Phosphorylation and activation of the atypical kinase p53-related protein kinase (PRPK) by Akt/PKB.

p53-related protein kinase (PRPK), the human homologue of yeast Bud32, belonging to a small subfamily of atypical protein kinases, is inactive unless it is previously incubated with cell lysates. Here we show that such an activation of PRPK is mediated by another kinase, Akt/PKB, which phosphorylates PRPK at Ser250. We show that recombinant PRPK is phosphorylated in vitro by Akt and its phospho-form is recognized by a Ser250-phospho-specific antibody; that cell co-transfection with Akt along with wild-type PRPK, but not with its Ser250Ala mutant, results in increased PRPK phosphorylation; and that the phosphorylation of p53 at Ser15, the only known substrate of PRPK, is markedly increased by co-transfection of Akt with wild-type PRPK, but not PRPK dead mutant, and is abrogated by cell treatment with the Akt pathway inhibitor LY294002. Our data disclose an unanticipated mechanism by which PRPK can be activated and provide a functional link between this enigmatic kinase and the Akt signaling pathway.

Large-scale phenotypic analysis reveals identical contributions to cell functions of known and unknown yeast genes.

Sequencing of the yeast genome has shown that about one-third of the yeast ORFs code for unknown proteins. Many other have similarity to known genes, but still the cellular functions of the gene products are unknown. The aim of the B1 Consortium of the EUROFAN project was to perform a qualitative phenotypic analysis on yeast strains deleted for functionally orphan genes. To this end we set up a simple approach to detect growth defects of a relatively large number of strains in the presence of osmolytes, ethanol, high temperature, inhibitory compounds or drugs affecting protein biosynthesis, phosphorylation level or nucleic acids biosynthesis. We have now developed this procedure to a semi-quantitative level, we have included new inhibitors, such as hygromycin B, benomyl, metals and additional drugs interfering with synthesis of nucleic acids, and we have performed phenotypic analysis on the deleted strains of 564 genes poorly characterized in respect to their cellular functions. About 30% of the deleted strains showed at least one phenotype: many of them were pleiotropic. For many gene deletions, the linkage between the deletion marker and the observed phenotype(s) was studied by tetrad analysis and their co-segregation was demonstrated. Co-segregation was found in about two-thirds of the analysed strains showing phenotype(s).

Inactivation of six genes from chromosomes VII and XIV of Saccharomyces cerevisiae and basic phenotypic analysis of the mutant strains.

Within the frame of the EUROFAN project, aimed at the functional analysis of the novel ORFs revealed by the systematic sequencing of the Saccharomyces cerevisiae genome, we have inactivated six ORFs encoding putative proteins with unknown function in the two S. cerevisiae strains FY1679 and W303-1B. Five ORFs are located on chromosome VII (YGR250c, YGR251w, YGR260w, YGR262c, YGR263c) and one on chromosome XIV (YNL234w). The genes have been inactivated in the FY1679 strain by a strategy that makes use of deletion cassettes containing the kanMX4 module, which confers resistance to geneticin to yeast cells, and short flanking regions homologous to the target locus (SFH). Tetrad dissection of heterozygous mutants and basic phenotypic analysis of the spores revealed that ORF YGR251w is an essential gene, while the disruption of YGR262c causes a severe slow-growth phenotype. Deletion of the remaining ORFs did not give rise to a detectable phenotype in the mutant strains. For each ORF we have cloned, in the pUG7 plasmid, a replacement cassette that possesses long flanking regions homologous to the target locus (LFH) and, in the pRS416 plasmid, the cognate wild-type gene. The LFH replacement cassettes were used to inactivate the respective genes in the W303-1B strain. This work has been performed in the framework of the B0 Consortium of the EUROFAN I project.

How to bring orphan genes into functional families.

In the framework of the B1 Consortium of the EUROFAN-1 project, we set up a series of simple phenotypic tests that can be performed on a large number of strains at a time. This methodological approach was intended to help assign functions of putative genes coding for unknown proteins to several specific aspects of cell biology. The tests were chosen to study phenotypes which should be affected by numerous genes. In this report, we examined the sensitivity/resistance or the adaptation of the cell to physical or chemical stresses (thermotolerance, osmotolerance and ethanol sensitivity), the effects of the alteration of the level of protein phosphorylation (sensitivity or resistance to compounds affecting the activity of protein kinases or phosphatases) and the effects of compounds interfering with synthesis of nucleic acids or proteins. Deletions in 66 genes of unknown function have been tested in 21 different conditions. In many deletant strains, phenotypes were observed and, for the most promising candidates, tetrad analysis was performed in order to verify co-segregation of the deletion marker with the phenotype.

