1887

Abstract

K28 killer strains of are permanently infected with a cytoplasmic persisting dsRNA virus encoding a secreted α/β heterodimeric protein toxin that kills sensitive cells by cell-cycle arrest and inhibition of DNA synthesis. processing of the 345 aa toxin precursor (preprotoxin; pptox) involves multiple internal and carboxy-terminal cleavage events by the prohormone convertases Kex2p and Kex1p. By site-directed mutagenesis of the preprotoxin gene and phenotypic analysis of its effects it is now demonstrated that secretion of a biological active virus toxin requires signal peptidase cleavage after Gly and Kex2p-mediated processing at the α subunit N terminus (after Glu-Arg), the α subunit C terminus (after Ser-Arg) and at the β subunit N terminus (after Lys-Arg). The mature C terminus of the β subunit is trimmed by Kex1p, which removes the terminal Arg residue, thus uncovering the toxin’s endoplasmic reticulum targeting signal (HDEL) which – in a sensitive target cell – is essential for retrograde toxin transport. Interestingly, both toxin subunits are covalently linked by a single disulfide bond between α-Cys and β-Cys, and expression of a mutant toxin in which β-Cys had been replaced by Ser resulted in the secretion of a non-toxic α/β heterodimer that is blocked in retrograde transport and incapable of entering the yeast cell cytosol, indicating that one important function of β-Cys might be to ensure accessibility of the toxin’s β subunit C terminus to the HDEL receptor of the target cell.

Loading

Article metrics loading...

/content/journal/micro/10.1099/00221287-148-5-1317
2002-05-01
2022-01-19
Loading full text...

Full text loading...

/deliver/fulltext/micro/148/5/1481317a.html?itemId=/content/journal/micro/10.1099/00221287-148-5-1317&mimeType=html&fmt=ahah

