The pleiotropic effects of the glutamate dehydrogenase (GDH) pathway in Saccharomyces cerevisiae.

Ammonium assimilation is linked to fundamental cellular processes that include the synthesis of non-essential amino acids like glutamate and glutamine. In Saccharomyces cerevisiae glutamate can be synthesized from α-ketoglutarate and ammonium through the action of NADP-dependent glutamate dehydrogenases Gdh1 and Gdh3. Gdh1 and Gdh3 are evolutionarily adapted isoforms and cover the anabolic role of the GDH-pathway. Here, we review the role and function of the GDH pathway in glutamate metabolism and we discuss the additional contributions of the pathway in chromatin regulation, nitrogen catabolite repression, ROS-mediated apoptosis, iron deficiency and sphingolipid-dependent actin cytoskeleton modulation in S.cerevisiae. The pleiotropic effects of GDH pathway in yeast biology highlight the importance of glutamate homeostasis in vital cellular processes and reveal new features for conserved enzymes that were primarily characterized for their metabolic capacity. These newly described features constitute insights that can be utilized for challenges regarding genetic engineering of glutamate homeostasis and maintenance of redox balances, biosynthesis of important metabolites and production of organic substrates. We also conclude that the discussed  pleiotropic features intersect with basic metabolism and set a new background for further glutamate-dependent applied research of biotechnological interest.

Functional analysis of the Saccharomyces cerevisiae YFR021w/YGR223c/YPL100w ORF family suggests relations to mitochondrial/peroxisomal functions and amino acid signalling pathways.

Saccharomyces cerevisiae YFR021w, YGR223c and YPL100w are paralogous ORFs of unknown function. Phenotypic analysis of overexpression, single-, double- and triple-ORF deletion strains under various growth conditions indicated mitochondria-related functions for all three ORFs. Two-hybrid screens of a yeast genomic library identified potentially interacting proteins for the three ORFs. Among these, the transcriptional activator Rtg3p interacted with both Yfr021wp and Ypl100wp and both ORF single deletions reduced the constitutive expression of the RTG-regulated CIT2 and DLD3 genes and caused typical retrograde response of CIT2 and DLD3 under growth conditions requiring functional mitochondria, indicating that YFR021w and YPL100w are also involved in unidentified mitochondrial functions. Ptr3p, a component of the amino acid sensor Ssy1p/Ptr3p, was also found as a two-hybrid interactant of Yfr021wp. Of the three single-ORF deletions, ypl100w Delta exhibited ptr3 Delta-similar phenotypes. These findings, combined with the fact that RTG-dependent expression is modulated by specific amino acids, suggested possible relations of Yfr021wp and Ypl100wp to amino acid signalling pathways. Under most conditions examined, the effects of the single- and double-ORF deletions indicated that YFR021w, YPL100w and YGR223c are not parts of the same pathway. We found no unique phenotype attributed to the deletion of YGR223c. However, its function interferes with the function of the other two ORFs, as revealed by the effects of double- and triple-ORF deletions.

The second cysteine-rich domain of Mac1p is a potent transactivator that modulates DNA binding efficiency and functionality of the protein.

Mac1p is a Saccharomyces cerevisiae DNA binding transcription factor that activates genes involved in copper uptake. A copper-induced N-C-terminal intramolecular interaction and copper-independent homodimerization affect its function. Here, we present a functional analysis of Mac1p deletion derivatives that attributes new roles to the second cysteine-rich (REPII) domain of the protein. This domain exhibits the copper-responsive potent transactivation function when assayed independently and, in the context of the entire protein, modulates the efficiency of Mac1p binding to DNA. The efficiency of binding to both copper-response promoter elements can determine the in vivo functionality of Mac1p independent of homodimerization.

Regulated expression of the Saccharomyces cerevisiae Fre1p/Fre2p Fe/Cu reductase related genes.

The Saccharomyces cerevisiae genome contains nine open reading frames (ORFs)--YLR214w (FRE1), YKL220c (FRE2), YOR381w, YNR060w, YOR384w, YLL051c, YOL152w, YGL160w and YLR047c--which, based on amino acid sequence similarity, fall in the category of iron/copper reductase-related genes. FRE1 and FRE2 are the first identified and studied genes of this family. They both encode for plasma membrane ferric/cupric reductases and their expression is regulated by iron and copper availability, mediated by the transcription factors Aft1p and Mac1p, respectively. We have studied the expression of the seven ORFs of unknown function by monitoring mRNA accumulation under different growth conditions, namely, their response to iron and copper availability in the medium, as well as the involvement of transcription factors Aft1p and Mac1p in their expression. A compilation of these results, together with sequence comparison data, permits a first classification of these genes under three major groups: genes mainly regulated by iron availability, genes mainly regulated by copper availability and genes not regulated by either metal.