Characterization of a new hemoprotein in the yeast Saccharomyces cerevisiae.

The Saccharomyces cerevisiae gene YNL234w encodes a 426-amino acid-long protein that shares significant similarities with the globin family. Compared with known globins from unicellular organisms, the Ynl234wp polypeptide is characterized by an unusual structure. In this protein, a central putative heme-binding domain of about 140 amino acids is flanked by two sequences of about 160 and 120 amino acids, respectively, which share no similarity with known polypeptides. Northern analysis indicates that YNL234w transcription is very low in cells grown under normal aerobic conditions but is induced by oxygen-limited growth conditions and by other stress conditions such as glucose repression, heat shock, osmotic stress, and nitrogen starvation. However, the deletion of the gene had no detectable effect on yeast growth. The Ynl234wp polypeptide has been expressed in Escherichia coli, and the hemoprotein nature of the recombinant protein was demonstrated by heme staining after SDS/polyacrylamide gel electrophoresis and spectroscopic analysis. Our data indicate that purified recombinant Ynl234wp possesses a noncovalently bound heme molecule that is predominantly found in a low spin form.

Evolution of mitochondrial DNA in yeast: gene order and structural organization of the mitochondrial genome of Saccharomyces uvarum.

We have determined the size, the restriction map and the gene order of the mitochondrial genome of the yeast Saccharomyces uvarum. Sequence analysis of the mitochondrial COXII gene confirmed the position of this yeast in the Saccharomyces cerevisiae-like group, near Saccharomyces cerevisiae and Saccharomyces douglasii. Most mitochondrial genes have been positioned on this approximately 57-kb long genome and three regions containing putative replication origins have been identified. The gene order of S. uvarum suggests that the mitochondrial genome of the S.cerevisiae-like yeasts could have evolved from an ancestral molecule, similar to that of S. uvarum, through specific genome rearrangements.

Biochemical evidence that Saccharomyces cerevisiae YGR262c gene, required for normal growth, encodes a novel Ser/Thr-specific protein kinase.

Saccharomyces cerevisiae YGR262c gene, whose disruption causes severely defective growth, encodes a putative protein kinase shorter than any other protein kinase biochemically characterized to date and lacking some of the conserved features of these enzymes. Here we show that the product of the YGR262c gene, piD261, expressed in E. coli with a C-terminal (His)6 tag, is a bona fide Ser/Thr protein kinase as judged from its capability to autophosphorylate and to phosphorylate casein and osteopontin in the presence of [gamma-32P]ATP. In contrast, no phosphorylation of histones, myelin basic protein, phosvitin, bovine serum albumin and poly(Glu/Tyr)4:1 could be detected. Mn2+ or, less effectively, Co2+ are required for piD261 catalytic activity, which is conversely undetectable in the presence of Mg2+, a behaviour unique among Ser/Thr protein kinases.

The nucleotide sequence of Saccharomyces cerevisiae chromosome VII.

The complete nucleotide sequence of Saccharomyces cerevisiae chromosome VII has 572 predicted open reading frames (ORFs), of which 341 are new. No correlation was found between G+C content and gene density along the chromosome, and their variations are random. Of the ORFs, 17% show high similarity to human proteins. Almost half of the ORFs could be classified in functional categories, and there is a slight increase in the number of transcription (7.0%) and translation (5.2%) factors when compared with the complete S. cerevisiae genome. Accurate verification procedures demonstrate that there are less than two errors per 10,000 base pairs in the published sequence.

Suppressors of cis-acting splicing-deficient mutations that affect the ribozyme core of a group II intron.

