The nucleotide sequence of the yeast PHO 5 gene : a putative precursor of

The nucleotide sequence of the PH05 gene of the yeast, Saccharomyces cerevlelae, which encodes repressible acid phosphatase (APase) was determined. Comparison of N-terainal amino acid sequence deduced from the nucleotide sequence with that of the purified repressible APase revealed the existence of a putative signal peptide in the precursor protein. The signal peptide was shown to contain 17 amino acid residues and its structural features were quite similar to those of higher eukaryotic and prokaryotlc signal peptides. The nucleotide sequence of 5' and 3' noncoding flanking regions of the PH05 gene are also discussed. INTRODUCTION The yeast Saccharomyces cerevisiae has two types of acid phosphatase (EC 3.1.3.2): one Is constitutive and the other is repressible by inorganic phosphate (Pi) (1). Two of the structural genes for these enzymes are genetically identified as PH03 and PHO5, respectively (2,3). The expression of these genes are regulated by a complex genetic system including both negative and positive regulatory factors (1,3,4). Mapping of these genes has revealed that the PH03 and PH05 genes are linked (5) and locate on chromosome II (1). Recently, the PHO3 and PH05 genes have been cloned from the yeast gene banks (6,7,8) and a partial nucleotide sequence of the PH05 gene were reported (7). The repressible acid phosphatase (APase) coded by the PHO5 gene is glycosylated during secretion across the membrane and is localized at the periplasmic space (9). Investigations on secreted proteins of prokaryotes and eukaryotes have revealed that a secreted protein Is synthesized as a precursor containing an extra peptide chain at the N-terminus of its mature form and the extra chain is cleaved during the secretion across the membrane (10). Characteristics of the signal peptides are well defined by Inouye and Halegoua (11) based on the study of an E_. coli envelope protein. In the case of £. cerevisiae, however, the existence of the signal peptide in the protein © IRL Prets Limited, Oxford, England. 1657 Nucleic Acids Research has been predicted for invertase, one of the proteins localized in the periplasmic space (12,13). To study the secretion mechanisms of proteins in yeast, we have attempted to characterize the structure of the repressible APase at both the gene and protein levels. This paper describes the nucleotide sequence of the PH05 gene encoding a putative precursor consisting of 467 amino acids. From the results of nucleotide sequence and N-terminal amino acid sequence of the mature protein, it is indicated that a peptide consisting of 17 amino acids is a signal peptide. In addition to that several features of 5' and 3' noncoding flanking region are discussed. MATERIALS AND METHODS Bacterial and yeast strains. Escherlchla coli strain JA221 (recAl leuB6 trpE5 hsdR hsdM lacY) (14) was used as a bacterial host. Saccharomyces cerevislae XP3-7A (MATa Ieu2 his4 trpl pho5 pho3) was constructed in our laboratory. Media. YPD medium contains 2 Z polypeptone, 1 Z yeast extract, 2 Z glucose. Low Inorganic phosphate (Pi) medium was prepared by treating YPD medium with MgSO, and aqueous ammonia (15). High Pi medium was prepared by supplementing low Pi medium with 0.2 Z KH.PO,. For purification of APase from the yeast, two folds basal medium (16) containing 10 Z glucose, 0.8 % casamino acids and 0.3 X KH-PO was used. The yeast minimal medium plates was based on Difco yeast Nitrogen Base without amino acids to which 2 Z glucose, the required amino acids and 2 Z agar were added. L-broth (pH 7.3) contained 1 X polypeptone, 0.5 Z yeast extract and 0.5 Z NaCl. Enzymes and Reagents. Restriction endonucleases except Clal and Avail were purchased from Takara Shuzo Co. Clal and Avail were purchased from Boehringer-Mannheim and Bethesda Research Laboratories, respectively. Zymolyase-60,000 was purchased from Kirin Brewery Co. Other enzymes and 32 reaction buffers were previously described (17). TP ATP (5000 Ci/m mole) and aP dNTP (3000 Ci/m mole) were from Amersham. Plasmlds. A yeast-E^. coll shuttle vector, pJDB219, which consists of pMB9 and 2 Jim plaamid containing the yeast LEU2 gene was kindly provided by J.D. Beggs. To obtain the plasmid carrying the PHO5 gene, the yeast gene bank was constructed from the genomic DNA of S_. cerevlslae SAT70-5B (18). The pPHOS plasmid was selected from the gene bank on the basis of the complementation of the pho5 mutation (R.S. Sidhu, A. Toh-e, S. Harashima and Y. Oshima, unpublished result).