Figure 1From: Human autoantibodies against the 54 kDa protein of the signal recognition particle block function at multiple stagesHuman sera containing autoantibodies directed against SRP inhibit protein translocation into the ER in vitro. The secretory precursor preprolactin (PPL) was synthesized as a 35S-radiolabelled protein using a cell-free system supplemented with signal recognition particle (SRP)-depleted endoplasmic reticulum (ER) membranes and purified SRP that had been preincubated with either buffer (lanes 1 and 2) or with no additions (lanes 3 and 4) to establish normal levels of prolactin (PL) translocation into ER-derived microsomes. The specificity of protein translocation was controlled for by performing experiments lacking exogenous SRP (lanes 5 and 6) or lacking salt-washed rough microsomal membranes (RM) (lanes 7 and 8). The complete translocation of signal-sequence-processed PL into the lumen of the ER microsomes was confirmed by showing resistance to digestion by proteinase K (cf. - and + PK). To investigate the ability of distinct autoantibodies to block function, SRP was preincubated with various anti-SRP-positive sera (S), IgG (I) or Fab (F) fractions prior to protein synthesis. TL, human control serum from a healthy individual, while 19-1, 17-1, 4-2 and 25-1 are human sera containing anti-SRP autoantibodies from polymyositis patients. Serum 5–15 is from a polymyositis patient without detectable myositis autoantibodies, and serum 1–24 is from a polymyositis patient with autoantibodies directed against histidyl-tRNA synthetase. PPL, unprocessed (signal-sequence containing) preprolactin that has not been translocated into the ER microsomes. PL, fully translocated, signal-cleaved prolactin located inside ER microsomes. Samples were analysed by SDS-PAGE on 10–15% gels and by fluorography.Back to article page