1887

Microbiology Society logo

Volume 143, Issue 8

The gene encoding a repressible acid phosphatase in the yeast cloning, sequencing and transcriptional analysis of the gene, and purification and properties of the enzyme Free

Abstract

Summary: A secreted phosphate-repressible acid phosphatase from 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, , encoding the protein has been isolated. The deduced protein, named KIPho5p, consists of 469 amino acids and has a molecular mass of 52 520 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 - and -binding sites are found in the 5′ region. Northern blot analysis of total cellular RNA indicates that the 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 gene of is able to functionally complement a mutation of Southern blot experiments, using the gene as probe, show that some reference strains lack repressible acid phosphatase, revealing a different gene organization for this kind of multigene family of proteins as compared to

  • Received:
  • Accepted:
  • Revised:
  • Published Online:
Keyword(s): acid phosphatase , Kluyveromyces lactis and transcriptional regulation
© Society for General Microbiology 1997
Loading

Article metrics loading...

/content/journal/micro/10.1099/00221287-143-8-2615
1997-08-01
2024-04-23
Loading full text...

Full text loading...

/deliver/fulltext/micro/143/8/mic-143-8-2615.html?itemId=/content/journal/micro/10.1099/00221287-143-8-2615&mimeType=html&fmt=ahah

References

  1. Arima K., Oshima T., Kubota I., Nakamura N., Mizunaga T., Toh-e A. 1983; The nucleotide sequence of the yeast PHO5 gene: a putative precursor of repressible acid phosphatase contains a signal peptide. Nucleic Acids Res 11:1657–1673
     [Google Scholar]
  2. Babczinski R. 1980; Partial purification, characterization and localization of the membrane associated invertase of yeast. Biochim Biophys Acta 614:121–127
     [Google Scholar]
  3. Bajwa W., Meyhack B., Rudolph H., Schweingruber A. M., Hinnen A. 1984; Structural analysis of the two tandemly repeated acid phosphatase genes in yeast. Nucleic Acids Res 12:7722–7738
     [Google Scholar]
  4. Barns S. M., Lane D. J., Sogin M. L., Bibeau C., Weisburg W. G. 1991; Evolutionary relationships among pathogenic Candida species and relatives. J Bacteriol 173:2250–2255
     [Google Scholar]
  5. Becker D. M., Guarente L. 1991; High efficiency transformation of yeast by electroporation. . In Methods Enzymol 194:182–187
     [Google Scholar]
  6. Bennetzen J. L., Hall B. D. 1982; Codon selection in yeast. J Biol Cbem 257:3026–3031
     [Google Scholar]
  7. Bergman L. W., McClinton D. C., Madden S. L., Preiss L. H. 1986; Molecular analysis of the DNA sequences involved in the transcriptional regulation of the phosphate-repressible acid phosphatase gene (PHO5) of Saccbaromyces cerevisiae. . Proc Natl Acad Sci USA 83:6070–6074
     [Google Scholar]
  8. Bianchi M. M., Falcone C., Chen X. J., Wesolowski-Louvel M., Frontali L., Fukuhara H. 1987; Transformation of the yeast Kluyveromyces lactis by new vectors derived from the 1.6 μm circular plasmid pkD1. Curr Genet 12:185–192
     [Google Scholar]
  9. Biggin M. D., Gibson T. J., Hong G. F. 1983; Buffer gradient gels and 35S label as an aid to rapid DNA sequence termination. Proc Natl Acad Sci USA 80:3963–3965
     [Google Scholar]
  10. Bostian K. A., Lemire J. A., Cannon L. E., Halvorson H. O. 1980; In vitro synthesis of repressible acid phosphatase: identification of multiple mRNAs and products. Proc Natl Acad Sci USA 77:4504–4508
     [Google Scholar]
  11. Boyer H., Roulland-Dussoix D. 1969; A complementary analysis of the restriction and modification of DNA in Escherichia coli. . J Mol Biol 41:459–72
