1887

Abstract

Pathogenic yeasts of the genus secrete aspartic proteinases (Sap) which are synthesized as preproenzymes. Expression of the gene lacking the propeptide-coding region in the methylotrophic yeast does not lead to the secretion of the enzyme into the culture supernatant, but results in an accumulation of recombinant protein in the cell. Co-expression in this system of the unattached propeptide from Sap1p, as well as from other Saps, restored Sap1p secretion. A deletion analysis revealed that only a 12 aa sequence in the propeptide, corresponding to a highly conserved region in all Sap propeptides, was necessary and sufficient to produce a large amount of Sap1p in culture supernatant. No Sap1p was secreted when Sap1p was produced with a propeptide carrying an F to D mutation in the identified 12 aa sequence. However, the simultaneous production of equivalent amounts of Sap1p and His-tagged Sap1p (H-Sap1p) with a mutated and a non-mutated propeptide, respectively, led to the secretion of both proteins in a ratio of approximately 1:2. The restoration of Sap1p secretion occurred at the expense of secretion of H-Sap1p since the total activity was comparable to that of strains producing only H-Sap1p with a non-mutated propeptide. In contrast, the proteolytic activity of strains secreting Sap1p and H-Sap1p both with a functional propeptide was twice that of strains producing either Sap1p or H-Sap1p alone, and the two enzymes were found in an equivalent amount in the culture supernatant. Altogether, these results show that the propeptide can only function once and that the maturation of recombinant secreted aspartic proteinase Sap1p is directed through a combination of intra- and inter-molecular pathways.

Loading

Article metrics loading...

/content/journal/micro/10.1099/00221287-146-11-2765
2000-11-01
2024-03-29
Loading full text...

Full text loading...

/deliver/fulltext/micro/146/11/1462765a.html?itemId=/content/journal/micro/10.1099/00221287-146-11-2765&mimeType=html&fmt=ahah

