TAILIEUCHUNG - Báo cáo khoa học:The isolation and characterization of temperature-dependent ricin A chain molecules in Saccharomyces cerevisiae

Ricin is a heterodimeric plant protein that is potently toxic to mammalian cells. Toxicity results from the catalytic depurination of eukaryotic ribo-somes by ricin toxin A chain (RTA) tBáo cáo khoa học: The isolation and characterization of temperature-dependehat follows toxin endocytosis to, and translocation across, the endoplasmic reticulum membrane. | ễFEBS Journal The isolation and characterization of temperature-dependent ricin A chain molecules in Saccharomyces cerevisiae Stuart C. H. Allen1 Katherine A. H. Moore1 Catherine J. Marsden1 Vilmos Fulop1 Kevin G. Moffat1 J. Michael Lord1 Graham Ladds2 and Lynne M. Roberts1 1 Department of BiologicalSciences University of Warwick Coventry UK 2 Division of ClinicalSciences Warwick Medical School University of Warwick Coventry UK Keywords ricin A chain yeast toxin temperaturedependent mutants Correspondence L. M. Roberts Department of Biological Sciences University of Warwick Coventry CV4 7AL UK Fax 44 2476 523568 Tel 44 2476 523558 E-mail Received 25 July 2007 revised 22 August 2007 accepted 30 August 2007 doi Ricin is a heterodimeric plant protein that is potently toxic to mammalian cells. Toxicity results from the catalytic depurination of eukaryotic ribosomes by ricin toxin A chain RTA that follows toxin endocytosis to and translocation across the endoplasmic reticulum membrane. To ultimately identify proteins required for these later steps in the entry process it will be useful to express the catalytic subunit within the endoplasmic reticulum of yeast cells in a manner that initially permits cell growth. A subsequent switch in conditions to provoke innate toxin action would permit only those strains containing defects in genes normally essential for toxin retro-translocation refolding or degradation to survive. As a route to such a screen several RTA mutants with reduced catalytic activity have previously been isolated. Here we report the use of Saccharomyces cerevisiae to isolate temperature-dependent mutants of endoplasmic reticulum-targeted RTA. Two such toxin mutants with opposing phenotypes were isolated. One mutant RTA RTAF108L L151P allowed the yeast cells that express it to grow at 37 C whereas the same cells did not grow at 23 C. Both mutations were required for temperature-dependent growth. .

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