     [Google Scholar]
  12. Breathnach R., Chambon P. 1981; Organization and expression of eukaryotic split genes coding for proteins. Annu Rev Biocbem 50:349–383
     [Google Scholar]
  13. Buckholz R. G., Gleeson A. G. M. 1991; Yeast systems for the commercial production of heterologous proteins. Biotechnology 9:1067–1072
     [Google Scholar]
  14. Chen X. J., Wesolowski-Louvel M., Tanguy-Rougeau C., Fukuhara H., Bianchi M. M., Fabiani L., Saliola M., Falcone C., Frontali L. 1988; A gene cloning system for Kluyveromyces lactis and isolation of a chromosomal gene required for killer production. J Basic Microbiol 28:211–220
     [Google Scholar]
  15. Cigan A. M., Donahue P. 1987; Sequence and structural features associated with translational initiator regions in yeast – a review. Gene 59:1–18
     [Google Scholar]
  16. Dimond R. L., Knecht D. A., Jordan K. B., Burns R. A., Livi G. P. 1983; Secretory mutants in the cellular slime mold Dictyo-stelium discoideum. . Methods Enzymol 96:815–826
     [Google Scholar]
  17. Domínguez A., López M. C., Santos B., Rodríguez C. 1991; Purification, glycosylation and secretion of a repressible acid phosphatase of Yarrowia lipolytica. . In Protein Glycosylation: Cellular, Biotechnological and Analytical Aspects (GBS Monographs vol. 15), pp. 117–124 . Edited by Conradt H. S. Weinheim, New York & Cambridge: VCH;
     [Google Scholar]
  18. Dubois N. K., Gilles K., Hamilton J. L., Rebers P. A., Smith F. 1956; Colorimetric method for determination of sugars and related substances. Anal Chem 28:350–356
     [Google Scholar]
  19. Elliott S., Chang C., Schweingruber M. E., Schaller, J„ Rickli E. E., Carbon J. 1986; Isolation and characterization of the structural gene for secreted acid phosphatase from Scbizo-saccbaromyces pombe. . J Biol Chem 261:2936–2941
     [Google Scholar]
  20. Erickson P. F., Minier L. N., Lasher R. S. 1982; Quantitative electrophoretic transfer of polypeptides from SDS polyacrylamide gels to nitrocellulose sheets: a method for their re-use in immunoradiographic detection of antigens. J Immunol Methods 51:241–249
     [Google Scholar]
  21. Fleer R., Yeh P., Amellal N., Maury I., Fournier A., Bacchetta F., Baduel P., Jung G., LʹHote H., Becquart J., Fukuhara H., Mayaux J. F. 1991; Stable multicopy vectors for high-level secretion of recombinant human serum albumin by Kluyveromyces yeasts. Biotechnology 9:968–975
     [Google Scholar]
  22. Goffrini P., Algeri A. A., Donnini C., Wesolowski-Louvel M., Ferrero I. 1989; RAG1 and RAG2: nuclear genes involved in the dependence/independence on mitochondrial regulatory functions for the growth on sugars. Yeast 5:99–106
     [Google Scholar]
  23. Haas J., Redl B., Friedlin E., Stoffler G. 1992; Isolation and analysis of the Penicillium chrysogenum phoA gene encoding a secreted phosphate-repressible acid phosphatase. Gene 113:129–133
     [Google Scholar]
  24. Haguenauer-Tsapis R., Hinnen A. 1984; A deletion that includes the signal peptidase cleavage site impairs processing, glycosylation, and secretion of cell surface yeast acid phosphatase. Mol Cell Biol 4:2668–2675
     [Google Scholar]
  25. Hahn S., Hoar E. T., Guarente L. 1985; Each of the three TATA elements specifies a subset of the transcription initiation sites at the CYC1 promoter of Saccbaromyces cerevisiae. . Proc Natl Acad Sci USA 82:8562–8566
     [Google Scholar]
  26. Hanahan D. 1983; Studies on transformation of Escherichia coli with plasmids. J Mol Biol 166:557–580
     [Google Scholar]
  27. von Heijne G. 1986; A new method for predicting signal sequence cleavage sites. Nucleic Acids Res 14:4683–4690
     [Google Scholar]
