Site of inhibitor actions on protein translation and translocation. a The translation cycle begins with ribosome assembly on the mRNA and initiation of polypeptide synthesis (bottom diagram). Ribosomes synthesizing secretory and membrane proteins are targeted to translocons (gray cylinders) at the ER membrane, where translocation occurs cotranslationally (middle diagram). Upon completion of translation, the translocon returns to its inactive state (right diagram). Translocation can be aborted prematurely by release of nascent polypeptides with puromycin (left diagram), a state shown previously to be conductive of ions and sugars across the ER membrane. Emetine inhibits elongation of translation by the ribosome, thereby stalling translocons in their engaged and active state. Pactamycin, by inhibiting initiation of translation, allows all translocating polypeptides to complete their synthesis normally and converts the translocons to their physiologically inactive state. b Effect of the protein synthesis inhibitors on GFP-calreticulin synthesis using a reticulocyte lysate system. Inhibitors were added either at time “0” (before the reaction was started) or after 5 min of reaction. Other details are provided in the figure and in the “” section
Several previous studies [ 20 , 33 ] have demonstrated that
premature release of nascent peptide chains from ribosomes with
puromycin to abruptly vacate engaged translocons (see Fig. 1
a) results in Ca 2+ release from the ER, a result that
we have also confirmed (data not shown).
Pactamycin inhibits protein synthesis at the initiation step
preventing assembly of the ribosome-translocon channel complex (see
Fig. 1 a).
Note that pactamycin inhibits synthesis of Crt-GFP completely only
when added at the beginning of the reaction, but not after
5 min (Fig. 1 b).
By contrast, both emetine and puromycin inhibit protein synthesis
completely irrespective of time of addition (Fig. 1 b).
The asterisk indicates the position of hemoglobin, a background
band often observed in reticulocyte lysate (Fig. 1 b). .
Emetine inhibits protein synthesis by irreversibly preventing
elongation of the peptide chain (see Fig. 1 ).
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