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Figure 5


Fig. 5. Chaperone-mediated pathways operate in parallel with SRP-mediated targeting. (A) Sec61βG was released from isolated RNCs by puromycin treatment in the presence of reticulocyte lysate ({diamond}), purified Hsp40 and Hsc70 with ATP ({blacksquare}) or purified SRP with GTP ({blacktriangleup}). Samples were incubated with membranes for 0 to 30 minutes, membrane-associated material resistant to extraction with alkaline sodium carbonate solution was recovered as described above, and relative integration efficiency measured by N-glycosylation was compared with the value obtained with reticulocyte lysate after 30 minutes. (B) Sec61βG or Syb2G were isolated as RNCs and the polypeptides released from the ribosomes by puromycin treatment in the presence of reticulocyte lysate, or depleted reticulocyte lysate supplemented with SRP, ATP and GTP, as indicated. Samples were then incubated with membranes for up to 30 minutes and relative integration was measured by N-glycosylation as compared to the level obtained with reticulocyte lysate after 30 minutes.





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