Sequence divergence of the RNA polymerase shared subunit ABC14.5 (Rpb8) selectively affects RNA polymerase III assembly in Saccharomyces cerevisiae.

ABC14.5 (Rpb8) is a eukaryotic subunit common to all three nuclear RNA polymerases. In Saccharomyces cerevisiae, ABC14.5 (Rpb8) is essential for cell viability, however its function remains unknown. We have cloned and characterised the Schizosaccharomyces pombe rpb8(+) cDNA. We found that S.pombe rpb8, unlike the similarly diverged human orthologue, cannot substitute for S.cerevisiae ABC14. 5 in vivo. To obtain information on the function of this RNA polymerase shared subunit we have used S.pombe rpb8 as a naturally altered molecule in heterologous expression assays in S.cerevisiae. Amino acid residue differences within the 67 N-terminal residues contribute to the functional distinction of the two yeast orthologues in S.cerevisiae. Overexpression of the S.cerevisiae largest subunit of RNA polymerase III C160 (Rpc1) allows S.pombe rpb8 to functionally replace ABC14.5 in S.cerevisiae, suggesting a specific genetic interaction between the S.cerevisiae ABC14.5 (Rpb8) and C160 subunits. We provide further molecular and biochemical evidence showing that the heterologously expressed S.pombe rpb8 molecule selectively affects RNApolymerase III but not RNA polymerase I complex assembly. We also report the identification of a S.cerevisiae ABC14.5-G120D mutant which affects RNA polymerase III.

The nucleotide sequence of Saccharomyces cerevisiae chromosome XV.

Chromosome XV was one of the last two chromosomes of Saccharomyces cerevisiae to be discovered. It is the third-largest yeast chromosome after chromosomes XII and IV, and is very similar in size to chromosome VII. It alone represents 9% of the yeast genome (8% if ribosomal DNA is included). When systematic sequencing of chromosome XV was started, 93 genes or markers were identified, and most of them were mapped. However, very little else was known about chromosome XV which, in contrast to shorter chromosomes, had not been the object of comprehensive genetic or molecular analysis. It was therefore decided to start sequencing chromosome XV only in the third phase of the European Yeast Genome Sequencing Programme, after experience was gained on chromosomes III, XI and II. The sequence of chromosome XV has been determined from a set of partly overlapping cosmid clones derived from a unique yeast strain, and physically mapped at 3.3-kilobase resolution before sequencing. As well as numerous new open reading frames (ORFs) and genes encoding tRNA or small RNA molecules, the sequence of 1,091,283 base pairs confirms the high proportion of orphan genes and reveals a number of ancestral and successive duplications with other yeast chromosomes.

Sequence analysis of a 33.2 kb segment from the left arm of yeast chromosome XV reveals eight known genes and ten new open reading frames including homologues of ABC transporters, inositol phosphatases and human expressed sequence tags.

The complete nucleotide sequence of a 33221 bp segment, contained in cosmid pEOA1044, derived from the left arm of chromosome XV of Saccharomyces cerevisiae, appears in public databases between coordinates 177013 and 210234 (http://speedy.mips.biochem.mpg.de/). Computer analysis of that sequence revealed the presence of the previously known genes IRA2, DEC1, NUF2, HST1, RTG1, RIB2 and HAL2, one previously partially sequenced open reading frame (ORF) of unknown function (SCORFAC) and ten newly identified ORFs. One of the new ORFs is similar to the Drosophila melanogaster white gene and other transmembrane ABC transporters, another one has similarities to inositol phosphatases and others are similar to ORFs of unknown function from various organisms, including human Expressed Sequence Tags (ESTs). Potential transmembrane regions, ATP/GTP-binding and WD motifs have also been identified. The existence of yeast ESTs for two of the newly identified ORFs indicates that they are transcribed.

The yeast Fre1p/Fre2p cupric reductases facilitate copper uptake and are regulated by the copper-modulated Mac1p activator.

