Protein insertion into the membrane of the endoplasmic reticulum: The architecture of the translocation site

In mammalian cells, most membrane proteins are inserted cotranslationally into the membrane of the endoplasmic reticulum (ER), and secretory proteins are translocated across this membrane (for review, see Rapoport 1992). These processes are initiated in the cytoplasm by binding of the signal sequence of the na-scent polypeptide chain to the signal recognition parti-cle (SRP) (Walter and Blobel 1981). The resulting ribosome nascent chain SRP complex is then targeted to the ER membrane by an interaction with the mem-brane-bound SRP receptor (docking protein) (Gilmore et al. 1982; Meyer et al. 1982). At the membrane, the signal sequence is released from SRP in a GTP-requiring step, and the nascent polypeptide is trans-ferred into the ER translocation site (Connolly an

Transfer of Proteins across Membranes II. Reconstitution of Functional RoughMicrosomes from Heterologous Components

The data presented in this paper demonstrate that native small ribosomal subunits from reticulocytes (containing initiation factors) and large ribosomal subunits derived from free polysomes of reticulocytes by the puromycin-KCI procedure can function with stripped microsomes derived from dog pancreas rough microsomes in a protein-synthesizing system in vitro in response to added lgG light chain mRNA so as to segregate the translation product in a proteolysis-resistant space. No such segregation took place for the translation product of globin mRNA. In addition to their ability to segregate the translation product of a specific heterologous mRNA, native dog pancreas rough microsomes as well as derived stripped microsomes were able to proteolytically process the larger, primary translation product in an apparently correct manner, as evidenced by the identical mol wt of the segregated translation product and the authentic secreted light chain. Segregation as well as proteolytic processing by native and stripped microsomes occurred only during ongoing translation but not after completion of translation. Attempts to solubilize the proteolytic processing activity, presumably localized in the microsomal membrane by detergent treatment, and to achieve proteolytic processing of the completed light chain precursor protein failed. Taken together, these results establish unequivocally that the information for segregation of a translation product is encoded in the mRNA itself, not in the protein-synthesizing apparatus; this provides strong evidence in support of the signal hypothesis

Signal Recognition Particle-dependent Membrane Insertion of Mouse Invariant Chain: A Membrane-spanning Protein with a Cytoplasmically Exposed Amino Terminus

Invariant (Ii) chain is a membrane-spanning protein that is found associated intracellularly with class II histocompatibility antigens. In the endoplasmic reticulum Ii chain spans the membrane and exposes the NH2 terminus on the cytoplasmic and the COOH terminus on the lumenal side. This orientation across the membrane is demonstrated directly with the monoclonal antibody In-l, which exclusively recognizes the NH2 terminal cytoplasmically exposed part of Ii chain. Membrane insertion of Ii chain requires signal recognition particle and docking protein. When tested in a wheat germ cell free system, signal recognition particle arrests translation of Ii chain. No signal sequence is cleaved from Ii chain upon membrane insertion