FIG. 4.

Extraction of p17MA from membranes. (A) Pr55^gag was synthesized in reticulocyte lysates, incubated with COS-1 cell membranes, and treated or not treated (C lanes) with HIV-1 protease for 10 min to generate membrane-bound p17MA. The products were fractionated by ultracentrifugation into soluble (S) and membrane bound. The membrane fraction was subsequently extracted with 0.1 M Na₂CO₃ (pH 9) (CO₃⁼ lanes) or with buffer containing 1 M NaCl (1 M NaCl lanes). The extraction mixtures were then refractionated into soluble (S′) and membrane bound (P′), and the products were resolved by SDS-PAGE and visualized by PhosphorImager analysis. Note that under these conditions, Pr55^gag was not completely digested and could be detected in the membrane fraction. A marker for ³⁵S-labeled p17MA is included in the last lane. (B) Membranes containing Pr55^gag or Gag69-DHFR were treated with HIV-1 protease and either carbonate or salt as described in panel A. Note that Gag69-DHFR did not undergo proteolysis and was not released from the membrane fraction (P′).