Fre1p and Fre2p are ferric reductases which account for the total plasma membrane associated activity, a prerequisite for iron uptake, in Saccharomyces cerevisiae. The two genes are transcriptionally induced by iron depletion. In this communication, we provide evidence that Fre2p has also cupric reductase activity, as has been previously shown for Fre1p (Hassett, R., and Kosman, D.J. (1995) J. Biol. Chem. 270, 128-134). Both Fre1p and Fre2p enzymes are functionally significant for copper uptake, as monitored by the accumulation of the copper-regulated CUP1 and CTR1 mRNAs in fre1Delta, fre2Delta, and fre1Deltafre2Delta mutant strains. However, only Fre1p activity is induced by copper depletion, even in the presence of iron. This differential copper-dependent regulation of Fre1p and Fre2p is exerted at the transcriptional level of the two genes. We have shown that Mac1p, known to affect the basal levels of FRE1 gene expression (Jungmann, J., Reins, H.-A., Lee, J., Romeo, A., Hassett, R., Kosman, D., and Jentsch, S. (1993) EMBO J. 12, 5051-5056), accounts for both the copper-dependent induction of FRE1 and down-regulation of FRE2 gene. Finally, Mac1p transcriptional activation function is itself modulated by the availability of copper.

Gene overexpression reveals alternative mechanisms that induce GCN4 mRNA translation.

The Saccharomyces cerevisiae GCN4 gene which encodes the transcription activator Gcn4, is under translational regulation. Derepression of GCN4 mRNA translation is mediated by the Gcn2 protein kinase which phosphorylates the alpha subunit of eIF-2, upon amino-acid starvation. Here, we report that overexpression of certain Saccharomyces cerevisiae genes generates intracellular conditions that alleviate the requirement for a functional Gcn2 kinase to induce GCN4 mRNA translation. Our findings, combined with the fact that Gcn2 kinase is dispensable during the initiation phase of the cellular response to amino-acid limitation, provide the grounds to further elucidate the mechanisms underlying the physiology of this homeostatic response.

Sequence analysis of a 40.7 kb segment from the left arm of yeast chromosome X reveals 14 known genes and 13 new open reading frames including homologues of genes clustered on the right arm of chromosome XI.

The complete nucleotide sequence of a 40.7 kb segment about 130 kb from the left end of chromosome X of Saccharomyces cerevisiae was determined from two overlapping cosmids. Computer analysis of that sequence revealed the presence of the previously known genes VPS35, INO1, SnR128, SnR190, MP12, YAK1, RPB4, YUR1, TIF2, MRS3 and URA2, three previously sequenced open reading frames (ORFs) of unknown function 5' of the INO1, 5' of the MP12 and 3' of the URA2 genes and 13 newly identified ORFs. One of the new ORFs is homologous to mammalian glycogenin glycosyltransferases and another has similarities to the human phospholipase D. Some others contain potential transmembrane regions or leucine zipper motifs. The existence of yeast expressed sequence tags for some of the newly identified ORFs indicates that they are transcribed. A cluster of six genes within 10 kb (YUR1, TIF2, two new ORFs, an RSP25 homologue and MRS3) have homologues arranged similarly within 28.5 kb on the right arm of chromosome XI.

Complete nucleotide sequence of Saccharomyces cerevisiae chromosome X.

The complete nucleotide sequence of Saccharomyces cerevisiae chromosome X (745 442 bp) reveals a total of 379 open reading frames (ORFs), the coding region covering approximately 75% of the entire sequence. One hundred and eighteen ORFs (31%) correspond to genes previously identified in S. cerevisiae. All other ORFs represent novel putative yeast genes, whose function will have to be determined experimentally. However, 57 of the latter subset (another 15% of the total) encode proteins that show significant analogy to proteins of known function from yeast or other organisms. The remaining ORFs, exhibiting no significant similarity to any known sequence, amount to 54% of the total. General features of chromosome X are also reported, with emphasis on the nucleotide frequency distribution in the environment of the ATG and stop codons, the possible coding capacity of at least some of the small ORFs (<100 codons) and the significance of 46 non-canonical or unpaired nucleotides in the stems of some of the 24 tRNA genes recognized on this chromosome.

Complete DNA sequence of yeast chromosome XI.

The complete DNA sequence of the yeast Saccharomyces cerevisiae chromosome XI has been determined. In addition to a compact arrangement of potential protein coding sequences, the 666,448-base-pair sequence has revealed general chromosome patterns; in particular, alternating regional variations in average base composition correlate with variations in local gene density along the chromosome. Significant discrepancies with the previously published genetic map demonstrate the need for using independent physical mapping criteria.

The complete sequencing of a 24.6 kb segment of yeast chromosome XI identified the known loci URA1, SAC1 and TRP3, and revealed 6 new open reading frames including homologues to the threonine dehydratases, membrane transporters, hydantoinases and the phospholipase A2-activating protein.

