Figure 6.

Sec61p complex–mediated export of CTA1. (a) Microsomes were loaded with CTA1 by in vitro translation, isolated by centrifugation, and resuspended in export buffer containing ATP. One half of the microsomes was incubated for 10 min at 30°C with saturating amounts (corresponding to a 100-μl translation reaction; see below) of CTA1Δ137, the other half with DHFRΔ112, which did not contain a signal peptide and was used as a control. After binding of the RNCs, the microsomes were used for an export reaction as described in Fig. 3 a. CTA1, CTAΔ137, and DHFRΔ112 were detected by autoradiography. Export rates per hour were <1% in the presence of CTA1Δ137 and 11% in the presence of DHFRΔ112, respectively. (b) Microsomes were incubated with increasing amounts of CTA1Δ137. After binding, the microsomes were high salt–washed to remove unspecifically bound RNCs. The reactions were separated by centrifugation in a pellet fraction containing the Sec61p complex–bound RNCs and a supernatant fraction containing the unbound RNCs. The amount of CTA1Δ137 isolated from a 60-μl translation reaction was sufficient to saturate the microsomes. (c) Microsomes reconstituted with purified CTA1 and lumenal ER proteins were incubated with RNCs and used for an export reaction exactly as described in a. CTA1 and CTA1Δ137 were detected by immunoblot using an anti–cholera toxin antiserum. Due to the immunodetection DHFRΔ112 is not visible. Export rates per hour were <1% in the presence of CTA1Δ137 and 16% in the presence of DHFRΔ112, respectively.