Opi1 mediates repression of phospholipid biosynthesis by phosphate limitation in the yeast Saccharomyces cerevisiae

Structural genes of phospholipid biosynthesis in the yeast Saccharomyces cerevisiae are transcribed when precursor molecules inositol and choline (IC) are limiting. Gene expression is stimulated by the heterodimeric activator Ino2/Ino4, which binds to ICRE (inositol/choline‐responsive element) promoter sequences. Activation is prevented by repressor Opi1, counteracting Ino2 when high concentrations of IC are available. Here we show that ICRE‐dependent gene activation is repressed not only by an excess of IC but also under conditions of phosphate starvation. While PHO5 is activated by phosphate limitation, INO1 expression is repressed about 10‐fold. Repression of ICRE‐dependent genes by low phosphate is no longer observed in an opi1 mutant while repression is still effective in mutants of the PHO regulon (pho4, pho80, pho81 and pho85). In contrast, gene expression with high phosphate is reduced in the absence of pleiotropic sensor protein kinase Pho85. We could demonstrate that Pho85 binds to Opi1 in vitro and in vivo and that this interaction is increased in the presence of high concentrations of phosphate. Interestingly, Pho85 binds to two separate domains of Opi1 which have been previously shown to recruit pleiotropic corepressor Sin3 and activator Ino2, respectively. We postulate that Pho85 positively influences ICRE‐dependent gene expression by phosphorylation‐dependent weakening of Opi1 repressor, affecting its functional domains required for promoter recruitment and corepressor interaction. Copyright © 2016 John Wiley & Sons, Ltd.     

Pho85 kinase, a yeast cyclin-dependent kinase, regulates the expression of UGP1 encoding UDP-glucose pyrophosphorylase

The PHO85 gene is a negative regulator of the PHO system in the yeast Saccharomyces cerevisiae and encodes a protein kinase (Pho85) highly homologous to the Cdc28 kinase (Cdc28). Ten cyclin-like proteins are known to interact with Pho85, and combination with different cyclins is believed to be responsible for distinct Pho85 functions, including phosphate metabolism, carbon source utilization and cell cycle regulation. However, only a limited number of substrates of Pho85 kinase, including Pho4, Gsy2 and Sicl, have so far been identified. To search for more targets of Pho85 and to clarify the genetic control mechanisms by Pho85 kinase in these cellular functions, we carried out a genome-wide analysis of the effect of a pho85Delta mutation on gene expression. We found that expression of various genes involved in carbon metabolism are affected by the mutation and that among them, UGP1 promoter activity was increased in the absence of Pho85 kinase. This increase in the promoter activity was not observed in a pho4Delta mutant or with a mutant UGP1 promoter that is devoid of putative Pho4 and Bas2 binding sites, suggesting that UGP1 expression is modulated by Pho85 through Pho4. We also found that expression of several Pho85-cyclin genes were altered by the carbon source, the growth phase and Pho85 kinase itself.     

Yeast Pho85 kinase is required for proper gene expression during the diauxic shift

The budding yeast Saccharomyces cerevisiae changes its gene expression profile when environmental nutritional conditions are changed. Protein kinases including cyclic AMP-dependent kinase, Snf1 and Tor kinases play important roles in this process. Pho85 kinase, a member of the yeast cyclin-dependent kinase family, is involved in the regulation of phosphate metabolism and reserve carbohydrates, and thus is implicated to function as a nutrient-sensing kinase. Upon depletion of glucose in the medium, yeast cells undergo a diauxic shift, accompanied by a carbon metabolic pathway shift, stimulation of mitochondrial function and downregulation of ribosome biogenesis and protein synthesis. We analysed the effect of a pho85Delta mutation on the expression profiles of the genes in this process to investigate whether Pho85 kinase participates in the yeast diauxy. We found that, in the absence of PHO85, a majority of mitochondrial genes were not properly induced, that proteasome-related and chaperonin genes were more repressed, and that, when glucose was still present in the medium, a certain class of genes involved in ribosome biogenesis (ribosomal protein and rRNA processing genes) was repressed, whereas those involved in gluconeogenesis and the glyoxylate cycle were induced. We also found that PHO85 is required for proper expression of several metal sensor genes and their regulatory genes. These results suggest that Pho85 is required for proper onset of changes in expression profiles of genes responsible for the diauxic shift.     

