Point mutations in the hydrophobic C-terminal region of ricin A chain indicate that Pro250 plays a key role in membrane translocation.
A series of mutations have been made in the carboxyl terminus of ricin A chain, centred on the hydrophobic region between amino acid residues Val245 and Val256. The mutant ricin A chains were expressed to a high level in an Escherichia coli system and the proteins purified to homogeneity. The enzymic activity of each of these A chain molecules was tested on rabbit reticulocyte ribosomes; in all cases, the activities were found to be comparable to wild-type recombinant ricin A chain. Following reassociation of these A chains to ricin B chain, Vero cells were challenged with these holotoxins and the cytotoxicities determined. Mutant ricin A chain with Ile247-->Ala was unable to reassociate and form holotoxin, indicating the importance of this residue in the interaction with ricin B chain. Mutant ricin A chain with Pro250-->Ala readily reassociated with ricin B chain, forming holotoxin with a 170-fold reduction in cytotoxicity to Vero cells. Other mutations in this region also produced A chain proteins which gave marked reductions in holotoxin cytotoxicity. We propose therefore that the C-terminal hydrophobic region of ricin A chain may be involved in membrane interactions prior to the translocation of this subunit into the cytosol, and that Pro250 plays a key role in one or both of these steps.
Simpson
J C
JC
Department of Biological Sciences, University of Warwick, Coventry, UK.
Lord
J M
JM
Roberts
L M
LM
D000595:Amino Acid Sequence; D000818:Animals; D002522:Chlorocebus aethiops; D004355:Drug Stability; D004926:Escherichia coli / Q000235:genetics; D066298:In Vitro Techniques; D007425:Intracellular Membranes / Q000378:metabolism; D008969:Molecular Sequence Data; D017354:Point Mutation; D011817:Rabbits; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism / Q000633:toxicity; D012156:Reticulocytes / Q000187:drug effects / Q000378:metabolism; D012270:Ribosomes / Q000187:drug effects / Q000378:metabolism; D012276:Ricin / Q000235:genetics* / Q000378:metabolism* / Q000633:toxicity; D014709:Vero Cells
D011994:Recombinant Proteins; D012276:Ricin