It

Fig. 5. Protease protection of translated proteins. Furin, PC1/3, or PC2 cRNAs were translated in the egg extract for 1 h. The translation reactions were then treated with buffer or proteinase K in the absence or presence of Triton X-100 before analysis by SDS-PAGE.

integral membrane protein on translocation into the ER, it is possible that membrane insertion requires other factors that are not present in the Xenopus egg extract. However, experiments like these illustrate the potential usefulness of the Xenopus egg extract translation system in determining membrane insertion and orientation.

4. Notes

1. Reducing the amount of GTP added helps incorporation of the cap analogue, which in turn increases translation of RNAs in the egg extract. However, it may also reduce the amount of RNA synthesized, and in some cases it may be better to add the same amount of GTP as the other nucleotides to improve RNA synthesis.

2. The reaction can continue for 2 h or more to increase the yield of RNA. Generally, after an incubation of 1 h, remove 2 ||L of the reaction and analyze on a 1.5% agarose gel. As RNA tends to diffuse easily out of these gels, it is best to run them for only a short period, say 15 min at 80 V, before visualizing the products by ethidium bromide staining. The RNA should be visible as a bright band running between the 500- and 1000-bp markers, depending on the size of the cDNA insert, with the DNA band barely visible. This gel is not a quantitative assessment of RNA produced but is merely a guide to show that the transcription reaction has been successful.

3. It is important to use 7 M ammonium acetate and not sodium acetate for precipitating the RNA to reduce the amount of unincorporated nucleotides precipitated. Generally leaving the precipitation reaction overnight at -70°C increases the amount of RNA recovered.

4. Addition of RRL to the translation reactions improves translation from extract that has been frozen (18). It is not necessary to add this if the extract is made and used immediately in translation reactions. The dilution of RRL in the egg extract is such that it is unlikely that translation will occur from the added RRL. However, to ensure that this does not occur, an S-100 fraction of the RRL can be made. Centrifuge the RRL at 100,000g for 2 h at 4°C in a Beckman TLA 100 Ultracentrifuge. Remove the supernatant, taking care not to disturb the pellet, and store the supernatant frozen in small aliquots at -70°C.

Furin PC 1/3

5. The translation reaction can be aliquoted into 10-|lL amounts at this stage, and up to 1 ||L of RNA can be added per 10 |L extract. This allows multiple RNAs to be translated separately with the same translation mix. In practice, for many RNAs made as described, it is possible to dilute the RNA in sterile water up to fivefold without loss of translation. Addition of more than 1 |L RNA to a 10-|lL aliquot of extract will reduce translation or translocation efficiency by overdilution of extract factors.

6. The volume used here depends on the volume of extract that was used in each translation. If 10-|lL aliquots of extract were used, addition of 10 |L of TES/Triton will allow sufficient membrane lysis to allow analysis by SDS-PAGE. If a full 50-|L aliquot of extract was used for the translation, then addition of 50 |L of TES/Triton should be sufficient.

7. The tripeptide Asn-Tyr-Thr is dissolved in DMSO to make a stock concentration of 500 mM. This can then be diluted in water to the 50 mM working solution, and 0.6 |L can be added to 30 |L of extract. For the control tube without tripeptide, 0.6 |L of DMSO, previously diluted 1 in 10 with water, should be added to 30 |L of extract.

8. This translation period is designated the pulse period and can vary between 2 h for PC2 and 30 min for furin and PC1/3 that undergo rapid proregion removal at neutral pH. For proprotein convertases with proregion removal that occurs rapidly, the translation period has to be reduced to observe primarily the proprotein. To prevent further translation and allow observation of processing to the mature form, cycloheximide can be added to the translation reactions to a concentration of 1 mg/mL at the end of the translation period.

9. The concentration of proteinase K required to give complete, or near-complete, digestion of translocated products in the presence of Triton X-100, without compromising the integrity of the extract membranes in the absence of Triton, may vary depending on the batch of extract. Thus, a range of concentrations of proteinase K will have to be tested with each batch of membranes.

Acknowledgments

I would like to thank Kathleen Scougall for carrying out the 7B2 work. This work was supported by grants from the Royal Society, the Medical Research Council, Diabetes UK, and the Biotechnology and Biological Sciences Research Council.

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