Bacterial plasmid pBR322 sequences serve as upstream activating sequences in Saccharomyces cerevisiae

The expression of acid phosphatase (APase) from PHO5 and MF alpha-PHO5 hybrid genes is regulated by inorganic phosphate and mating type locus respectively, as well as the PHO4 and MAT alpha 1 gene products respectively. When PHO5 and MF alpha-PHO5 hybrid genes were cloned in the BamHI site of the pBR322 sequence of the yeast shuttle vectors (YRp7 or YEp9T), in one orientation they were regulated normally but in the other orientation their expression was not regulated but expressed constitutively. The pBR322 sequences present upstream of the inserted genes are responsible for the constitutive expression. By replacing the PHO5 upstream activating sequences (UAS) element with pBR322 fragments, we have identified three pBR322 sequences, from nucleotides 376 to 650, 2068 to 2116 and 2136 to 2247, which were able to promote expression of APase. A comparison of these three pBR322 fragments revealed 5' ATCGCGCGAG 3' and 5' CGGTGATGNCGG 3' to be the common sequences likely to act as UASs in Saccharomyces cerevisiae. By using synthetic oligonucleotides, it was found that both sequences are required for maximum expression of APase activity.     

A strong carbon source effect is mediated by pUC plasmid sequences in a series of classical yeast vectors designed for promoter characterization

While using YIp356 and YEp356R lacZ reporter plasmids, we found lacZ expression driven by the ARG2 promoter to be much higher in cells grown on a non‐glucose carbon source than in glucose‐grown cells (5–10‐fold higher on galactose and up to 40‐fold higher on ethanol). Furthermore, expression increased 30‐fold upon shifting from a high‐glucose to a low‐glucose medium. This carbon source regulation requires Snf1p and possibly Ssn6p. It appears, however, to be artefactually mediated by plasmid sequences located upstream from the multicloning site. This emerged from the following observations: (a) the derepressive effect disappears if any extra piece of DNA is inserted upstream from the ARG2 promoter; and (b) similar derepression on low glucose is observed with another yeast promoter (ARG11), provided that the flanking 5′ region is short. We determined that the cis‐elements responsible for this physiologically irrelevant glucose regulation are located between positions 636 and 879 of the pUC18 DNA sequence. Copyright © 1999 John Wiley & Sons, Ltd.     

The sequence of 36·8 kb from the left arm of chromosome XIV reveals 24 complete open reading frames: 18 correspond to new genes,one of which encodes a protein similar to the human myotonic dystrophy kinase

We have determined the complete nucleotide sequence of a 36·8 kb segment from the left arm of chromosome XIV carried by the cosmid 14–11. The sequence encodes the 5′ coding region of the PSD1 gene, the 3′ coding region of an unknown gene and 24 complete open reading frames, of which 18 correspond to new genes and six (SKO1, SCL41A, YGP1, YCK2, RPC31 and MFA2) have been sequenced previously. Of the 24 new genes, five show significant similarities to sequences present in the databanks. These include elongation factors 2 and the human myotonic dystrophy kinase. The sequence has been deposited in the EMBL databank under the Accession Number X92517.     

II. Yeast sequencing reports. The sequence of 12·5 kb from the right arm of chromosome II predicts a new N-terminal sequence for the IRA1 protein and reveals two new genes,one of which is a DEAD-box helicase

We have determined the complete nucleotide sequence of a 12·5 kb segment from the right arm of chromosome II carried by the cosmid α20. The sequence encodes the 5′ end of the IRA1 gene. Two complete new open reading frames and the 3′ non-coding region of the SUP1 (SUP45) gene. A comparison of our sequence with the data bank reveals a 154 amino acid extension at the N-terminus of Ira1p compared to the previously predicted sequence. According to the 11th edition of the Saccharomyces cerevisiae genetic map, our sequence should encode the MAK5 gene, which is necessary for the maintenance of dsRNA killer plasmids. One of the two new open reading frames, YBR1119, is predicted to encode an RNA helicase, thus YBR1119 may correspond to the MAK5 gene. The sequence has been deposited in the EMBL data library under Accession Number X78937.     

