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Biomedical subjects

A Hinnen

Publications and source records attributed to A Hinnen.

At least 37 records · Page 2Linked to original sources
PubMed1990

The yeast phosphatase system.

Yeast cells produce a set of enzymes which are involved in the metabolism of phosphate, and include acid and alkaline phosphatases as well as permeases. Most of these enzymes are synthesized in response to the presence or absence of inorganic phosphate. In the past few years a considerable amount of genetic and molecular evidence has accumulated and a rather precise overall picture emerges which describes the mechanism of phosphate control at the level of gene activation. This mini-review summarizes these data. The main focus lies on the regulatory features associated with the control of transcription of PHO5, a gene coding for most of the regulated acid phosphatase activity produced by yeast cells.

Acid Phosphatase↗
PubMed1989

Functional analysis of the signal-sequence processing site of yeast acid phosphatase.

A systematic study of the signal peptidase cleavage site of the main cell-wall-repressible Saccharomyces cerevisiae acid phosphatase encoded by the PHO5 gene is presented. The last amino acid of the signal sequence, the chromosomally encoded alanine of the wild-type gene, was changed by any of 19 other amino acids in the chromosomal DNA by using in vitro mutagenesis in Escherichia coli and the technique of gene replacement. Processing and secretion are normal when the amino acid at this position is a small neutral amino acid, i.e. alanine, glycine, cysteine, serine or threonine. Processing glycosylation, and secretion of regulated acid phosphatase are distinctly affected with other amino acid substitutions and core-glycosylated protein accumulates in the cell. Surprisingly, PHO5 protein is still secreted to the cell wall and into the growth medium but at a lower rate and without cleavage of the signal sequence. The same features are exhibited by a mutated acid phosphatase with a deletion of four amino acids at the end of the signal peptide (-7 to -4 relative to the processing site) thus preserving the important -3 to -1 region.

Acetylglucosaminidase↗
PubMed1989

Interpathway regulation of the TRP4 gene of yeast.

Two regulatory proteins, PHO2 and the general control regulator GCN4, bind in vitro to the promoter of the tryptophan biosynthetic TRP4 gene; the TRP4 gene product catalyses the phosphoribosylation of anthranilate. PHO2 binds specifically to the TRP4 promoter, but does not bind to any other TRP promoter. PHO2 and GCN4 proteins bind in a mutually exclusive manner to the same sequence, UAS1, one of two GCN4 binding sites in the TRP4 promoter. UAS1 is the major site for GCN4-dependent TRP4 activation. The second GCN4 binding site, UAS2, interacts with GCN4 alone. PHO2 binding interferes with the general control response of TRP4 under low phosphate conditions and simultaneous amino acid starvation and thus the PHO2 regulatory protein connects phosphate metabolism and amino acid biosynthesis in yeast. The GCN4 protein mediates the response of the transcriptional apparatus to the environmental signal 'amino acid limitation', while PHO2 seems to be the phosphate sensor that adjusts the response to the availability of phosphate precursors.

Base Sequence↗
PubMed1989

The two positively acting regulatory proteins PHO2 and PHO4 physically interact with PHO5 upstream activation regions.

The repressible acid phosphatase gene PHO5 of Saccharomyces cerevisiae requires the two positively acting regulatory proteins PHO2 and PHO4 for expression. pho2 or pho4 mutants are not able to derepress the PHO5 gene under low-Pi conditions. Here we show that both PHO2 and PHO4 bind specifically to the PHO5 promoter in vitro. Gel retardation assays using promoter deletions revealed two regions involved in PHO4 binding. Further characterization by DNase I footprinting showed two protected areas, one located at -347 to -373 (relative to the ATG initiator codon) (UASp1) and the other located at -239 to -262 (UASp2). Exonuclease III footprint experiments revealed stops at -349 and -368 (UASp1) as well as at -245 and -260 (UASp2). Gel retardation assays with the PHO2 protein revealed a binding region that lay between the two PHO4-binding sites. DNase I footprint analysis suggested a PHO2-binding site covering the region between -277 and -296.

Acid Phosphatase↗
PubMed1988

A 28-bp segment of the Saccharomyces cerevisiae PHO5 upstream activator sequence confers phosphate control to the CYC1-lacZ gene fusion.

Two regions within the Saccharomyces cerevisiae PHO5 upstream activator sequence (UAS) are involved in phosphate dependent transcription activation [Rudolph and Hinnen, Proc. Natl. Acad. Sci. USA 84 (1987) 1340-1344]. In experiments carried out in vivo we showed that one of these can compensate for the CYC1 UAS and expresses the heterologous CYC1-lacZ gene in response to phosphate starvation. A 28-bp segment is very efficient in gene activation, and a 19-bp subsegment that corresponds to the UASp consensus sequence brings about a weak but still detectable activation. As was observed with other UAS, gene activation is obtained with either orientation of the element, and tandem copies yield double lacZ activity compared to a single copy. No gene activation is observed in a pho4 and in a pho2 mutant. Absence of PHO2 reduces the basal expression of CYC1.

