Figure 7.

Processing of P4501A1 by a cytosolic endoprotease. (A) P4501A1, +33/1A1, and 1A1M32/33 (V32A and T33I) translation products in reticulocyte lysate (25 μl) were incubated with 15 μg of (NH₄)₂SO₄-fractionated rat liver cytosolic protein. Incubation was carried out for 30 min at 30°C, and 3-μl aliquots each were electrophoresed on a 14–16% gradient SDS– polyacrylamide gel. A mixture of ³⁵S-labeled +5/1A1 and +33/1A1 were run as markers. (B) P4501A1 translation product was incubated with control cytosolic extract (C) or extract incubated at 95°C for 5 min (B) in the presence or absence of added EDTA (5 mM) or protein inhibitor mix (to a final concentration of 1 mM PMSF and 20 μg/ml each of leupeptin, pepstatin, and chymostatin), as described in Materials and Methods. The incubation and electrophoresis conditions were as described in A. (C) The effects of membrane association of P4501A1 on the processing activity of the cytosolic protein extract was tested. 1A1 protein was translated in the presence of added canine pancreatic ER (2.5 Eq/50 μl reaction), and the membrane-bound fraction (pellet) was isolated by centrifugation over a layer of 0.5 M sucrose containing 100 mM KCl, 50 mM Hepes, pH 7.4, and 5 mM MgOAc₂ at 100,000 g for 30 min. The soluble fraction remaining over the sucrose cushion was aspirated and used as the supernate fraction. Details of incubation with the control and heated cytosolic protein fractions were as described in A.