References

  1. Baker D., Sohl J. L., Agard D. A. 1992; A protein-folding reaction under kinetic control. Nature 356:263–265 [CrossRef]
    [Google Scholar]
  2. Borg-von Zepelin M., Beggah S., Boggian K., Sanglard D., Monod M. 1998; The expression of the secreted aspartyl proteinases Sap4 to Sap6 from Candida albicans in murine macrophages. Mol Microbiol 28:543–554 [CrossRef]
    [Google Scholar]
  3. Bradford M. M. 1976; A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254 [CrossRef]
    [Google Scholar]
  4. Drocourt D., Calmels T. P. G., Reynes J. P., Baron M., Tiraby G. 1990; Casettes of the Streptoalloteichus hindustanus ble gene for transformation of lower and higher eucaryotes to phleomycine resistance. Nucleic Acids Res 18:4009 [CrossRef]
    [Google Scholar]
  5. Eder J., Fersht A. R. 1995; Pro-sequence-assisted protein folding. Mol Microbiol 16:609–614 [CrossRef]
    [Google Scholar]
  6. Fabre E., Nicaud J. M., Lopez M. C., Gaillardin C. 1991; Role of the proregion in the production and secretion of the Yarrowia lipolytica alkaline extracellular protease. J Biol Chem 266:3782–3790
    [Google Scholar]
  7. Fabre E., Tharaud C., Gaillardin C. 1992; Intracellular transit of a yeast protease is rescued by trans-complementation with its prodomain. J Biol Chem 267:15049–15055
    [Google Scholar]
  8. Fallon K., Bausch K., Noonan J., Huguenel E., Tamburini P. 1997; Role of aspartic proteases in disseminated Candida albicans infection in mice. Infect Immun 65:551–556
    [Google Scholar]
  9. Fukuda R., Horiuchi H., Ohta A., Takagi M. 1994; The prosequence of Rhizopus niveus aspartic proteinase-I supports correct folding and secretion of its mature part in Saccharomyces cerevisiae. J Biol Chem 269:9556–9561
    [Google Scholar]
  10. Fukuda R., Umebayashi K., Horiuchi H., Ohta A., Takagi M. 1996; Degradation of Rhizopus niveus aspartic proteinase-I with mutated prosequences occurs in the endoplasmic reticulum of Saccharomyces cerevisiae. J Biol Chem 271:14252–14255 [CrossRef]
    [Google Scholar]
  11. Gatignol A., Durand H., Tiraby G. 1988; Bleomycin resistance conferred by a drug-binding protein. FEBS Lett 230:171–175 [CrossRef]
    [Google Scholar]
  12. van den Hazel H., Kielland-Brandt M. C., Winther J. R. 1994; The propeptide is required for in vivo formation of stable active yeast proteinase A and can function even when not covalently linked to the mature region. J Biol Chem 268:18002–18007
    [Google Scholar]
  13. Helenius A., Marquart T., Braakman I. 1992; The endoplasmic reticulum as a protein-folding compartment. Trends Cell Biol 2:227–231 [CrossRef]
    [Google Scholar]
  14. Hu X., Haghjoo K., Jordan F. 1996; Further evidence for the structure of the subtilisin propeptide and for its interactions with mature subtilisin. J Biol Chem 271:3375–3384 [CrossRef]
    [Google Scholar]
  15. Hube B., Turver C. J., Odds F. C., Eiffert H., Boulnois G. J., Kochel H., Rüchel R. 1991; Sequence of the Candida albicans gene encoding the secretory aspartate proteinase. J Med Vet Mycol 29:129–132 [CrossRef]
    [Google Scholar]
  16. Inouye M. 1991; Intramolecular chaperone: the role of the pro-peptide in protein folding. Enzyme 45:314–321
    [Google Scholar]
  17. Julius D., Brake A., Blair L., Kunisawa R., Thorner J. 1984; Isolation of the putative structural gene for the lysine-arginine-cleaving endopeptidase required for processing of yeast prepro-α-factor. Cell 37:1075–1089 [CrossRef]
    [Google Scholar]
  18. Laemmli U. K. 1970; Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685 [CrossRef]
    [Google Scholar]
  19. Lerner C. G., Kobayashi T., Inouye M. 1990; Isolation of subtilisin prosequence mutations that affect formation of active protease by localized random polymerase chain reaction mutagenesis. J Biol Chem 265:2085–2086
    [Google Scholar]
  20. McIver K. S., Kessler E., Olson J. C., Ohman D. E. 1995; The elastase propeptide functions as an intramolecular chaperone required for elastase activity and secretion in Pseudomonas aeruginosa. Mol Microbiol 18:877–889 [CrossRef]
    [Google Scholar]
  21. Monod M., Togni G., Hube B., Sanglard D. 1994; Multiplicity of genes encoding secreted aspartic proteases in Candida species. Mol Microbiol 13:357–368 [CrossRef]
    [Google Scholar]
  22. Monod M., Hube B., Hess D., Sanglard D. 1998; Differential regulation of SAP8 and SAP9, which encode two new members of the secreted aspartic proteinase family in Candida albicans. Microbiology 144:2731–2737 [CrossRef]
    [Google Scholar]
  23. Newport G., Agabian N. 1997; KEX2 influences Candida albicans proteinase secretion and hyphal formation. J Biol Chem 272:28954–28961 [CrossRef]
    [Google Scholar]
  24. Ohta Y., Hojo H., Aimoto S., Kobayashi T., Zhu X., Jordan F., Inouye M. 1991; Pro-peptide as an intermolecular chaperone: renaturation of denaturated subtilisin E with a synthetic pro-peptide. Mol Microbiol 5:1507–1510 [CrossRef]
    [Google Scholar]
  25. Pfeffer S. R., Rothman J. E. 1987; Biosynthetic protein transport and sorting by the endoplasmic reticulum and Golgi. Annu Rev Biochem 56:829–852 [CrossRef]
    [Google Scholar]
  26. Schaller M., Schäfer W., Korting H. C., Hube B. 1998; Differential expression of secreted aspartyl proteinases in a model of human oral candidosis and in patient samples from the oral cavity. Mol Microbiol 29:605–615 [CrossRef]
    [Google Scholar]
  27. Schaller M., Korting H. C., Schäfer W., Bastert J., WenChieh C., Hube B. 1999; Secreted aspartic proteinase (Sap) activity contributes to tissue damage in a model of human oral candidosis. Mol Microbiol 34:169–180 [CrossRef]
    [Google Scholar]
  28. Shinde U. P., Liu J. J., Inouye M. 1997; Protein memory through altered folding mediated by intramolecular chaperones. Nature 389:520–522 [CrossRef]
    [Google Scholar]
  29. Togni G., Sanglard D., Falchetto R., Monod M. 1991; Isolation and nucleotide sequence of the extracellular acid protease gene (ACP) from the yeast Candida tropicalis. FEBS Lett 286:181–185 [CrossRef]
    [Google Scholar]
  30. Togni G., Sanglard D., Quadroni M., Foundling S. I., Monod M. 1996; Acid proteinase secreted by Candida tropicalis: functional analysis of preproregion cleavages in C. tropicalis and Saccharomyces cerevisiae. Microbiology 142:493–503 [CrossRef]
    [Google Scholar]
  31. de Viragh P. A., Sanglard D., Togni G., Falchetto R., Monod M. 1993; Cloning and sequencing of two Candida parapsilosis genes encoding acid proteases. J Gen Microbiol 139:335–342 [CrossRef]
    [Google Scholar]
  32. Yaffe M. P., Schatz G. 1984; Two nuclear mutations that block mitochondrial protein import in yeast. Proc Natl Acad Sci USA 81:4819–4823 [CrossRef]
    [Google Scholar]
  33. Zhu X., Ohta Y., Jordan F., Inouye M. 1989; Pro-sequence of subtilisin can guide the refolding of denaturated subtilisin in an intermolecular process. Nature 339:483–484 [CrossRef]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/00221287-146-11-2765
Loading
/content/journal/micro/10.1099/00221287-146-11-2765
Loading

Data & Media loading...

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