extract. With this method, we have determined some kinetic parameters for the physiological substrates of both enzymes, and investigated the inhibition exerted by different amino acids on these activities. Off all natural amino acids tested, only threonine inhibits effectively both enzymatic activities, although to a different degree. We did not find the reported inhibition by L‐homoserine over the asparate kinase. Altogether the data point to be asparate kinase and to the threonine as the key factors in the regulation of this route.

Isolation of a mutant allele that deregulates the threonine biosynthesis in Saccharomyces cerevisiae.

We have cloned the yeast allele HOM3-R2, that codes for a mutant aspartate kinase which is insensitive to feedback inhibition by threonine, by gap-repair. A strain carrying this allele in a multicopy plasmid, or integrated into the genome, accumulates 14-times and 8-times more threonine than the wild-type, respectively. The sequence of the mutant allele differs from that of the wild-type in a single base pair change, namely a G by an A, at position 1355 in the open reading frame. The fact that the presence of this mutant allele in a cell induces threonine overproduction points to aspartate kinase as the key enzyme in the regulation of threonine biosynthesis in yeast.

Mutations that cause threonine sensitivity identify catalytic and regulatory regions of the aspartate kinase of Saccharomyces cerevisiae.

The HOM3 gene of Saccharomyces cerevisiae encodes aspartate kinase, which catalyses the first step in the branched pathway leading to the synthesis of threonine and methionine from aspartate. Regulation of the carbon flow into this pathway takes place mainly by feedback inhibition of this enzyme by threonine. We have isolated and characterized three HOM3 mutants that show growth inhibition by threonine due to a severe, threonine‐induced reduction of the carbon flow into the aspartate pathway, leading to methionine limitation. One of the mutants has an aspartate kinase which is 30‐fold more strongly inhibited by threonine than the wild‐type enzyme. The predicted amino acid substitution in this mutant, A406T, is located in a region associated with the modulation of the enzymatic activity. The other two mutants carry an aspartate kinase with reduced affinity for its substrates, aspartate and ATP. The corresponding amino acid substitutions, K26I and G25D, affect residues located in the vicinity of a highly conserved lysine‐phenylalanine‐glycine‐glycine (KFGG) stretch present in the N‐terminal part of the aspartate kinase, to which no function has so far been assigned. We suggest that this region is involved in substrate binding. Mutagenesis of a HOM3 region centred in the KFGG‐coding triplets generated alleles that determine threonine sensitivity or auxotrophy for threonine and methionine, but not a phenotype associated with a feedback‐resistant aspartate kinase, indicating that this region is not involved in the allosteric response of the enzyme. Copyright

Isolation of a DNA fragment that is expressed as an amber suppressor when present in high copy number in yeast

A plasmid carrying a DNA sequence conferring amber suppression has been isolated from yeast pool 35 DNA. The plasmid YEp13-SUP suppresses the amber mutations rad50-1 and trp1-289 as well as other known amber mutations. This sequence is located on the 2.5-kb insert and is expressed only when the plasmid bearing it is present in high copy number. The suppressor sequence was shown to integrate close to the MAL4 gene located on the right arm of chromosome XI.

LOCUS-SPECIFIC SUPPRESSION OF ILV1 IN SACCHAROMYCES CEREVISIAE BY DEREGULATION OF CHA1 TRANSCRIPTION

Abstract The ILV1 gene of Saccharomyces cerevisiae encodes the anabolic threonine deaminase, which catalyzes the first committed step in isoleucine biosynthesis. Strains devoid of a functional Ilv1p have a requirement for isoleucine. Threonine can also be deaminated by a second serine/threonine deaminase encoded by the CHA1 gene. CHA1 is regulated by transcriptional induction by serine and threonine, and enables yeast to utilize the hydroxyamino acids as sole nitrogen source. Phenotypic suppression of ilv1 can occur by inducer-mediated transcriptional activation of the CHA1 gene. To identify mutations in putative trans-acting factors regulating CHA1 expression, we have isolated and characterized three extragenic suppressors of ilv1. A dominant mutation, SIL4 (suppressor of ilv1), is allelic to HOM3. It increases the size of the threonine pool, by 15- to 20-fold, which is sufficient to induce CHA1 transcription, thereby creating a metabolic bypass of ilv1. A second dominant mutation, SIL3, and a recessive mutation, sil2, both suppress ilv1 by causing inducer-independent, constitutive transcription of CHA1. Importantly, sil2 and SIL3 increase the expression of a CHA1p–lacZ translational gene fusion, demonstrating that they exert their action through the CHA1 promoter. Genetic analysis showed that both SIL3 and sil2 are alleles of CHA4, a positive regulator of CHA1, i.e., they convert Cha4p to a constitutive activator.