Many of the cis-dominant mutations that lead to respiratory deficiency by preventing maturation of specific yeast mitochondrial transcripts are found to affect the ribozyme core of group I and group II introns. We have searched for suppressors of mutations in the ribozyme-encoding sections of a group II intron, the first intron in the COX1 gene of Saccharomyces cerevisiae, which was independently subjected to in vitro site-directed mutagenesis. Three of the original mutants bore multiple mutations, which act synergistically, since for most individual mutations, suppressors could be obtained that ensured at least partial recovery of respiratory competence and splicing. Out of a total of ten suppressor mutations that were identified, three were second-site substitutions that restored postulated base-pairings in the ribozyme core. Remarkably, and as is observed for group I introns, at least half of the cis-dominant mutations in the first two group II introns of the COX1 gene affect sites that have been shown to participate in RNA tertiary interactions. We propose that this bias reflects cooperativity in the formation of ribozyme tertiary but not secondary structure, on the one hand, and the need for synergistic effects in order to generate a respiratory-deficient phenotype in the laboratory on the other. Finally, a novel in vivo splicing product of mutant cells is attributed to bimolecular splicing at high concentrations of defective transcripts.

Analysis of a 17.9 kb region from Saccharomyces cerevisiae chromosome VII reveals the presence of eight open reading frames, including BRF1 (TFIIIB70) and GCN5 genes.

We report the nucleotide sequence of a 17,893 bp DNA segment from the right arm of Saccharomyces cerevisiae chromosome VII. This fragment begins at 482 kb from the centromere. The sequence includes the BRF1 gene, encoding TFIIIB70, the 5' portion of the GCN5 gene, an open reading frame (ORF) previously identified as ORF MGA1, whose translation product shows similarity to heat-shock transcription factors and five new ORFs. Among these, YGR250 encodes a polypeptide that harbours a domain present in several polyA binding proteins. YGR245 is similar to a putative Schizosaccharomyces pombe gene, YGR248 shows significant similarity with three ORFs of S. cerevisiae situated on different chromosomes, while the remaining two ORFs, YGR247 and YGR251, do not show significant similarity to sequences present in databases.

Analysis of an 11.6 kb region from the right arm of chromosome VII of Saccharomyces cerevisiae between the RAD2 and the MES1 genes reveals the presence of three new genes.

Sequence analysis of an 11,628 bp DNA segment from the right arm of Saccharomyces cerevisiae chromosome VII revealed the presence of the 5' end of the RAD2 gene, the MES1 gene and six open reading frames (ORFs) each longer than 300 bp. Four of these ORFs are expressed genes, as indicated by transcript analysis. One of them, YGR261c, which specifies a putative beta-adaptine, corresponds to gene YKS5, which has recently been identified as a suppressor of loss of casein kinase 1 function. The remaining three ORFs are new genes; of these, YGR260w encodes a protein showing similarity to the S. cerevisiae allantoate permease and YGR262c specifies a putative protein kinase.

Comparative analysis of the region of the mitochondrial genome containing the ATPase subunit 9 gene in the two related yeast species Saccharomyces douglasii and Saccharomyces cerevisiae.

We have determined the nucleotide sequence of a region of the mitochondrial genome of the yeast Saccharomyces douglasii which contains the ATPase subunit 9 gene and part of the intergenic sequences that surround it. The gene is 228 nucleotides long and encodes a polypeptide of 76 aa. A comparison of the coding sequence with that of S. cerevisiae reveals the presence of three silent transitions. A high level of similarity is also found between regions involved in the initiation of transcription and mRNA processing. More interestingly, a region of similarity situated outside the known regulatory regions has been identified. As the intergenic regions are generally highly divergent, the remarkable conservation of these non-coding sequences suggests that their structure may be relevant to the expression of this region of the mitochondrial DNA.

A putative serine/threonine protein kinase gene on chromosome III of Saccharomyces cerevisiae.

We have sequenced a gene on chromosome III of Saccharomyces cerevisiae which codes for a putative serine/threonine protein kinase of 726 amino acids (calculated molecular weight 82 kDa). We have called this gene KIN82. The amino acid sequence of KIN82 is most similar to the cyclic nucleotide-dependent protein kinase subfamily and the protein kinase C subfamily. Gene disruption of KIN82 did not produce any phenotype when tested under a variety of conditions. Reduced stringency hybridizations revealed the presence of another genomic sequence with high homology to the carboxy-terminal catalytic domain of KIN82.

The sequence of a 6.3 kb segment of yeast chromosome III reveals an open reading frame coding for a putative mismatch binding protein.

