Characterization of secretory protein translocation: ribosome-membrane interaction in endoplasmic reticulum.

Abstract

Abstract. Secretory proteins are synthesized on ribosomes bound to the membrane of the endoplasmic reticulum (ER). After the selection of polysomes synthesizing secretory proteins and their direction to the membrane of the ER via signal recognition particle (SRP) and docking protein respectively, the polysomes become bound to the ER membrane via an unknown, protein-mediated mechanism. To identify proteins involved in protein translocation, beyond the (SRPdocking protein-mediated) recognition step, controlled proteolysis was used to functionally inactivate rough microsomes that had previously been depleted of docking protein. As the membranes were treated with increasing levels of protease, they lost their ability to be functionally reconstituted with the active cytoplasmic fragment of docking protein (DPf). This functional inactivation did not correlate with a loss of either signal peptidase activity, nor with the ability of the DPf to reassociate with the membrane. It did correlate, however, with a loss of the ability of the microsomes to bind ribosomes.Ribophorins are putative ribosome-binding proteins.Immunoblots developed with monoclonal antibodies against canine ribophorins I and II demonstrated that no correlation exists between the protease-induced inability to bind ribosomes and the integrity of the ribophorins. Ribophorin I was 85 % resistant and ribophorin II 100% resistant to the levels of protease needed to totally eliminate ribosome binding. Moreover, no direct association was found between ribophorins and ribosomes; upon detergent solubilization at low salt concentrations, ribophorins could be sedimented in the presence or absence of ribosomes. Finally, the alkylating agent N-ethylmaleimide was shown to be capable of inhibiting translocation (beyond the SRP-docking protein-mediated recognition step), but had no affect on the ability of ribosomes to bind to ER membranes. We conclude that potentially two additional proteinaceous components, as yet unidentified, are involved in protein translocation. One is protease sensitive and possibly involved in ribosome binding, the other is N-ethylmaleimide sensitive and of unknown function.

Cell Biology of the Endoplasmic Reticulum and the Golgi Apparatus through Proteomics

“…In one such study, Dejgaard and coworkers succeeded in separating and characterizing the native ribosome -translocon complex from canine pancreas microsomes (Dejgaard et al 2010). The native complex identified was larger than that predicted, and confirmed earlier work by Meyer and others (Hortsch et al 1986;Savitz and Meyer 1990), showing that the p180/ribosome receptor is indeed a constituent of the ribosometranslocon complex. Additional components such as CLIMP63, glucosidase I, and BAP31 were also assigned to the ribosome -translocon complex giving rise to a native size of 4 MDa rather than the previously predicted size of 2 MDa.…”

Induction of expression of protein disulphide‐isomerase during lymphocyte maturation stimulated by bacterial lipopolysaccharide

“…It detected a single protein band on Western blots of microsomal membranes from rat liver and dog pancreas, and was used in a detailed study of the distribution of several proteins between various endo-membranes in the dog pancreas [18]. The anti-bovine PDI antiserum cross-reacted with a protein of 59 kDa in preparations of 'reticuloplasm' [19] (i.e., e.r.…”

The Mammalian Staufen Protein Localizes to the Somatodendritic Domain of Cultured Hippocampal Neurons: Implications for Its Involvement in mRNA Transport

“…The following antibodies were used in the indicated dilutions: monoclonal antibody against M AP2 (Sigma, St. L ouis, MO) at 1:500 (De Hoop et al, 1994); the monoclonal antibody against tau-1 (Boehringer Mannheim, Mannheim, Germany) at 1:200 or 1:500 (Binder et al, 1985); and the monoclonal antibody against ribophorin I (courteously provided by D. Meyer, University of C alifornia at L os Angeles) at 1:100 (Hortsch et al, 1986). As secondary antibodies, a rhodamine-conjugated goat anti-rabbit whole Ig (1:100; 55674; Organon Teknika-C appel, Durham, NC), a C y5-conjugated donkey anti-rabbit whole Ig (1:300; 711 175 152; Dianova, Hamburg, Germany) or FI TC -conjugated sheep anti-mouse whole Ig (1:50; N1031; Amersham, Arlington Heights, IL) were used.…”