The KIPHO5 gene encoding a repressible acid phosphatase in the yeast Kluyveromyces lactis: cloning, sequencing and transcriptional analysis of the gene, and purification and properties of the enzyme.
A secreted phosphate-repressible acid phosphatase from Kluyveromyces lactis has been purified and the N-terminal region and an internal peptide have been sequenced. Using synthetic oligodeoxyribonucleotides based on the sequenced regions, the genomic sequence, KIPHO5, encoding the protein has been isolated. The deduced protein, named KIPho5p, consists of 469 amino acids and has a molecular mass of 52520 Da (in agreement with the data obtained after treatment of the protein with endoglycosidase H). The purified enzyme shows size heterogeneity, with an apparent molecular mass in the range 90-200 kDa due to the carbohydrate content (10 putative glycosylation sites were identified in the sequence). A 16 amino acid sequence at the N-terminus is similar to previously identified signal peptides in other fungal secretory proteins. The putative signal peptide is removed during secretion since it is absent in the mature secreted acid phosphatase. The gene can be induced 400-600-fold by phosphate starvation. Consensus signals corresponding to those described for Saccharomyces cerevisiae PHO4- and PHO2-binding sites are found in the 5' region. Northern blot analysis of total cellular RNA indicates that the KIPHO5 gene codes for a 1.8 kb transcript and that its expression is regulated at the transcriptional level. Chromosomal hybridization indicated that the gene is located on chromosome II. The KIPHO5 gene of K. lactis is able to functionally complement a pho5 mutation of Sacch. cerevisiae. Southern blot experiments, using the KIPHO5 gene as probe, show that some K. lactis reference strains lack repressible acid phosphatase, revealing a different gene organization for this kind of multigene family of proteins as compared to Sacch. cerevisiae.
Fermiñán
Encarnación
E
Departamento de Microbiología Genética, Universidad de Salamanca, 37071 Salamanca, Spain.
Domínguez
Angel
A
Departamento de Microbiología Genética, Universidad de Salamanca, 37071 Salamanca, Spain.
D000135:Acid Phosphatase / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D000595:Amino Acid Sequence; D001483:Base Sequence; D002874:Chromosome Mapping; D003001:Cloning, Molecular; D004790:Enzyme Induction; D005656:Fungal Proteins / Q000378:metabolism; D015966:Gene Expression Regulation, Fungal; D005800:Genes, Fungal; D007716:Kluyveromyces / Q000201:enzymology / Q000235:genetics*; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000096:biosynthesis; D017422:Sequence Analysis, DNA; D013045:Species Specificity; D014158:Transcription, Genetic
D005656:Fungal Proteins; D011994:Recombinant Proteins; D000135:Acid Phosphatase