A variety of protein toxins, such as the bacterial toxins Pseudomonas toxin and

Question

A variety of protein toxins, such as the bacterial toxins Pseudomonas toxin 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 inhibiting protein synthesis in cells that have been attacked by this toxin. Amazingly, only one molecule of A subunit when introduced into the cytosol is sufficient to kill a cell. The B subunit targets Shiga toxin to the ER by binding to a glycolipid GM3 on the cell surface, which acts as the Shiga toxin internalization receptor. Shiga toxin is internalized into endosomes, from endosomes it is transferred to the Golgi complex, and from the Golgi complex it goes to the ER where the A and B subunits dissociate. Finally, the free A subunit of Shiga toxin is transferred into the cytosol from the lumen of the ER by the Sec61 protein translocon.

In a series of experiments designed to compare the mechanisms of Pseudomonas toxin 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

a) From inspection of these sequences, what is the probable targeting receptor for transfer of Pseudomonas toxins from the Golgi apparatus to the ER? (2 pts.)

The picture is for part b

5. cont. To test this prediction directly, investigators experimentally characterized the role of COPI coat proteins and KDEL receptors in intoxication. Monkey cells were microinjected 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 [3SS]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. -400 Anti-COPI 300 Anti-KDEL receptor 200 100 Pseudomonas Shiga toxin Control toxin b) How do these results support your sequence-based predictions and the known role of COPI coat protein in retrograde transport? Can you formulate a hypothesis for how Shiga toxin is transported from the Golgi to the ER? (3 pts)

Explanation / Answer

a) Proteins destined to be in ER carry sorting signal i.e., Lys - Asp - Glu - Leu (KDEL) at ther C-terminus. The KDEL sequence is recognized and bound by the KDEL receptor found on the ER and cis-glogi. The KDEL receptor retrieves soluble proteins containing KDEL sequence. The targeting receptor for transfer of Pseudomonas toxins from the Golgi apparatus to the ER is KDEL receptor.

b) The toxin is internalized into the endosomes, trasferred to glogi body so for the inhibition of protein synthesis the toxin has to be trasported to the ER. This is done by COP I, which forms vesicles for both intra golgi transport and retrograde transport from the golgi to ER. So if Anti COP I binds to COP I then the transport of the toxin will be inhibited and the protein synthesis will continue. As seen in controlled, in the absence of Anti COP I, the toxin gets trasported to ER and thus lowers the rate of protein synthesis.

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