A variety of protein toxins, such as the bacterial toxins

| March 14, 2016

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 Shigatoxin internalization receptor. Shigatoxin 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 Shigatoxin 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 Shigatoxin 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? (10 points)

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 [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 Shigatoxin without antibody injection.

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? (5 points)

To explore further whether or not Shiga toxin transfer from the Golgi apparatus to the ER depends on COPI coat proteins, investigators prepared two different fluorescent dye–conjugated Shiga toxin B subunits and then assessed by fluorescence microscopy transport of the B subunits from the Golgi complex to the ER. The first preparation was Cy3-conjugated wild-type B subunit. The second preparation was Cy3-conjugated B subunit in which the C terminus was extended by the four amino acids KDEL (B-KDEL). Cells were microinjected with antibody directed against COPI coat proteins. Following microinjection, cells were incubated with fluorescent B subunit for various periods of time and B subunit distributions scored. The results are shown in the figure below. (Anti-EAGE is the WT and the noninjected/B-KDEL, and anti-EAGE/BDEL is the one that with the extended KDEL sequence.)

c) What evidence do these results provide for or against transport of wild-type Shiga toxin B subunit from the Golgi complex to the ER in COPI-coated vesicles? What is the importance of the results with B-KDEL in interpreting the overall results of these experiments? (5 points)

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