Figure 3.

Insertion of p28 into ER microsomes. Pre–β-lactamase (lanes 1–4) and p28 (lanes 5–8) mRNA was translated in a rabbit reticulocyte lysate system in the presence of [³⁵S]methionine, in the presence (lanes 2–4 and 6–8) or absence (lanes 1 and 5) of ribosome-stripped canine pancreas microsomes (Walter and Blobel, 1983). At the end of the reaction, microsomes were recovered and analyzed by SDS-PAGE and fluorography either directly (lanes 2 and 6) or after isolation of alkali-insoluble (NaCO₃, pH 11.5) product (lanes 3 and 7; Nguyen et al., 1993), or after treatment with proteinase K (lanes 4 and 8; McBride et al., 1992). The positions of p28, pre–β-lactamase (pre-β-L), and processed β-lactamase (β-L) are indicated, as is the gel front. c, marker translation product. The schematic shown below the fluorogram depicts the deduced topology of p28 in the ER membrane (see text).