References

  1. Bevan A., Brenner C., Fuller R. S. 1998; Quantitative assessment of enzyme specificity in vivo : P2 recognition by Kex2 protease defined in a genetic system. Proc Natl Acad Sci USA 95:10384–10389 [CrossRef]
    [Google Scholar]
  2. Boone C., Bussey H., Greene D., Thomas D. Y., Vernet T. 1986; Yeast killer toxin: site-directed mutations implicate the precursor protein as the immunity component. Cell 46:105–113 [CrossRef]
    [Google Scholar]
  3. Bostian K. A., Rogers D. T., Tipper D. J. 1983; A glycosylated protoxin in killer yeast: models for its structure and maturation. Cell 32:169–180 [CrossRef]
    [Google Scholar]
  4. Bourbonnais Y., Ash J., Daigle M., Thomas D. Y. 1993; Isolation and characterization of S. cerevisiae mutants defective in somatostatin expression: cloning and functional role of a yeast gene encoding an aspartyl protease in precursor processing at monobasic cleavage sites. EMBO J 12:285–294
    [Google Scholar]
  5. Brenner C., Fuller R. S. 1992; Structural and enzymatic characterization of a purified prohormone-processing enzyme: secreted, soluble Kex2 protease. Proc Natl Acad Sci USA 89:922–926 [CrossRef]
    [Google Scholar]
  6. Bryant N. J., Boyd A. 1993; Immunoisolation of Kex2p-containing organelles from yeast demonstrates colocalisation of three processing proteinases to a single Golgi compartment. J Cell Sci 106:815–822
    [Google Scholar]
  7. Bussey H., Sacks W., Galley D., Saville D. 1982; Yeast killer plasmid mutations affecting toxin secretion and activity and toxin immunity function. Mol Cell Biol 2:346–354
    [Google Scholar]
  8. Chaudhary V. K., Jinno Y., FitzGerald D., Pastan I. 1990; Pseudomonas exotoxin contains a specific sequence at the carboxyl terminus that is required for cytotoxicity. Proc Natl Acad Sci USA 87:308–312 [CrossRef]
    [Google Scholar]
  9. Dignard D., Whiteway M., Germain D., Tessier D., Thomas D. Y. 1991; Expression in yeast of a cDNA copy of the K2 killer toxin gene. Mol Gen Genet 227:127–136 [CrossRef]
    [Google Scholar]
  10. Dmochowska A., Dignard D., Henning D., Thomas D. Y., Bussey H. 1987; Yeast KEX1 gene encodes a putative protease with a carboxypeptidase B-like function involved in killer toxin and alpha-factor precursor processing. Cell 50:573–584 [CrossRef]
    [Google Scholar]
  11. Egel-Mitani M., Flygenring H. P., Hansen M. T. 1990; A novel aspartyl protease allowing KEX2-independent MF alpha propheromone processing in yeast. Yeast 6:127–137 [CrossRef]
    [Google Scholar]
  12. Eisfeld K., Riffer F., Mentges J., Schmitt M. J. 2000; Endocytotic uptake and retrograde transport of a virally encoded killer toxin in yeast. Mol Microbiol 37:926–940 [CrossRef]
    [Google Scholar]
  13. Fuller R. S., Brake A. J., Thorner J. 1989; Intracellular targeting and structural conservation of a prohormone-processing endoprotease. Science 246:482–486 [CrossRef]
    [Google Scholar]
  14. Horton R. M., Hunt H. D., Ho S. N., Pullen J. K., Pease L. R. 1989; Engineering hybrid genes without the use of restriction enzymes: gene splicing by overlap extension. Gene 77:61–68 [CrossRef]
    [Google Scholar]
  15. Ledgerwood E. C., Brennan S. O., Cawley N. X., Loh Y. P., George P. M. 1996; Yeast aspartic protease 3 (Yap3) prefers substrates with basic residues in the P2, P1 and P2′ positions. FEBS Lett 383:67–71 [CrossRef]
    [Google Scholar]
  16. Martinac B., Zhu H., Kubalsky A., Zhou X.-L., Culbertson M., Bussey H., Kung C. 1990; Yeast K1 killer toxin forms ion channels in sensitive yeast spheroplasts and in artificial liposomes. Proc Natl Acad Sci USA 87:6228–6232 [CrossRef]
    [Google Scholar]
  17. Park C. M., Bruenn J. A., Ganesa C., Flurkey W. F., Bozarth R. F., Koltin Y. 1994; Structure and heterologous expression of the Ustilago maydis viral toxin KP4. Mol Microbiol 11:155–164 [CrossRef]
    [Google Scholar]
  18. Pelham H. R. B., Roberts L. M., Lord J. M. 1992; Toxin entry: how reversible is the secretory pathway?. Trends Cell Biol 2:183–185 [CrossRef]
    [Google Scholar]
  19. Redding K., Holcomb C., Fuller R. S. 1991; Immunolocalization of Kex2 protease identifies a putative late Golgi compartment in the yeast Saccharomyces cerevisiae . J Cell Biol 113:527–538 [CrossRef]
    [Google Scholar]
  20. Schiestl R. H., Gietz R. D. 1989; High efficiency transformation of intact yeast cells using single stranded nucleic acids as a carrier. Curr Genet 16:339–346 [CrossRef]
    [Google Scholar]
  21. Schmitt M. J. 1995; Cloning and expression of a cDNA copy of the viral K28 killer toxin gene in yeast. Mol Gen Genet 246:236–246 [CrossRef]
    [Google Scholar]
  22. Schmitt M. J., Eisfeld K. 1999; Killer viruses in S. cerevisiae and their general importance in understanding eucaryotic cell biology. Recent Res Devel Virol 1:525–545
    [Google Scholar]
  23. Schmitt M. J., Tipper D. J. 1990; K28, a unique double-stranded RNA killer virus of Saccharomyces cerevisiae . Mol Cell Biol 10:4807–4815
    [Google Scholar]
  24. Schmitt M. J., Tipper D. J. 1992; Genetic analysis of maintenance and expression of L and M double-stranded RNAs from yeast killer virus K28. Yeast 8:373–384 [CrossRef]
    [Google Scholar]
  25. Schmitt M. J., Tipper D. J. 1995; Sequence of the M28 dsRNA: preprotoxin is processed to an alpha/beta heterodimeric protein toxin. Virology 213:341–351 [CrossRef]
    [Google Scholar]
  26. Schmitt M., Brendel M., Schwarz R., Radler F. 1989; Inhibition of DNA synthesis in Saccharomyces cerevisiae by yeast killer toxin KT28. J Gen Microbiol 135:1529–1535
    [Google Scholar]
  27. Schmitt M. J., Klavehn P., Wang J., Schönig I., Tipper D. J. 1996; Cell cycle studies on the mode of action of yeast K28 killer toxin. Microbiology 142:2655–2662 [CrossRef]
    [Google Scholar]
  28. Steiner D. F., Smeekens S. P., Ohagi S., Chan S. J. 1992; The new enzymology of precursor processing endoproteases. J Biol Chem 267:23435–23438
    [Google Scholar]
  29. Sturley S. L., Elliot Q., Le Vitre J., Tipper D. J., Bostian K. A. 1986; Mapping of functional domains within the Saccharomyces cerevisiae type 1 killer preprotoxin. EMBO J 5:3381–3389
    [Google Scholar]
  30. Tao J., Ginsberg I., Banerjee N., Held W., Koltin Y., Bruenn J. A. 1990; Ustilago maydis KP6 killer toxin: structure, expression in Saccharomyces cerevisiae , and relationship to other cellular toxins. Mol Cell Biol 10:1373–1381
    [Google Scholar]
  31. Tipper D. J., Schmitt M. J. 1991; Yeast dsRNA viruses: replication and killer phenotypes. Mol Microbiol 5:2331–2338 [CrossRef]
    [Google Scholar]
  32. von Heijne G. 1986; A new method for predicting signal sequence cleavage sites. Nucleic Acids Res 14:4683–4690 [CrossRef]
    [Google Scholar]
  33. Wickner R. B. 1992; Double-stranded and single-stranded RNA viruses of Saccharomyces cerevisiae . Annu Rev Microbiol 46:347–375 [CrossRef]
    [Google Scholar]
  34. Yoshida T., Chen C., Zhang M., Wu H. C. 1991; Disruption of the Golgi apparatus by brefeldin A inhibits the cytotoxicity of ricin, modeccin, and Pseudomonas toxin. Exp Cell Res 192:389–395 [CrossRef]
    [Google Scholar]
  35. Zhu H., Bussey H., Thomas D. Y., Gagnon J., Bell A. W. 1987; Determination of the carboxyl termini of the alpha and beta subunits of yeast K1 killer toxin: requirement of a carboxypeptidase B-like activity for maturation. J Biol Chem 262:10728–10732
    [Google Scholar]
  36. Zhu Y. S., Zhang X. Y., Cartwright C. B., Tipper D. J. 1992; Kex2-dependent processing of yeast K1 killer preprotoxin includes cleavage at ProArg-44. Mol Microbiol 6:511–520 [CrossRef]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/00221287-148-5-1317
Loading
/content/journal/micro/10.1099/00221287-148-5-1317
Loading

Data & Media loading...

Most cited this month 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