26 Jun A variety of protein toxins, such as the bacterial toxins Pseudomonas
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? (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 Shiga toxin 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 wildtype 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 antiEAGE/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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