We report the entire sequence of a 26.4 kb segment of chromosome XI of Saccharomyces cerevisiae. Identification of the known loci URA1, TRP3 and SAC1 revealed a translocation compared to the genetic map. Additionally, six unknown open reading frames have been identified. One of them is similar to catabolic threonine dehydratases. Another one contains characteristic features of membrane transporters. A third one is homologous in half of its length to the prokaryotic hydantoinase HyuA and in the other half to hydatoinase HyuB. A fourth one is homologous to the mammalian phospholipase A2-activating protein. A fifth one, finally, is homologous to the hypothetical open reading frame YCR007C of chromosome III. The sequence has been deposited in the EMBL data library under Accession Number X75951.

Two distinctly regulated genes are required for ferric reduction, the first step of iron uptake in Saccharomyces cerevisiae.

Iron uptake in Saccharomyces cerevisiae involves at least two steps: reduction of ferric to ferrous ions extracellularly and transport of the reduced ions through the plasma membrane. We have cloned and molecularly characterized FRE2, a gene which is shown to account, together with FRE1, for the total membrane-associated ferric reductase activity of the cell. Although not similar at the nucleotide level, the two genes encode proteins with significantly similar primary structures and very similar hydrophobicity profiles. The FRE1 and FRE2 proteins are functionally related, having comparable properties as ferric reductases. FRE2 expression, like FRE1 expression, is induced by iron deprivation, and at least part of this control takes place at the transcriptional level, since 156 nucleotides upstream of the initiator AUG conferred iron-dependent regulation when fused to a heterologous gene. However, the two gene products have distinct temporal regulation of their activities during cell growth.

Sequencing of a 13.2 kb segment next to the left telomere of yeast chromosome XI revealed five open reading frames and recent recombination events with the right arms of chromosomes III and V.

We report the entire sequence of a 13.2 kb segment next to the left telomere of chromosome XI of Saccharomyces cerevisiae. A 1.2 kb fragment near one end is 91% homologous to the right arm of chromosome III and 0.7 kb of that are 77% homologous to the right arm of chromosome V. Five open reading frames are included in the sequenced segment. Two of them are almost identical to the known YCR104W and YCR103C hypothetical proteins of chromosome III. A third one contains a region homologous to the Zn (2)-Cys (6) binuclear cluster pattern of fungal transcriptional activators. The fourth one, part of which is similar to the mammalian putative transporter of mevalonate, has the structure of membrane transporters. The fifth one is similar to yeast ferric reductase.

The complete DNA sequence of yeast chromosome III.

The entire DNA sequence of chromosome III of the yeast Saccharomyces cerevisiae has been determined. This is the first complete sequence analysis of an entire chromosome from any organism. The 315-kilobase sequence reveals 182 open reading frames for proteins longer than 100 amino acids, of which 37 correspond to known genes and 29 more show some similarity to sequences in databases. Of 55 new open reading frames analysed by gene disruption, three are essential genes; of 42 non-essential genes that were tested, 14 show some discernible effect on phenotype and the remaining 28 have no overt function.

Multiple cis-acting elements modulate the translational efficiency of GCN4 mRNA in yeast.

The expression of the GCN4 gene in yeast is regulated at the translational level: growth of yeast cells under amino acid starvation conditions results in an increase in the translational efficiency of GCN4 mRNA. A sequence within the 5' untranslated region of this mRNA, which contains four small open reading frames, acts in cis to suppress translation when growth occurs in rich media. In this report, we have analyzed the effects on translation of a series of deletion, insertion, and substitution mutations in the 5' untranslated region of GCN4 mRNA. This analysis showed that at least two distinct cis elements located within the region of the small upstream open reading frames are required, in conjunction with trans positive elements, for the translational activation of GCN4 mRNA. We propose that the translational efficiency of GCN4 mRNA is modulated by the rate of translation initiation at the upstream AUG codons.

Expression of alpha- and beta-tubulin genes during development of sea urchin embryos.

