Figure 5
Autoantibodies against SRP54N and SRP54G domains inhibit release of the signal sequence from SRP54 at the endoplasmic reticulum membrane. A 103-residue fragment of a model membrane protein derived from regions of the invariant chain of the MHC class II complex, multiple colony-stimulating factor and chloroamphenicol transferase (IMC-CAT103) was synthesized in vitro as a 35S-radiolabelled polypeptide in the presence of SRP and ε-4-(3-trifluoromethyldiazarino) benzoyl-lysine tRNA to yield a stable ribosome/nascent chain/signal recognition particle complex. Cycloheximide was added to prevent further chain elongation and the complex was incubated with sera (lanes 1–6), with no additions (lane 7), with affinity-purified antibodies (Aff, lanes 8 and 9) or with their corresponding unbound fractions (Sup, lanes 10 and 11). Following the addition of endoplasmic reticulum membranes and the activation of the crosslinking reaction by UV irradiation, membrane-targeted nascent chains were isolated by sedimentation through a high-salt/sucrose cushion and the resulting samples were analysed by SDS-PAGE on 10–15% gels and by fluorography. The locations of the IMC-CAT103 polypeptide (IMC-CAT103), of IMC-CAT103 crosslinked to SRP54 (IMC-CAT103/SRP54) and of IMC-CAT103 crosslinked to Sec61α (IMC-CAT103/Sec61α) are indicated.