Release of Nascent Polypeptide Chains

We discussed earlier that one mechanism for the transfer of particular translation products across the membrane barriers is the process of vectorial translation as carried out by polysomes attached to the rough endoplasmic reticulum of secretory cells. An in vitro assay for this process has been devised by Redman and Sabatini. In order to carry out this assay, it is necessary to release labeled, nascent polypeptide chains from membrane-bound ribosomes by reaction with puromycin and assess their distribution between the medium and the membrane compartment. We followed this procedure to assay for vectorial release of nascent chains from bound 80 S polysomes attached to the outer mitochondrial membrane. 

From these data, we calculated that about half of the puromycin-released nascent chains were discharged vectorially. This result is in fair agreement with the values for the extent of vectorial translation obtained by others with preparations of rough endoplasmic reticulum from rat liver.  

As an independent approach to determining if nascent polypeptide chains are released from attached ribosomes to the mitochondrial membrane, we labeled nascent chains as above and then determined the amount of radioactivity that could be recovered with mitochondria after removal of attached ribosomes. As shown in Table IV, when mitochondria were washed several times with buffer containing Mg in order to maintain the integrity of the ribosome-membrane interactions, 67% of the total incorporated radioactivity was recovered in the washed mitochondrial pellet. However, if the attached cytoplasmic ribosomes were removed by treatment with RNase and EDTA and then washed with PPi-citrate, there 

Range of total incorporation (DPM) (A) - Puromycin -Triton (B) -Puromycin +Triton (C) + Puromycin -Triton (D) + Puromycin +Triton % Puromycin release % Vectorial 

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