JCS020321 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Authentic RAMP4 is also crosslinked to p40 in the presence or absence of nucleotides (NXP). After termination of RAMP4 synthesis in the RRL, small molecules were removed by gel filtration and 2 mM nucleotides added as indicated. After the cro sslinking by either DSS or BMH, RAMP4 was immunoprecipitated using the anti-RAMP4 antibody and proteins separated by SDS-PAGE and analyzed by autoradiography.

• Supplemental Figure S2 -

  Fig. S2. Ribosomes are efficiently separated from other cytosolic components of RRL. Ribosomes were pelleted by high-speed centrifugation. Proteins from total RRL (T), the resulting supernatant (Sn) and pellet (P) were separated by SDS-PAGE and analysed by western blotting using an antibody against the ribosomal protein L23a. A cytosolic protein that crossreacts with the anti-L23a antibody is marked with ‘x’.

• Supplemental Figure S3 -

  Fig. S3. High-salt concentrations do not prevent RAMP4op crosslinking to p40. After synthesis of RAMP4op in the RRL, KOAc was added to give the final potassium (K) concentrations indicated on top of the figure. According to the manufacturer’s specifications, endogenous concentration of K⁺ in RRL is 80 mM. After crosslinking by BMH and immunoprecipitation with the anti-opsin antibody, proteins were characterized by SDS-PAGE and autoradiography.

• Supplemental Figure S4 -

  Fig. S4. Post-translational membrane insertion of R4op, S61βop, b5op) and Ii. In vitro translation and membrane insertion of the TA proteins R4op, S61βop, b5op and the type II membrane protein Ii. Proteins were synthesized in the rabbit reticulocyte lysate (RRL), in the absence (lanes 1, 3, 4, 6, 7, 9, 10, 12) or presence of RMs (co) (lanes 2, 5, 8, 11). To samples shown in lanes 3, 6, 9 and 12, RMs were added after completion of translation (post). Proteins were separated by SDS-PAGE and visualized by autoradiography. g, glycosylated form of the protein

• Supplemental Figure S5 -

  Fig. S5. Membrane insertion of R4op under different redox conditions in the presence or absence of ATP. After synthesis of ³⁵S-labeled R4op in the RRL (lane 1), RMs were added and the insertion reaction incubated for 30 minutes at 30°C (lane 2). To test redox conditions required for membrane insertion of R4op, small molecules were removed from the lysates by gel filtration and different concentrations of H₂O₂ or DTT added as indicated. To test the dependence on ATP, 3 mM ATP was added (upper panel) or no ATP (lower panel) and reactions were incubated with RMs. Proteins were separated by SDS-PAGE and visualized by autoradiography. Glycosylated R4op (glyc) generated in the presence or absence of ATP was quantified in percent (bottom panel).

• Supplemental Figure S6 -

  Fig. S6. Crosslinking of R4op in the absence or presence of RMs under oxidizing or reducing conditions and immunoprecipitation by an anti-Asna1 antibody. After synthesis of R4op in the RRL, small molecules were removed by gel filtration. The lysates were then adjusted to 2 mM H₂O₂ (lanes 1-8) or 2mM DTT (lanes 9-16), 3mM ATP and as indicated incubated with RMs. After the membrane insertion small molecules were removed by gel filtration and BMH crosslinking was induced. Proteins were immunoprecipitated using anti-Asna1 antibodies, separated by SDS-PAGE and visualized by autoradiography.