Cloning, Molecular↗
PubMed1987

The sequence of the Saccharomyces cerevisiae gene PHO2 codes for a regulatory protein with unusual aminoacid composition.

A new centromere vector for the construction of a Saccharomyces cerevisiae gene library, allowing direct selection for DNA insert, will be described. From that library the gene for the regulatory protein PHO2 involved in PHO5 induction has been cloned by complementation of a pho2 mutation. The complementing activity was shown to be located on a 3.6 kb HindIII fragment. This fragment was used to evict the genomic copy and with appropriate genetic crosses we proved, that the cloned gene is PHO2. The DNA sequence of PHO2 was determined. Analysis of the sequence data uncovered striking homology regions with PHO4, another protein necessary for the induction of PHO5. The relevance of the observed homology will be discussed.

Amino Acid Sequence↗
PubMed1987

PHO5 upstream sequences confer phosphate control on the constitutive PHO3 gene.

To identify the sequences involved in the regulation of the yeast acid phosphatase gene (PHO5) we constructed a series of hybrid promoters. Increasing lengths of 5'-flanking sequences of the PHO5 gene were placed in front of the TATA-box of constitutively expressed acid phosphatase gene (PHO3). The PHO5/PHO3 promoter constructions were used to replace the entire PHO5, PHO3 gene cluster on chromosome II. Depending on the length of PHO5 5'-flanking sequences present the PHO3 gene driven by the hybrid promoter could now be derepressed in response to inorganic phosphate (low Pi) exactly as the PHO5 wild type gene. A critical regulatory element was located between position -402 to -351 (upstream from ATG) and sequences further downstream (from -351 to -300) could increase transcriptional activation. The transcription levels of PHO3 were determined by northern blot analysis, under repressed (high Pi) and derepressed (low Pi) conditions which was paralleled by an increase in extra-cellular acid phosphatase activity. Fully regulated promoter hybrids showed a 40-fold induction of mRNA levels, comparable to wild type PHO5 promoter. Sl-nuclease protection experiments revealed that the PHO5 5'-flanking sequences, placed in front of PHO3, did not change the PHO3 transcription initiation site/s.

Acid Phosphatase↗
PubMed1987

The yeast PHO5 promoter: phosphate-control elements and sequences mediating mRNA start-site selection.

Transcription of PHO5 is strongly regulated in response to the level of inorganic phosphate (Pi) present in the growth medium. We have identified elements required for PHO5 expression by analyzing small deletions in the PHO5 promoter on chromosome II. The results reveal three functionally different components of the PHO5 promoter: regulatory regions, a "TATA" element, and specific mRNA initiation sites. The regulatory regions contain related 19-base-pair (bp) dyad sequences acting as phosphate-controlled upstream activation sites (UASpS). These UASpS mediate the transcriptional activation of PHO5 observed in low Pi conditions. The unlinked but coordinately regulated PHO11 promoter contains a single copy of an almost identical dyad sequence, suggesting that there is a common regulatory UASp for both genes. A TATA element is absolutely required for detectable PHO5 transcription. Specific purine-pyrimidine motifs (RRYRR) (R = purine and Y = pyrimidine) serve as PHO5 mRNA initiation sites, but only if they lie 55-110 bp downstream of a functional TATA element. Such an "initiation window" is not found in higher eukaryotes and implies mechanistic differences in the transcription machineries between yeast and higher eukaryotes.

Base Sequence↗
PubMed1987

The yeast acid phosphatase can enter the secretory pathway without its N-terminal signal sequence.

The repressible Saccharomyces cerevisiae acid phosphatase (APase) coded by the PHO5 gene is a cell wall glycoprotein that follows the yeast secretory pathway. We used in vitro mutagenesis to construct a deletion (delta SP) including the entire signal sequence and four amino acids of the mature sequence of APase. An APase-deficient yeast strain was transformed with a high-copy-number plasmid carrying the PHO5/delta SP gene. When expressed in vivo, the PHO5/delta SP gene product accumulated predominantly as an inactive, unglycosylated form located inside the cell. A large part of this unglycosylated precursor underwent proteolytic degradation, but up to 30% of it was translocated, core glycosylated, and matured by the addition of mannose residues, before reaching the cell wall. It appears, therefore, that the signal sequence is important for efficient translocation and core glycosylation of yeast APase but that it is not absolutely necessary for entry of the protein into the yeast secretory pathway. mRNA obtained by in vitro transcription of PHO5 and PHO5/delta SP genes were translated in vitro in the presence of either reticulocyte lysate and dog pancreatic microsomes or yeast lysate and yeast microsomes. The PHO5 gene product was translocated and core glycosylated in the heterologous system and less efficiently in the homologous system. We were not able to detect any translocation or glycosylation of PHO5/delta SP gene product in the heterologous system, but a very small amount of core suppression of glycosylated material could be evidenced in the homologous system.

