FIGURE 13-3 Secretory proteins enter the ER lumen. Labeling experiments demonstrated that secretory proteins are localized to the ER lumen shortly after synthesis. Cells are incubated for a brief time with radiolabeled amino acids so that only newly synthesized proteins become labeled. The cells are then homogenized, fracturing the plasma membrane and shearing the rough ER into small vesicles called microsomes. Because they have bound ribosomes, microsomes have a much greater buoyant density than other membranous organelles and can be separated from them by a combination of differential and sucrose density-gradient centrifugation (see Chapter 4). The purified microsomes are treated with a protease in the presence or absence of a detergent. The labeled secretory proteins associated with the microsomes are digested by the protease only if the microsomal membrane is first destroyed by treatment with detergent. This finding indicates that the newly made proteins are inside the microsomes, equivalent to the lumen of the rough ER.