  28. Henikoff S. 1987; Unidirectional digestion with exonuclease III in DNA sequence analysis. Methods Enzymol 155:156–165
     [Google Scholar]
  29. Higgins D. G., Sharp P. M. 1988; clustal:.a package for performing multiple sequence alignments on a microcomputer. Gene 73:237–244
     [Google Scholar]
  30. Hinnen A., Meyhack B., Heim J. 1989; Heterologous gene expression in yeast. . In Yeast Genetic Engineering , pp. 193–213 . Edited by Barr P. J., Brake A. J., Valenzuela P. Boston: Butterworths;
     [Google Scholar]
  31. Johnston M., Carlson M. 1992; Regulation of carbon and phosphate utilization. . In The Molecular and Cellular Biology of the Yeast Saccbaromyces: Gene Expression pp. 193–281 . Edited by Strathern J. N., Jones E. W., Broach J. R. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
     [Google Scholar]
  32. Kim S., Mellor J., Kingsman A. J., Kingsman S. M. 1986; Multiple control element in the TRP1 promoter of Saccbaromyces cerevisiae. . Mol Cell Biol 6:4251–4258
     [Google Scholar]
  33. Kozak M. 1987; An analysis of 5ʹ-noncoding sequences from 699 vertebrate mRNAs. Nucleic Acids Res 15:8125–8148
     [Google Scholar]
  34. Kyte J., Doolittle R. F. 1982; A simple method for displaying the hydropathy of a protein. J Mol Biol 157:105–132
     [Google Scholar]
  35. Laemmli U. K. 1970; Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685
     [Google Scholar]
  36. López M. C. 1989 Purificación y caracterización de una fosfatasa ácida repremible de Yarrowia lipollytica. Estudio del procesamiento y aislamiento de una secuencia genica PhD thesis University of Salamanca;
     [Google Scholar]
  37. López M. C., Domfnguez A. 1988; Purification and properties of a glycoprotein acid phosphatase from the yeast form of Yarrowia lipolytica. . J Basic Microbiol 28:249–263
     [Google Scholar]
  38. MacRae W. D., Buxton F. P., Sibley S., Garven S., Gwynne D. I., Davies R. W., Arst H. N. 1988; A phosphate-repressible acid phosphatase gene from Aspergillus niger: its cloning, sequencing and transcriptional analysis. Gene 71:339–348
     [Google Scholar]
  39. Meissner P. S., Sisk W. P., Berman M. L. 1987; Bacteriophage cloning system for the construction of directional cDNA libraries. Proc Natl Acad Sci USA 84:4171–4175
     [Google Scholar]
  40. Mizunaga T. 1979; Some properties of phosphatase-repressible and constitutive acid phosphatases of baker’s yeast. Agric Biol Chem 43:1211–1218
     [Google Scholar]
  41. Moreno S., Sánchez Y., Rodríguez L. 1990; Purification and characterization of the invertase from Schizosaccharomyces pombe. . Biochem J 267:697–702
     [Google Scholar]
  42. Morrissey J. M. 1981; Silver stain for proteins in polyacrylamide gels: a modified procedure with enhanced uniform sensitivity. Anal Biochem 117:307–310
     [Google Scholar]
  43. Oshima Y. 1982; Regulatory circuits for gene expression: the metabolism of galactose and phosphate. . In The Molecular Biology of the Yeast Saccharomyces: Metabolism and Gene Expression pp. 159–180 . Edited by Strathern J. N., Jones E. W., Broach J. R. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
     [Google Scholar]
  44. Payne W. E., Gannon P. M., Kaiser C. A. 1995; An inducible acid phosphatase from the yeast Pichia pastoris: characterization of the gene and its product. Gene 163:19–26
     [Google Scholar]
  45. Pearson W. R., Lipman D. J. 1988; Improved tools for biological sequence comparison. Proc Natl Acd Sci USA 85:2444–2448
     [Google Scholar]
  46. Percival-Smith A., Segall J. 1984; Isolation of DNA sequences preferentially expressed during sporulation in Saccharomyces cerevisiae. . Mol Cell Biol 6:2443–2451
     [Google Scholar]
