Figure 1.

Ribosome-stripped ER membranes are permeable to small molecules. (A) Ribosomes carrying a radiolabeled fragment of ppαF (86mer) with a single cysteine at position 56 were incubated with ribosome-stripped microsomes (PK-RM). After treatment with puromycin to release the nascent chains into the ER lumen, the samples were incubated with 0.1 mM MB or 0.075 mM MPB for 10 min on ice, either in the absence of detergent, or in the presence of 0.5% TX-100 or 0.1% digitonin, as indicated. One-half of the samples was analyzed after precipitation with TCA (total), the other was subjected to incubation with streptavidin beads, and the bound (beads) and unbound (supernatant) fractions were analyzed. All samples were analyzed by SDS-PAGE and autoradiography. The stars indicate molecules with one to three carbohydrate chains. (B) Full-length ppαF with a single cysteine at position 56 was imported into untreated RMs. Membranes were pelleted before the modification reaction was carried out. The samples were then treated for 10 min on ice with the indicated concentrations of MB in the absence or presence of 0.5% Triton X-100. One half of the sample was analyzed directly (total), the other was subjected to incubation with streptavidin beads and the bound material was analyzed (beads). All samples were subjected to SDS-PAGE and autoradiography. ppαF, gppαF, pro-α-factor without or with one to three carbohydrate chains, respectively. Molecular masses (in kilodaltons) are indicated on the right side.