Figure S6.
EMC and Sec61 Complex Act at Different Steps during β1AR Insertion, Related to Figure 5
(A) Immunoblotting of proteoliposomes (PLs) reconstituted from total ER proteins (Tot.) or Sec61-depleted ER proteins (ΔSec) shows that under conditions where even 5% of total PLs show readily detectable Sec61, none is seen in ΔSec PLs. EMC levels are comparable.
(B) Ribosome-nascent chain complexes of constructs containing the indicated TMD1 regions (see diagram, Figure S5A) truncated ∼60 residues beyond the TMD (corresponding to residue 116 in the β1AR-TMD1 construct) were produced in RRL. They were incubated without anything, with liposomes, or with PLs from total ER proteins (Tot.) or Sec61-depleted ER proteins (ΔSec). An aliquot of the sample was analyzed directly (-PK) or subjected to digestion with proteinase K (+PK). An aliquot of the PK-digested sample was subsequently immunoprecipitated via the N-terminal HA tag after RNase digestion (N-term. IPs). FL indicates full length product protected from protease, indicative of successful insertion. CTFs indicate C-terminal fragments from non-inserted products.
(C) The total IVT products from panel B shown from an overexposed autoradiograph to visualize the minor glycosylated product (+glyc). Glycosylation is relatively inefficient in PLs compared to native microsomes.
(D) The PLs from Figure 5B were analyzed by immunoblotting for Sec61 and EMC to verify no Sec61 contamination of either EMC or SRP receptor (SR) PLs.
(E) The two-TMD β1AR construct (see Figure 5C) was analyzed in the indicated proteoliposome preparations or canine-pancreas derived microsomes (cRM) by the protease-protection assay. Samples were analyzed directly without immunoprecipitation. The left panel shows the experiment when membranes are present during the translation reaction (co-translational; reproduced from Figure 5D), while the right panel shows the experiment when incubation with membranes was post-translational. Red asterisks indicate ubiquitinated products, green arrow indicates the glycosylated product, “1+2” indicates the protected product indicative of the double-spanning topology, and “1 only” indicates the single-spanning topology.