Functional differences among the six Saccharomyces cerevisiae tRNATrp genes

The Saccharomyces cerevisiae haploid genome includes six copies of the gene encoding tRNA^Trp which are scattered on five chromosomes. Other, non-functional tDNA^Trp fragments also occur in the genome. The segments of all six genes which encode the 72-nucleotide mature tRNAas well as a 34-nucleotide intervening sequence, are identical. However, the 5′ and 3′ flanking sequences diverge virtually at the boundaries of the coding region. We have used an assay based on suppression of UGA mutations by multi-copy clones of tDNA^Trp to search for functional differences among these genes. Previous studies with one tDNA^Trp had demonstrated that moderate suppression of a UGA mutation, leu2-2, resulted from introduction of a multi-copy clone of the gene. Attempts to use this assay to select tDNA^Trp clones from a yeast genomic library yielded only four of the six different clones. The other two genes were amplified by PCR and cloned in pRS202, a 2 μ vector also used for the genomic library. Plasmids bearing the six tRNA genes were transformed into S. cerevisiae strain JG369.3B and scored for their ability to suppress the leu2-2 mutation as well as his4-260, another UGA marker. Two of the six tRNA^Trp clones were unable to suppress either marker, two evidenced weak suppression of the Leu auxotrophy, and two were able to suppress both markers. Growth rates in liquid media requiring suppression were measured for cell lines carrying each of the clones. Differences greater than 50-fold were observed in media lacking histidine. An evolutionary tree based on 5′-flanking sequence corresponds reasonably well with suppressor activity, while a similar analysis of 3′-flanking sequence does not. This suggests that the functional differences are based on divergence in the 5′-flanking sequences of the tRNA^Trp genes. © 1997 John Wiley & Sons, Ltd.     

Metabolism of extracellular inositol hexaphosphate (phytate) by Saccharomyces cerevisiae

Iron and zinc deficiencies are global problems, frequently leading to severe illness in vulnerable human populations. Addition of phytases can improve the bioavailability of iron and zinc in food. Saccharomyces cerevisiae would be an ideal candidate as a bioavailability improving food additive if it demonstrates significant phytase activity. The purpose of the paper was to study yeast phytase activity to obtain information required to improve strains. All yeasts tested readily degraded extracellular inositol hexaphosphate (phytate; IP6) in media with IP6 as the sole phosphorous source. Phosphate (Pi) addition yielded repression consistent with the PHO system. However, repression of IP6-degrading enzymes was not only dependent on level of Pi, but also on pH and medium composition. In complex medium, containing Pi at a concentration previously suggested to yield full repression of the secretory acid phosphatases (SAPs; e.g., [Mol. Biol. Cell 11 (2000) 4309]), and at relatively high pH, repression of phytate-degrading enzymes was weak. The capacity to degrade phytate, irrespective of Pi addition or not, was highest at the pH most distant from the pH optimum of the SAPs [Microbiol. Res. 151 (1996) 291], suggesting that expression rather than enzyme activity was affected by pH. In synthetic medium, repression was strong and pH-independent (no IP6 degradation within the range tested). The distinct difference between media shows that, in addition to known regulatory role of Pi for the PHO system, additional factors may be involved. Using a deletion strain, we further demonstrate that the main secretory acid phosphatase Pho5p is not essential for intact phytate-degrading capacity and growth without Pi, neither is Pho3p. However, when constitutively overexpressing PHO5 an increased net phytase activity was obtained, in repressing and non-repressing conditions. This proves that, although redundant in a wild type, Pho5p can catalyze hydrolysis of IP6 and that at least one more enzyme is capable of effective hydrolysis of IP6 (sufficient to provide the cell with phosphorous at a rate yielding maximum growth). Finally, a bread dough experiment showed that the typical concentrations of Pi during leavening exceed levels shown to repress phytate degradation by a wild-type S. cerevisiae.