Ricin is a potent plant cytotoxin composed of an A-chain [RTA (ricin A-chain)] connected by a disulfide bond to a cell binding lectin B-chain [RTB (ricin B-chain)]. After endocytic uptake, the toxin is transported retrogradely to the ER (endoplasmic reticulum) from where enzymatically active RTA is translocated to the cytosol. This transport is promoted by the EDEM1 (ER degradation-enhancing α-mannosidase I-like protein 1), which is also responsible for directing aberrant proteins for ERAD (ER-associated protein degradation). RTA contains a 12-residue hydrophobic C-terminal region that becomes exposed after reduction of ricin in the ER. This region, especially Pro250, plays a crucial role in ricin cytotoxicity. In the present study, we introduced a point mutation [P250A (substitution of Pro250 with alanine)] in the hydrophobic region of RTA to study the intracellular transport of the modified toxin. The introduced mutation alters the secondary structure of RTA into a more helical structure. Mutation P250A increases endosomal–lysosomal degradation of the toxin, as well as reducing its transport from the ER to the cytosol. Transport of modified RTA to the cytosol, in contrast to wild-type RTA, appears to be EDEM1-independent. Importantly, the interaction between EDEM1 and RTAP250A is reduced. This is the first reported evidence that EDEM1 protein recognition might be determined by the structure of the ERAD substrate.
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Research Article|
May 13 2011
A single point mutation in ricin A-chain increases toxin degradation and inhibits EDEM1-dependent ER retrotranslocation
Iwona Sokołowska;
Iwona Sokołowska
*Department of Molecular Biology, University of Gdańsk, Kładki 24, 80-822 Gdańsk, Poland
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Sébastien Wälchli;
Sébastien Wälchli
†Department of Immunology, Institute for Cancer Research, The Norwegian Radium Hospital, Oslo University Hospital, Montebello, N-0310 Oslo, Norway
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Grzegorz Węgrzyn;
Grzegorz Węgrzyn
*Department of Molecular Biology, University of Gdańsk, Kładki 24, 80-822 Gdańsk, Poland
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Kirsten Sandvig;
Kirsten Sandvig
‡Department of Biochemistry, Institute for Cancer Research, The Norwegian Radium Hospital, Oslo University Hospital, Montebello, N-0310 Oslo, Norway
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Monika Słomińska-Wojewódzka
Monika Słomińska-Wojewódzka
1
*Department of Molecular Biology, University of Gdańsk, Kładki 24, 80-822 Gdańsk, Poland
1To whom correspondence should be addressed (email slominsk@biotech.ug.gda.pl).
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Publisher: Portland Press Ltd
Received:
September 16 2010
Revision Received:
March 07 2011
Accepted:
March 09 2011
Accepted Manuscript online:
March 09 2011
Online ISSN: 1470-8728
Print ISSN: 0264-6021
© The Authors Journal compilation © 2011 Biochemical Society
2011
Biochem J (2011) 436 (2): 371–385.
Article history
Received:
September 16 2010
Revision Received:
March 07 2011
Accepted:
March 09 2011
Accepted Manuscript online:
March 09 2011
Citation
Iwona Sokołowska, Sébastien Wälchli, Grzegorz Węgrzyn, Kirsten Sandvig, Monika Słomińska-Wojewódzka; A single point mutation in ricin A-chain increases toxin degradation and inhibits EDEM1-dependent ER retrotranslocation. Biochem J 1 June 2011; 436 (2): 371–385. doi: https://doi.org/10.1042/BJ20101493
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