Acid Phosphatase↗
PubMed1986

Removal of positioned nucleosomes from the yeast PHO5 promoter upon PHO5 induction releases additional upstream activating DNA elements.

The chromatin fine structure in the promoter region of PHO5, the structural gene for a strongly regulated acid phosphatase in yeast, was analyzed. An upstream activating sequence 367 bp away from the start of the coding sequence that is essential for gene induction was found to reside in the center of a hypersensitive region under conditions of PHO5 repression. Under these conditions three related elements at positions -469, -245 and -185 are contained within precisely positioned nucleosomes located on both sides of the hypersensitive region. Upon PHO5 induction the chromatin structure of the promoter undergoes a defined transition, in the course of which two nucleosomes upstream and two nucleosomes downstream of the hypersensitive site are selectively removed. In this way approximately 600 bp upstream of the PHO5 coding sequence become highly accessible and all four elements are free to interact with putative regulatory proteins. These findings suggest a mechanism by which the chromatin structure participates in the functioning of a regulated promoter.

Acid Phosphatase↗
PubMed1985

One-step gene replacement in yeast by cotransformation.

A general method to replace chromosomal DNA sequences of Saccharomyces cerevisiae by any in vitro modified DNA sequence has been developed and was applied to the PHO5 locus on chromosome II. A recipient strain was constructed in which part of the chromosomal PHO5 sequence was substituted by the URA3 gene. Replacement of this pho5-URA3 substitution by pho5 mutant alleles was achieved in one step by cotransformation with a pho5 DNA fragment and the self-replicating plasmid YEp13, which contains the LEU2 gene as a selectable marker. Leu+ transformants were selected, and the replacement events at the PHO5 locus were detected by their Ura- phenotype (1-4% of the Leu+ were Ura-). In a similar way the PHO5 coding sequence was replaced by the sequence coding for human tissue-type plasminogen activator (t-PA).

Alleles↗
PubMed1984

Structural analysis of the two tandemly repeated acid phosphatase genes in yeast.

We have sequenced the genetically linked genes for repressible (PHO5) and and constitutive (PHO3) acid phosphatase from S. cerevisiae. Both genes are located on a 3.91 Kb BamHI and HpaI fragment, in the order (5') PHO5, PHO3 (3'). The mRNA transcripts have been analysed by S1-nuclease mapping. They show heterogenous initiation sites. Each of the PHO5 and PHO3 genes codes for 467 amino acids as deduced from the DNA sequence. The coding regions of the two genes show homology both at the nucleotide (82%) and the amino acid (87%) level. In the coding sequences, long stretches of homologous regions are flanked by small non-homologous regions. The nucleotide homology (65%) extends to some length into the 5' and 3' non-coding flanking sequences. Further upstream sequences are unrelated. The comparison of the NH2-terminal amino acid sequence deduced from the nucleotide sequence, with that of purified repressible acid phosphatase revealed the presence of a putative signal peptide.

Acid Phosphatase↗
PubMed1984

A deletion that includes the signal peptidase cleavage site impairs processing, glycosylation, and secretion of cell surface yeast acid phosphatase.

We transformed Saccharomyces cerevisiae with a high-copy-number plasmid carrying either the wild-type gene coding for a repressible cell surface acid phosphatase or two modified genes whose products lack a 13- or 14-amino-acid segment spanning or immediately adjacent to the signal peptidase cleavage site. The wild-type gene product underwent proteolytic cleavage of the signal peptide, core glycosylation, and outer chain glycosylation. The deletion spanning the signal peptidase cleavage site led to an unprocessed protein. This modified protein exhibited core glycosylation, whereas its outer chain glycosylation was severely inhibited. Secretion of the deleted protein was impaired, and active enzyme accumulated within the cell. The deletion immediately adjacent to the signal peptidase cleavage site exhibited only a small decrease in the efficiency of processing and had no effect on the efficiency of secretion.

Acid Phosphatase↗
PubMed1982

Two yeast acid phosphatase structural genes are the result of a tandem duplication and show different degrees of homology in their promoter and coding sequences.

We have cloned the structural genes for a regulated ( PHO5 ) and a constitutive ( PHO3 ) acid phosphatase from yeast by transformation and complementation of a yeast pho3 , pho5 double mutant. Both genes are located on a 5.1-kb BamHI fragment. The cloned genes were identified on the basis of genetic evidence and by hybrid selection of mRNA coupled with in vitro translation and immunoprecipitation. Subcloning of partial Sau3A digests and functional in vivo analysis by transformation together with DNA sequence analysis showed that the two genes are oriented in the order (5') PHO5 , PHO3 (3'). While the nucleotide sequences of the two coding regions are quite similar, the putative promoter regions show a lower degree of sequence homology. Partly divergent promoter sequences may explain the different regulation of the two genes.

Acid Phosphatase↗