Detection and Analysis of Genetic Variation in Salicornieae (Chenopodiaceae) Using Random Amplified Polymorphic DNA (RAPD) Markers

Salicornia L., Sarcocornia A. J. Scott and Arthrocnemum Moq. are halophyte genera belonging to the tribe Salicornieae Dumort. (Chenopodiaceae). They are taxonomically very close and share several morphological features, such as articulate, fleshy stems and opposite scale-like leaves. Some of their ecological, caryological and palynological characteristics also coincide, but others do not. The three genera are widely distributed in coastal habitats and inland salt marshes in many parts of the world. In coastal habitats, populations of each genus may grow under very different tidal regimes, ranging from the seaward edge that is inundated twice daily to the landward fringe that is flooded only by certain spring tides (Davy & Costa, 1992). On sand and low-lying mud deposits in estuaries of south-western Spain, the different species of Salicornia and Sarcocornia perennis (Mill.) A. J. Scott are found mostly in flooded marshes (at levels between mean high tide and mean high water), whereas Sarcocornia fruticosa (L.) A. J. Scott and Arthrocnemum macrostachyum (Moric.) Moris grow in non-flooded marshes (at levels between mean high water and mean spring tides). They all play an important role in salt marsh succession (Castellanos & al., 1994), showing a clear zonation with relation to tides. The taxonomic treatment of these genera by various authors and the delimitation and position of the species differ. The only species of Arthrocnemum present in Spain, A. macrostachyum, was included in Salicornia by early authors (Moricand, 1820; Lagasca, 1817), but later separated as a distinct genus based on differences in habit (Salicornia is annual while Arthrocnemum is perennial) and other characters. The two Spanish representatives of Sarcocornia usually recognized, S. fruticosa and S. perennis, were placed in Salicornia by Linnaeus (1753) and Miller (1768), respectively, then in Arthrocnemum by Moquin-Tandon (1840) and others, as again lately

Effect of gene amplification on threonine production by yeast.

In this work, we have studied the effect of amplifying different alleles involved in the threonine biosynthesis on the amino acid production by Saccharomyces cerevisiae. The genes used were wild‐type HOM3, HOM2, HOM6, THR1, and THR4, and two mutant alleles of HOM3 (namely HOM3‐R2 and HOM3‐R6), that code for feedback‐insensitive aspartate kinases. The results show that only the amplification of the HOM3 alleles leads to threonine and, in some instances, to homoserine overproduction. In terms of the regulation of the pathway, the data indicate that the main control is exerted by inhibition of the aspartate kinase and that, probably, a second and less important regulation takes place at the level of the homoserine kinase, the THR1 gene product. However, amplification of THR1 in two related Hom3‐R2 strains does not increase the amount of threonine but, in one of them, it does induce accumulation of more homoserine. This result probably reflects differences between these strains in some undetermined genetic factor/s related with threonine metabolism. In general, the data indicate that the common laboratory yeast strains are genetically rather heterogeneous and, thus, extrapolation of conclusions must be done carefully.

Effect of the use of commercial Saccharomyces strains in a newly established winery in Ronda (Malaga, Spain)

An ecological study of the yeasts present in a spontaneous and an inoculated fermentation in red wine was carried out in 2005 vintage in a winery located in the Denomination of Origin “Sierras de Málaga” (Málaga, southern of Spain). The winery operated by the first time with the 2003 vintage and since then, has used commercial yeast inocula to start alcoholic fermentation. Yeast isolates were identified by PCR-RFLP analysis of the 5.8S-ITS region from the ribosomal DNA and by mitochondrial DNA RFLP analysis. Except for non-Saccharomyces yeasts found in the fresh must before fermentation, all the isolates were found to be commercial Saccharomyces cerevisiae strains employed by the winery during the successive vintages; thus, no indigenous Saccharomyces yeasts were isolated during fermentation. The same four restriction patterns were found in non inoculated and inoculated vats, although with different frequencies. The use of commercial yeast starter in a new established winery seems to have prevented the development of a resident indigenous Saccharomyces flora.