The isolation and characterisation of temperature-dependent Ricin A chain molecules in Saccharomyces cerevisiae.

Article English OPEN
Allen, Stuart C. H. ; Moore, Katherine A. H. ; Marsden, Catherine J. ; Fülöp, Vilmos ; Moffat, Kevin G. ; Lord, Mike ; Ladds, Graham ; Roberts, L. M. (Lynne M.) (2007)

Ricin is a heterodimeric plant protein that is potently toxic to mammalian cells. Toxicity\ud results from the catalytic depurination of eukaryotic ribosomes by ricin A chain (RTA)\ud that follows toxin endocytosis to, and translocation across, the endoplasmic reticulum\ud (ER) membrane. To ultimately identify proteins required for these later steps in the entry\ud process, it will be useful to express the catalytic subunit within the ER of yeast cells in a\ud manner that initially permits cell growth. A subsequent switch in conditions to provoke\ud innate toxin action would permit only those strains containing defects in genes normally\ud essential for toxin retro-translocation, refolding or degradation to survive. As a route to\ud such a screen, several RTA mutants with reduced catalytic activity have previously been\ud isolated. Here we report the use of Saccharomyces cerevisiae to isolate temperaturedependent\ud mutants of endoplasmic reticulum-targeted RTA. Two such toxin mutants\ud with opposing phenotypes were isolated. One mutant RTA (RTAF108L/L151P) allowed the\ud yeast cells that express it to grow at 37°C while the same cells did not grow at 23ºC. Both\ud mutations were required for temperature-dependent growth. The second toxin mutant\ud (RTAE177D) allowed cells to grow at 23°C but not at 37°C. Interestingly, RTAE177D has\ud been previously reported to have reduced catalytic activity, but this is the first\ud demonstration of a temperature-sensitive phenotype. To provide a more detailed\ud characterisation of these mutants we have investigated their N-glycosylation, stability,\ud catalytic activity and, where appropriate, a three dimensional structure. The potential\ud utility of these mutants is discussed.
  • References (42)
    42 references, page 1 of 5

    1. Lord, M. J., Jolliffe, N. A., Marsden, C. J., Pateman, C. S., Smith, D. C., Spooner, R. A., Watson, P. D. & Roberts, L. M. (2003) Ricin. Mechanisms of cytotoxicity, Toxicol Rev. 22, 53-64.

    2. Wales, R., Roberts, L. M. & Lord, J. M. (1993) Addition of an endoplasmic reticulum retrieval sequence to ricin A chain significantly increases its cytotoxicity to mammalian cells, J Biol Chem. 268, 23986-90.

    3. Simpson, J. C., Dascher, C., Roberts, L. M., Lord, J. M. & Balch, W. E. (1995) Ricin cytotoxicity is sensitive to recycling between the endoplasmic reticulum and the Golgi complex, J Biol Chem. 270, 20078-83.

    4. Rapak, A., Falnes, P. O. & Olsnes, S. (1997) Retrograde transport of mutant ricin to the endoplasmic reticulum with subsequent translocation to cytosol, Proc Natl Acad Sci U S A. 94, 3783-8.

    5. Lord, J. M. & Roberts, L. M. (1998) Toxin entry: retrograde transport through the secretory pathway, J Cell Biol. 140, 733-6.

    6. Wesche, J., Rapak, A. & Olsnes, S. (1999) Dependence of ricin toxicity on translocation of the toxin A-chain from the endoplasmic reticulum to the cytosol, J Biol Chem. 274, 34443-9.

    7. Simpson, J. C., Roberts, L. M., Romisch, K., Davey, J., Wolf, D. H. & Lord, J. M. (1999) Ricin A chain utilises the endoplasmic reticulum-associated protein degradation pathway to enter the cytosol of yeast, FEBS Lett. 459, 80-4.

    8. Lord, J. M., Roberts, L. M. & Lencer, W. I. (2005) Entry of protein toxins into mammalian cells by crossing the endoplasmic reticulum membrane: co-opting basic mechanisms of endoplasmic reticulum-associated degradation, Curr Top Microbiol Immunol. 300, 149-68.

    9. Endo, Y., Mitsui, K., Motizuki, M. & Tsurugi, K. (1987) The mechanism of action of ricin and related toxic lectins on eukaryotic ribosomes. The site and the characteristics of the modification in 28 S ribosomal RNA caused by the toxins, J Biol Chem. 262, 5908-12.

    10. Moazed, D., Robertson, J. M. & Noller, H. F. (1988) Interaction of elongation factors EF-G and EF-Tu with a conserved loop in 23S RNA, Nature. 334, 362-4.

  • Bioentities (1)
    1ift Protein Data Bank
  • Metrics
    views in OpenAIRE
    views in local repository
    downloads in local repository

    The information is available from the following content providers:

    From Number Of Views Number Of Downloads
    Warwick Research Archives Portal Repository - IRUS-UK 0 22
Share - Bookmark