  47. Piddington C. S., Houston C. S., Paloheimo M., Cantrell M., Miettinen-Oinonen A., Nevalainen H., Rambosek J. 1993; The cloning and sequencing of the genes encoding phytase (phy) and pH 2.5-optimum acid phosphatase (aph) from Aspergillus niger var. awamori. . Gene 133:55–62
     [Google Scholar]
  48. Raeder U., Broda P. 1985; Rapid preparation of DNA from filamentous fungi. Lett Appl Microbiol 1:17–20
     [Google Scholar]
  49. Riederer M. A., Hinnen A. 1991; Removal of N-glycosylation sites of the yeast acid phosphatase severely affects protein folding. J Bacterial 173:3539–3546
     [Google Scholar]
  50. Rudolph H., Hinnen A. 1987; The yeast PHO5 promoter: phosphate-control elements and sequences mediating mRNA start site selection. Proc Natl Acad Sci USA 84:1340–1344
     [Google Scholar]
  51. Sambrook J. E., Fritsch E. F., Maniatis T. 1989 Molecular Cloning: a Laboratory Manual, 2nd edn.. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
     [Google Scholar]
  52. Sánchez M., Iglesias F. J., Santamaría C., Domínguez A. 1993; Transformation of Kluyveromyces lactis by electroporation. Appl Environ Microbiol 59:2087–2092
     [Google Scholar]
  53. Sanger F., Nicklen S., Coulson A. R. 1977; DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74:5463–5467
     [Google Scholar]
  54. Schurr A., Yagil E. 1971; Regulation and characterization of acid and alkaline phosphatase in yeast. J Gen Microbiol 65:291–303
     [Google Scholar]
  55. Schweingruber M. E. 1987; Acid and alkaline phosphatases in yeast. Adv Protein Phosphatases 4:77–93
     [Google Scholar]
  56. Sherman F., Fink G. R., Lawrence C. 1977 Methods in Yeast Genetics Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
     [Google Scholar]
  57. Steensma H. Y., de, Jonge P., de, Kaptein A., Kaback D. B. 1989; Molecular cloning of chromosome I DNA from Saccharomyces cerevisiae: localization of a repeated sequence containing an acid phosphatase gene near a telomere of chromosome I and chromosome VIII. Curr Genet 16:131–137
     [Google Scholar]
  58. Swinkels B. W., van Ooyen A. J. J., Bonekamp F. J. 1993; The yeast Kluyveromyces lactis as an efficient host for heterologous gene expression. Antonie Leeuwenhoek 64:187–201
     [Google Scholar]
  59. Towbin H., Staehelin T., Gordon J. 1979; Electrophoretic transfer of protein from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 76:4350–4354
     [Google Scholar]
  60. Vogel K., Hinnen A. 1990; The yeast phosphatase system. Mol Microbiol 4:2013–2017
     [Google Scholar]
  61. Vogel K., Hȣrz W., Hinnen A. 1989; The two positively acting regulatory proteins Pho2 and Pho4 physically interact with PHO5 upstream activation regions. Mol Cell Biol 9:2050–2057
     [Google Scholar]
  62. Wesolowski-Louvel M., Fukuhara H. 1995; A map of Kluyveromyces lactis genome. . Yeast 11:211–218
     [Google Scholar]
  63. Wickerham L. J. 1946; A critical evaluation of the nitrogen assimilation tests commonly used in the classification of yeast. J Bacteriol 52:293–301
     [Google Scholar]
  64. Yang J., Schweingruber E. 1990; The structural gene coding for thiamine-repressible acid phosphatase in Schizosaccharomyces pombe. . Curr Genet 18:269–272
     [Google Scholar]
  65. Zaret K. S., Sherman F. 1982; DNA sequence required for efficient transcription termination in yeast. Cell 28:265–273
     [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/00221287-143-8-2615
Loading
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
Microbiology 143, 2615 (1997); https://doi.org/10.1099/00221287-143-8-2615
/content/journal/micro/10.1099/00221287-143-8-2615
/content/journal/micro/10.1099/00221287-143-8-2615
Loading

Data & Media loading...

Most cited Most Cited RSS feed

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error