Journal
BIOCHEMICAL JOURNAL
Volume 436, Issue -, Pages 371-385Publisher
PORTLAND PRESS LTD
DOI: 10.1042/BJ20101493
Keywords
endoplasmic reticulum degradation-enhancing alpha-mannosidase I-like protein l (EDEM1); endoplasmic reticulum (ER); endosomal-lysosomal degradation; P250A mutation; retrotranslocation; ricin A-chain (RTA)
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Funding
- Ministry of Science and Higher Education [3682/P01/2006/32]
- Foundation for Polish Science ['Homing' HOM/12/2007]
- University of Gdansk [1480-5-0344-6]
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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 EDEM I (ER degradation-enhancing alpha-mannosidase 1-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 Pro(250), plays a crucial role in ricin cytotoxicity. In the present study, we introduced a point mutation [P250A (substitution of Pro(250) 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 RTA(p250A) 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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