Giovanna Carignani

Analysis of the interaction between piD261/Bud32, an evolutionarily conserved protein kinase of Saccharomyces cerevisiae, and the Grx4 glutaredoxin.

The Saccharomyces cerevisiae piD261/Bud32 protein and its structural homologues, which are present along the Archaea-Eukarya lineage, constitute a novel protein kinase family (the piD261 family) distantly related in sequence to the eukaryotic protein kinase superfamily. It has been demonstrated that the yeast protein displays Ser/Thr phosphotransferase activity in vitro and contains all the invariant residues of the family. This novel protein kinase appears to play an important cellular role as deletion in yeast of the gene encoding piD261/Bud32 results in the alteration of fundamental processes such as cell growth and sporulation. In this work we show that the phosphotransferase activity of Bud32 is relevant to its functionality in vivo, but is not the unique role of the protein, since mutants which have lost catalytic activity but not native conformation can partially complement the disruption of the gene encoding piD261/Bud32. A two-hybrid approach has led to the identification of several proteins interacting with Bud32; in particular a glutaredoxin (Grx4), a putative glycoprotease (Ykr038/Kae1) and proteins of the Imd (inosine monophosphate dehydrogenase) family seem most plausible interactors. We further demonstrate that Grx4 directly interacts with Bud32 and that it is phosphorylated in vitro by Bud32 at Ser-134. The functional significance of the interaction between Bud32 and the putative protease Ykr038/Kae1 is supported by its evolutionary conservation.

Critical sequences within mitochondrial introns: Cis-dominant mutations of the “cytochrome-b-like” intron of the oxidase gene

We have established the DNA sequence of two cis-dominant mutations located in the fourth intron, a14, of the yeast mitochondrial gene oxi3. These mutations prevent the synthesis of subunit I of cytochrome oxidase. Both mutations affect a very short DNA sequence located several hundred base pairs from the intron-exon junctions. An identical sequence is found in the cob-box gene; and this sequence is critical for the excision of the cytochrome b intron. Our interpretation is that this short sequence represents a common signal that must be recognized by the box7-encoded mRNA maturase, in conjunction with the mitochondrial ribosome, to splice out the introns in the two nonhomologous genes, cob-box and oxi3.

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 [γ‐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.

Functional homology between yeast piD261/Bud32 and human PRPK: both phosphorylate p53 and PRPK partially complements piD261/Bud32 deficiency.

Yeast piD261/Bud32 belongs to the piD261 family of atypical protein kinases structurally conserved, from Archaea to human. The disruption of its gene is causative of severely defective growth. Its human homologue, PRPK, interacts with and phosphorylates the oncosuppressor p53 protein, which is lacking in yeast. Here we show that on one hand piD261/Bud32 interacts with and phosphorylates human p53 in vitro, on the other hand PRPK can partially complement the phenotype of yeast lacking the gene encoding piD261/Bud32. These data indicate that, despite considerable structural divergence, members of the piD261 family from distantly related organisms display a remarkable functional conservation.

Structure-function analysis of yeast piD261/Bud32, an atypical protein kinase essential for normal cell life

The Saccharomyces cerevisiae YGR262c/BUD32 gene, whose disruption causes a severe pleiotropic phenotype, encodes a 261-residue putative protein kinase, piD261, whose structural homologues have been identified in a variety of organisms, including humans, and whose function is unknown. We have demonstrated previously that piD261, expressed in Escherichia coli as a recombinant protein, is a Ser/Thr kinase, as judged by its ability to autophosphorylate and to phosphorylate casein. Here we describe a mutational analysis showing that, despite low sequence similarity, the invariant residues representing the signature of protein kinases are conserved in piD261 and in its structural homologues, but are embedded in an altered context, suggestive of unique mechanistic properties. Especially noteworthy are: (i) three unique inserts of unknown function within the N-terminal lobe, (ii) the lack of a lysyl residue which in all other Ser/Thr kinases participates in the catalytic event by interacting with the transferred ATP gamma-phosphate, and which in piD261 is replaced by a threonine, and (iii) an exceedingly short activation loop including two serines, Ser-187 and Ser-189, whose autophosphorylation accounts for the appearance of an upshifted band upon SDS/PAGE. A mutant in which these serines are replaced by alanines was devoid of the upshifted band and displayed reduced catalytic activity. This would include piD261 in the category of protein kinases activated by phosphorylation, although it lacks the RD (Arg-Asp) motif which is typical of these enzymes.

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). Copyright

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. Copyright

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

SummaryWe 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 introns ORE Most of the mutations do not affect the splicing of the following intervening sequences; only in the case of mutations in the all 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 all IVS. (Moreover, the aI1 maturase, which accumulates in some mutants, can efficiently splice aI2 IVS when the translation of the latters proper maturase cannot occur).

The cytochrome oxidase subunit I split gene in Saccharomyces cerevisiae: genetic and physical studies of the mtDNA segment encompassing the 'cytochrome b-homologous' intron.

SummaryWe have constructed a refined genetic and physical map of 38 oxi3 mutations. With the help of the rho- clones derived from ‘short’ and ‘long’ genes, pairwise crosses between mutants, estimations of their reversion frequencies and analyses of mitochondrially synthesized proteins, we have characterized and localized several mutants in the exon A4 and in the intron aI4. We present genetic and physical evidence that in the ‘long’ gene the exon A5 is split into at least three quite distinct exons, A5-1, A5-2 and A5-3 where numerous mutations are localized. We suggest that a novel 56 Kd polypeptide, which accumulates in some cis-dominant oxi3- mutants results from the translation of the upstream exons and the downstream aI4 intron.

Petite deletion map of the mitochondrial oxi3 region in Saccharomyces cerevisiae.

SummaryFifty eight mitochondrial mutants (p+ mit- mutants), all deficient in cytochrome oxidase activity and previously assigned to the genetic region oxi3 on the mitochondrial DNA, were mapped by the method of “petite deletion mapping”.This procedure resulted in the identification of at least twenty one different classes of oxi3 mutants, which could be arranged in a linear order.Moreover, it provided a set of twenty three p- petite mutants, each containing a differentially deleted mit DNA segment included in the oxi3 region. The two sets of mutants, p+oxi3- and p-oxi3+, will be of interest for a further genetic and physical analysis of this mitochondrial DNA segment which spans over about ten thousand base pairs and controls the subunit I of cytochrome oxidase.