JCS002410 Supplementary Material

Files in this Data Supplement:

• Supplemental Table S1 -

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• Supplemental Figure S1 -

  Fig. S1. (A,B) Western blot analysis of canine pancreatic rough microsomes and ribosome nascent. Rough microsomes, before (W-RM) and after (K-RM) extraction with high-salt buffer, reticulocyte lysate (RL), ribosome−nascent-chain complexes (RNC) purified from cell-free translations primed with mRNA encoding Sec61β (Sec61βRNC) or with H₂O (Mock RNC), purified signal recognition particle (SRP) or purified Hsc70 (0.2 mg) were loaded on a 12% acrylamide gel transferred onto a PVDF membrane and analysed using a rabbit anti-SRP 54 antiserum at a 1:400 dilution (A) or mouse anti-Hsc70 antiserum at a 1/1000 dilution (B). To provide a loading control, the rough microsomes were also analysed with a rabbit antserum recognizing the ER integral membrane protein Sec62p (1:1000) and the RNCs were analysed with a rabbit antiserum recognising the large ribosomal subunit protein L17 (1:5000). Whereas detectable SRP54 was present on the W-RMs that had not been extracted with high salt, no SRP54 was found associated with either the K-RMs or the RNCs (A). Likewise, no detectable Hsc70 was found with either microsome preparation. By contrast, both ribosomal pellets contained significant levels of Hsc70 (panel B, RNC lanes). Antisera were kindly provided by B. Dobberstein, ZMBH, Heidelberg, Germany (anti-SRP54), M. Pool, University of Manchester, UK (anti-L17) and R. Zimmerman, University of Saarbrucken, Germany (anti-Sec62p). Anti-Hsc70 was from Stressgen.

• Supplemental Figure S2 -

  Fig. S2. Purified Hsc70 and C-BAG preparations. Bovine Hsc70 and the C-terminal domain of human Bag-1M (C-BAG) were prepared as described in the Materials and methods. Samples were resolved by SDS-PAGE and stained with Coomassie Blue. The relative migration of molecular mass standards (kDa) are indicated.

• Supplemental Figure S3 -

  Fig. S3. Analysing membrane integration using N-glycosylation. (A) The removal of ribosomal subunits following the puromycin-dependant release of the nascent chain does not affect membrane integration as assessed by N-glycosylation. Sec61βOPG ribosome−nascent-chain complexes were treated with puromycin as described in the Material and Methods and incubated directly with K-RMs (lane 1). Alternatively, the sample was centrifuged at 213,000 g for 20 minutes after puromycin treatment and then incubated with K-RMs (lane 2). In each case the proportion of the total membrane associated protein that was N-glycosylated was quantified by phosphorimaging and is indicated below the appropriate lane (% N-glycosylation). (B) Extraction with alkaline sodium carbonate solution has little effect on the proportion of N-glycosylated Sec61βOPG recovered in the membrane pellet. Following the post-translational integration of Sec61βOPG into K-RMs, the membrane fraction was isolated by centrifugation through a high-salt−sucrose cushion and then resuspended in 200 ml of either ice cold 0.1 M Na₂CO₃ (lane 2) or low-salt−sucrose cushion (LSC) buffer (lane 1) and incubated for 15 minutes on ice before being re-pelleted by centrifugation at 100,000 g for 10 minutes. The proportion of the total membrane associated products that were N-glycosylated is indicated in each case.