Solved by a verified expert: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
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?
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?
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
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)