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Figure 4


Fig. 4. Specific chaperones stimulate the membrane integration of TA proteins. (A) Sec61βG was released from isolated RNCs by puromycin treatment for 5 minutes in the presence of ATP (except for –ATP) and various molecular chaperones or reticulocyte lysate (RL) as shown. Samples were incubated with ER-derived microsomes (K-RM) for 30 minutes, and membrane-associated material was isolated as before. N-glycosylated material was quantified after extraction with alkaline sodium carbonate solution and standardised relative to the sample incubated with reticulocyte lysate (set to 100). Of the membrane associated products recovered, 10% were N-glycosylated for the control sample (lane 12). (B) Sec61βOPG was used to analyse the role of molecular chaperones as described in A. In this case, the membrane-associated material was analysed directly after the isolation of the membrane fraction through a high-salt sucrose cushion because a comparison with subsequent alkaline extraction revealed that the two procedures give similar results with this precursor (supplementary material Fig. S3B). Combinations of chaperones were added together with ATP and integration efficiency was analysed on the basis of N-glycosylation efficiency in four independent experiments. One such experiment is presented together with the average level of stimulation for the different treatments and the ±s.e.m. For the experiment shown, 42% of the membrane-associated products recovered were N-glycosylated when the sample was incubated with reticulocyte lysate (lane 6). **P<0.01 for these chaperone combinations causing a stimulation of membrane integration when compared to the control (lane 7). (C) Sec61βG was treated as described for A, except that varying concentrations of CBAG, a C-terminal fragment of Bag1, were included. N-glycosylation was used to measure membrane integration, and the values were standardised relative to those obtained with Hsp40 and Hsc70 alone.





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