The PH084 gene specifies Pi-transport in Saccharomyces cerevisiae. A DNA fragment bearing the PH084 gene was cloned by its ability to complement constitutive synthesis of repressible acid phosphatase of pho84 mutant cells. Its nucleotide sequence predicted a protein of 596 amino acids with a sequence homologous to that of a superfamily of sugar transporters. Hydropathy analysis suggested that the secondary structure of the PH084 protein consists of two blocks of six transmembrane domains separated by 74 amino acid residues. The cloned PH084 DNA restored the Pi transport activity of pho84 mutant cells. The PH084 transcription was regulated by P1 like those of the PHOS, PHO8, and PHO81 genes. A PH084-lacZ fusion gene produced j}-galactosidase activity under the regulation of Pi, and the activity was suggested to be bound to a membrane fraction. Gene disruption of PH084 was not lethal. By comparison of nucleotide sequences and by tetrad analysis with GAL80 as a standard, the PH084 locus was mapped at a site beside the TUB3 locus on the left arm of chromosome XIII.

We have cloned a DNA fragment complementing the aarl mutation defective in the al-a2 repression of the al cistron and haploid-specific genes in Saccharomyces cerevisiae. Nucleotide sequence and mapping data indicated that the AAR] gene is identical with TUPI, which is allelic to the SF12, FLKI, CYC9, UMR7, AMMI, and AER2 genes, whose mutations are known to confer a variety of phenotypes, such as thymidine uptake, flocculation, insensitivity to glucose repression, a defect in UV-induced mutagenesis, and a defect in ARS plasmid maintenance. The TUP1/AER2 protein is known to have significant similarity with the subunits of G proteins in the C-terminal half, in two glutamine-rich domains in the N-terminal half, and in a central region rich in serine and threonine residues. Disruption of the chromosomal AAR] gene in a and a/a cells conferred the nonmating phenotype, and the a/a diploids could not sporulate. The AAR1/TUP1 gene is transcribed into a 2.5-kb mRNA independently of the mating-type information of the cell. These observations and mRNA analysis of cell-type-specific genes indicated that the AAR1/TUP1 protein is also indispensable for al-a2 repression of RMEI and for a2 repression of a-specific genes.

Sterile mutants of Saccharomyces cerevisiae were isolated from ca* cells having the a/a aarl-6 genotype (exhibiting a mating ability and weak a mating ability as a result of a defect in al-a2 repression). Among these sterile mutants, we found two steS mutants together with putative ste7, stell, and stel2 mutants of the signal transduction pathway of mating pheromones. The amino acid sequence of the Ste5p protein predicted from the nucleotide sequence of a cloned STE5 DNA has a domain rich in acidic amino acids close to its C terminus, a cysteine-rich sequence, resembling part of a zinc finger structure, in its N-terminal half, and a possible target site of cyclic AMP-dependent protein kinase at its C terminus. Northern (RNA) blot analysis revealed that STE5 transcription is under al-ai2-Aarlp repression. The AL4Tor cistron has a single copy of the pheromone response element in its 5' upstream region, and its basal level of transcription was reduced in these ste mutant cells. However, expression of the AL4TaJ cistron was not enhanced appreciably by pheromone signals. One of the ste5 mutant alleles conferred a sterile phenotype to a/ca aarl-6 cells but a mating ability to MATa cells.

The a2 protein, the product of the MATa2 cistron, represses various genes specific to the a mating type (a2 repression), and when combined with the MATal gene product, it represses MATaJ and various haploidspecific genes (al-a2 repression). One target of al-a2 repression is RME1, which is a negative regulator of a/a-specific genes. We have isolated 13 recessive mutants whose al-a2 repression is defective but which retain a2 repression in a genetic background of ho MATa HMLa HMRa sir3 or ho MATa HMRa HMRa sir3. These mutations can be divided into three different classes. One class contains a missense mutation, designated hmla2-102, in the a2 cistron of HML, and another class contains two missense mutations, mata2-201 and mata2-202, in the MATa locus. These three mutants each have an amino acid substitution of tyrosine or phenylalanine for cysteine at the 33rd codon from the translation initiation codon in the a2 cistron of HMLa or MATa. The remaining 10 mutants make up the third class and form a single complementation group, having mutations designated aarl (al-a2 repression), at a gene other than MAT, HML, HMR, RMEI, or the four SIR genes. Although a diploid cell homozygous for the aarl and sir3 mutations and for the MATa, HMLa, and HMRa alleles showed a mating type, it could sporulate and gave rise to asci containing four a mating-type spores. These facts indicate that the domain for a2 repression is separable from that for al-a2 protein interaction or complex formation in the a2 protein and that an additional regulatory gene, AAR], is associated with the al-a2 repression of the al cistron and haploid-specific genes.Cell types of Saccharomyces cerevisiae are determined by the codominant MATa and MATa alleles at the mating-type locus, MAT, on the right arm of chromosome III (for reviews, see references 16 and 37). A haploid MATa cell has the a mating type and can mate with a cell having the MATa genotype. Conjugation of a and a cells produces an ala diploid cell which has the third cell type, nonmater. The a/a diploid cells undergo meiosis and form asci, and each ascus contains four ascospores bearing a haploid tetrad nucleus. Besides cell-type control, the MAT locus regulates various other genes such as HO for homothallic switching of the MAT locus (19) and the transcription of Ty elements (8,9).In addition to the MAT locus, complete but unexpressed copies of MAT information are encoded at two other loci, HML and HMR, on the same chromosome (16, 37). Homothallic switching of information at the MATa locus to a or MATa to a is achieved by transposition of the a or a cassette from the HMR or HML locus to the MAT locus. In general, HMR encodes the a information and HML has a, but their expression is normally repressed by the function of the SIR genes.A hypothesis concerning the regulatory function of the MAT genes, known as the al-a2 hypothesis (39), proposes that MATa consists of two cistrons, al and a2. The al cistron encodes a positive regulator for a-specific genes, and the a2 cistron encodes a negative regulat...

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