Cloning and characterization of the Pichia pastoris PRC1 gene encoding carboxypeptidase Y.
We purified a 58 kDa serine protease from culture-supernatant of Pichia pastoris and found that the NH2-terminal amino acid sequence of this protease is closely homologous to that of mature protein of Saccharomyces cerevisiae carboxypeptidase Y (CPY), which is encoded by the PRC1 gene. Using the S. cerevisiae PRC1 gene as a hybridization probe, a cross-hybridizing fragment of P. pastoris genomic DNA was identified and the gene, PRC1, encoding CPY, was cloned. The open reading frame of the P. pastoris PRC1 gene consists of 1569 bp encoding a protein of 523 amino acids. The molecular mass of the protein is calculated to be 59.44 kDa without sugar chains. The protein comprises 20 amino acids of pre (signal)-peptide, 87 amino acids of pro-peptide and 416 amino acids of mature peptide, and has four N-glycosylation sites. The NH2-terminal amino acid sequence of mature peptide is completely identical with that of the protease purified from the culture-supernatant. There is 61% identity between the amino acid sequences of P. pastoris Prc1p and S. cerevisiae Prc1p. Chromosomal disruption of the PRC1 gene resulted in the loss of CPY activity. Over-expression of the PRC1 gene under regulation of the P. pastoris AOX1 promoter resulted in accumulation of a large amount of active CPY in the intracellular fraction, and secretion of a slightly larger molecule that is thought to be pro-CPY. The nucleotide sequence data reported in this paper will appear in the EMBL Nucleotide Sequence Databases under the Accession Number X87987.
Ohi
H
H
Central Research Laboratory, Green Cross Corporation, Osaka 573, Japan.
Ohtani
W
W
Okazaki
N
N
Furuhata
N
N
Ohmura
T
T
D000595:Amino Acid Sequence; D001483:Base Sequence; D002268:Carboxypeptidases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D043402:Cathepsin A; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D005800:Genes, Fungal; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010843:Pichia / Q000201:enzymology* / Q000235:genetics*; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity
D017931:DNA Primers; D002268:Carboxypeptidases; D043402:Cathepsin A