A variety of protein toxins, such as the bacterial toxin Pseudomonas and Shiga toxin and the plant toxin ricin, are heteromeric proteins consisting of A and B subunits. The A subunit is catalytic. For Shiga toxin, the proximal cause of food poisoning due to bacterially contaminated hamburger, the A subunit is an N-glycosidase and specifically cleaves 28S ribosomal RNA, thereby intoxicating cells by inhibiting protein synthesis. Amazingly, only one molecule of A subunit when introduced into the cytosol is sufficient to kill a cell. Interestingly the A subunit of Shiga toxin is transferred into the cytosol from the lumen of the ER by the Sec61 protein translocon. The B subunit targets Shiga toxin to the ER by binding to a glycolipid GM3 on the cell surface that acts as the Shiga toxin internalization receptor. Shiga toxin is internalized into endosomes, from en-dosomes is transferred to the Golgi complex, and from the Golgi complex goes to the ER where the A and B subunits dissociate, permitting the A subunit to translocate into the cytosol.
In a series of experiments designed to characterize the comparative mechanisms of Pseudomonas and Shiga toxin transfer from the Golgi complex to the ER, investigators first sequenced the respective targeting subunits. The C-terminal 24 amino acids of the B subunits of Pseudomonas toxin and Shiga toxin are shown below:
C-terminal 24 amino acids of Pseudomonas toxin B subunit KEQAISALPD YASQPGKPPR KDEL
C-terminal 24 amino acids of Shiga toxin B subunit TGMTVTIKTN ACHNGGGFSE VIFR
From inspection of these sequences, what is the probable targeting receptor for transfer of Pseudomonas toxins from the Golgi apparatus to the ER?
To test this prediction directly, investigators experimentally characterized the role of COPI coat proteins and KDEL receptors in intoxication. Monkey cells were mi-croinjected with antibodies directed against either COPI coat proteins or the cytosolic domain of KDEL receptors. Cells then were incubated with Pseudomonas or Shiga toxin for 4 h. Protein synthesis was determined following a 30-minute pulse labeling with [35S]methionine. Results are shown in the accompanying figure, with controls showing the low level of protein synthesis caused by incubation with either Pseudomonas or Shiga toxin without antibody injection.
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