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. 2003 Apr 18;4(5):505–510. doi: 10.1038/sj.embor.embor826

Figure 1.

Figure 1

Lumenal endoplasmic reticulum proteins, trapped in proteoliposomes, stimulate translocation efficiency. (A–F) Proteoliposomes (PL) were formed in the absence (−RP) or presence (+RP) of added lumenal endoplasmic reticulum (ER) proteins (reticuloplasm), as described in the Methods. (E) For a third preparation, proteoliposomes that had been formed in the absence of added lumenal ER proteins (−RP) were supplemented with lumenal ER proteins after dialysis, but before separation by centrifugation (+RP after dialysis). (A,B) Proteoliposomes (−RP and +RP) were subjected to sequestration analysis. Proteins were separated by SDS–polyacrylamide gel electrophoresis (SDS–PAGE) and blotted onto polyvinylidene difluoride membranes. Blots were incubated with rabbit anti-PDI (protein disulphide isomerase) antibodies (StressGen), rabbit anti-Sec61-α antibodies and peroxidase-conjugated goat anti-rabbit IgG secondary antibodies. The antibodies were visualized by incubation of the blots in ECL chemiluminescence reagents and exposure to X-ray film. We note that reticuloplasm did not affect the reconstitution (B) and protease accessibility (not shown) of Sec61-α. (C–E) Preprolactin (ppl) was synthesized in reticulocyte lysate in the presence of [35S]methionine with buffer, microsomes (RM) or the proteoliposomes indicated. After incubation for 60 min at 30 °C, the reactions were subjected to sequestration analysis. (F) Preprocecropin A (ppCecA) was synthesized in reticulocyte lysate in the presence of [3H]proline for 15 min. After inhibition of protein synthesis, buffer, microsomes or proteoliposomes were added as indicated. After incubation for 30 min at 30 °C, the reactions were subjected to sequestration analysis. The samples were separated by SDS–PAGE and were analysed by phosphorimaging or fluorography. The sequestration efficiency was calculated as the amount of protease-resistant prolactin as a percentage of the total prolactin. Average values and standard errors of the mean (s.e.m.) are presented, and were based on n = 13 (D) and n = 2 (E). pCecA, procecropin A; PK, proteinase K; pl, prolactin; T, trypsin; TX, Triton X-100.