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

By Stuart C. H. Allen, Katherine A. H. Moore, Catherine J. Marsden, Vilmos Fülöp, Kevin G. Moffat, Mike Lord, Graham Ladds and L. M. (Lynne M.) Roberts

Abstract

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

Topics: QK, QD, RS
Publisher: Blackwell
Year: 2007
OAI identifier: oai:wrap.warwick.ac.uk:108

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