Mature unfertilized eggs of the sea urchin Lytechinus pictus contain multiple alpha-tubulin mRNAs, which range in size from 1.75 to 4.8 kb, and two beta-tubulin mRNAs, 1.8 and 2.25 kb. These mRNAs were found at similar levels throughout the early cleavage stages. RNA gel blot hybridizations showed that prominent quantitative and qualitative changes in tubulin mRNAs occurred between the early blastula and hatched blastula stages. The overall amounts of alpha- and beta-tubulin mRNAs increased two- to fivefold between blastula and pluteus. These increases were due mainly to a rise in a 1.75-kb alpha RNA and a new 2.0-kb beta RNA. Other, minor changes also occurred during subsequent development. All size classes of alpha- and beta-tubulin RNAs in early and late embryos contained poly(A)+ translatable sequences. As reported earlier, some of each of the alpha RNAs, but neither of the beta RNAs, are translated in the egg and a small portion of each of the stored alpha and beta RNAs is recruited onto polysomes within 30 min of fertilization. In the work described here, subsequent development up to the morula stage was accompanied by a gradual recruitment of tubulin mRNAs into polysomes. By the early blastula stage, most of the maternal tubulin sequences were associated with polysomes. In contrast to the gradual recruitment of maternal sequences throughout cleavage, the tubulin mRNAs which appeared at the blastula stage showed no delay in entering polysomes. The exact fraction of each mRNA that was translationally active at later stages varied somewhat among the individual mRNAs. From the differential hybridization patterns of egg, embryo, and testis RNAs to various tubulin cDNA and genomic DNA probes, it is concluded that at least one gene producing maternal alpha mRNA is different from a second one which is expressed only in testis. Each of the three embryonic beta RNAs is encoded by a different beta gene; at least two of these different beta genes are also expressed in testis.

Multiple polymorphic alpha- and beta-tubulin mRNAs are present in sea urchin eggs.

Multiple alpha- and beta-tubulin RNAs were found in the mature unfertilized eggs of the sea urchin Lytechinus pictus. The alpha-tubulin RNAs were polymorphic in number, size, and relative amounts in the eggs of different females. Five to seven different size classes [1.75-4.2 kilobases (kb)] were detected on RNA gel blots. All egg preparations contained variable amounts of 1.8- and 2.25-kb beta-tubulin RNAs, and a few of them contained an additional 2.9-kb beta-tubulin RNA. The total amount of alpha-tubulin RNA did not always parallel that of beta-tubulin RNA. A portion of all of the various alpha- and beta-tubulin RNAs were polyadenylylated. RNase H digestions ruled out the possibility that some of these RNAs represented a single transcript bearing different lengths of 3' poly(A). One class of alpha-tubulin RNAs (2.4-2.65 kb) was reduced to 2 kb by RNase H, suggesting the presence of internal oligo(A) regions. All of the egg beta-tubulin RNAs sedimented as free ribonucleoprotein particles. Only a small portion of the 1.75- to 3.6-kb alpha-tubulin RNAs, but most of the 4.2-kb alpha-tubulin RNA, were found on polysomes before fertilization. In the 30-min embryo, small amounts of each of the various alpha- and beta-tubulin RNAs were recruited onto polysomes. Thus, each of the multiple polymorphic alpha- and beta-tubulin RNAs in the egg represent translationally competent mRNA.

Evolution of alpha q- and beta-tubulin genes as inferred by the nucleotide sequences of sea urchin cDNA clones.

Evolutionary studies on the tubulin multigene families were initiated by nucleotide sequence analysis of cDNA clones complementary to sea urchin (Lytechinus pictus) testis alpha- and beta-tubulin cDNA clones (p beta 1, p beta 2, p beta e) demonstrated the existence of tubulin mRNA heterogeneity. p beta 2 and p beta 3 contain identical tubulin-coding regions and extremely similar 3' untranslated sequences, including a polyadenylation signal (AAUAAA). However, p beta 2 contains an additional region of 3' untranslated sequence which includes a second polyadenylation signal. These two sequences may be allelic, representing products of alternative transcription termination or processing pathways. p beta 1 and p beta 2 (or p beta 3) cDNAs almost certainly correspond to transcripts of distinct but evolutionarily related genes. Examination of the available coding portions showed that they differ only by a few silent nucleotide substitutions and the deletion/insertion of one codon; most of the differences are clustered within the last 15 3'-end codons. In contrast, their 3' untranslated sequences are considerably divergent. Nucleotide alignment in this region was feasible by considering specific point and segmental mutations, mainly T in equilibrium or formed from C transitions and small deletions/insertions associated with small direct repeats. The sea urchin alpha- and beta-tubulin cDNA and corresponding protein sequences were compared with previously described tubulin cDNA and protein sequences from other organisms. Both alpha and beta tubulins are very conserved proteins, evolving with a rate comparable to that of histones. Analysis of the nucleotide divergence of the coding cDNA regions showed that replacement sites have changed with a rate 20-175 times lower than that of the silent sites. Among the 177 codons compared between the sea urchin testis and chick brain beta-tubulin cDNAs, there are 7 conservative amino acid replacements and the deletion/insertion of two codons. Most of these changes are clustered near the C-terminus. The 161-amino acid portion of chick brain, rat and porcine alpha-tubulin sequences differs by 3 conservative amino acid replacements from the corresponding sea urchin testis alpha-tubulin sequence. The compared interspecies 3' untranslated sequences are very divergent.