We report the sequence of a 6.3 kb segment of DNA mapping near the end of the right arm of chromosome III of Saccharomyces cerevisiae. The sequence reveals a major open reading frame coding for a putative protein of 1047 amino acids with a striking similarity to the bacterial proteins involved in recognition of mismatched DNA base pairs. This is particularly interesting as the existence of a yeast mismatch repair system similar to that of bacteria has been postulated for some years, but a yeast protein homologous to the bacterial mismatch binding protein had not been identified. The results of a comparison of the putative yeast mismatch binding protein with the bacterial mismatch binding proteins and with two cognate mammalian sequences, support the idea that a similar mismatch repair system may be present also in mammalian cells. The possibility that all of these proteins may have evolved from a common ancestral gene is also discussed.

Antibodies against a fused gene product identify the protein encoded by a group II yeast mitochondrial intron.

In the mitochondrial genome of Saccharomyces cerevisiae, introns aI1 and aI2 of the gene encoding the COX1 subunit are the only group II introns with open reading frames (ORFs); these can be translated into two homologous proteins, the maturase aI1 and aI2. These proteins are structurally related to viral reverse transcriptases and have been shown genetically to be involved in pre-mRNA splicing and in the deletion of introns from mitochondrial DNA. To identify these mitochondrial proteins and study their properties more directly, we raised antibodies against a part of the intron aI2 ORF translation product. For this purpose, we constructed series of fusion genes, by joining parts of the genes for protein A or lacZ to different portions of the intron aI2. These were expressed in Escherichia coli as hybrid polypeptides, which were used for the production and identification of specific antibodies against the yeast mitochondrial protein. The antibodies recognized the 57 kDa protein (maturase aI2) that accumulates in two yeast mutants deficient in the splicing of aI2. This protein corresponds to the translation product of the 3' part of intron aI2 and accumulates unaltered in the two cis-acting mutants.

A controversy concerning the structure of the mitochondrial genome of a yeast petite mutant: resolution by sequence analysis.

Sequence analysis was used to define the repeat unit that constitutes the mitochondrial genome of a petite (rho-) mutant of the yeast Saccharomyces cerevisiae. This mutant has retained and amplified in tandem a 2,547 bp segment encompassing the second exon of the oxi3 gene excised from wild-type mtDNA between two direct repeats of 11 nucleotides. The identity of the mtDNA segment retained in this petite has recently been questioned (van der Veen et al., 1988). The results presented here confirm the identity of this mtDNA segment to be that determined previously by restriction mapping (Carignani et al., 1983).

Expression of the mitochondrial split gene coding for cytochrome oxidase subunit I in S. cerevisiae: RNA splicing pathway.

We have studied the splicing pathway leading to the synthesis of cytochrome oxidase subunit I (COX I) mRNA, by analysing the transcription pattern of several oxi3- splicing deficient mutants located in the first four introns of the gene. The four introns contain long open reading frames (ORFs) in phase with the upstream exons. All the mutations block the excision of the mutated intervening sequence (IVS) from the pre-mRNA, and accumulate characteristic novel polypeptides of sizes which could correspond to the translation products of the intron's ORF. Most of the mutations do not affect the splicing of the following intervening sequences; only in the case of mutations in the aI1 intron is a polar effect observed on the splicing of the second intron, aI2. Our results indicate that the splicing of these two intervening sequences which both belong to the class II of introns described by Michel et al. (1982), is controlled by the activity of the maturases encoded by their respective ORFs and that the translation of the aI2 maturase depends on the previous excision of aI1 IVS. (Moreover, the aI1 maturase, which accumulates in some mutants, can efficiently splice aI2 IVS when the translation of the latter's proper maturase cannot occur).

An mRNA maturase is encoded by the first intron of the mitochondrial gene for the subunit I of cytochrome oxidase in S. cerevisiae.

We have localized ten oxi3- mutations in the first, al1, intron of the coxl gene. All are splicing deficient, being unable to excise the intron. Complementation experiments disclose several domains in the intron al1: the 5'-proximal and 3'-proximal domains harbor cis-dominant mutations, while trans-recessive ones are located in the intron's open reading frame. Comprehensive analyses of allele-specific polypeptides accumulating in mutants show that they result from the translation of the intron's ORF. We conclude that a specific mRNA maturase involved in splicing of oxidase mRNA is encoded by the intron al1 in a manner similar to the cytochrome b mRNA maturase.