Proteins of rough microsomal membranes related to ribosome binding. II. Cross-linking of bound ribosomes to specific membrane proteins exposed at the binding sites.

Two proteins (ribophorins I and II), which are integral components of rough microsomal membranes and appear to be related to the bound ribosomes, were shown to be exposed on the surface of rat liver rough microsomes (RM) and to be in close proximity to the bound ribosomes. Both proteins were labeled when intact RM were incubated with a lactoperoxidase iodinating system, but only ribophorin I was digested during mild trypsinization of intact RM. Ribophorin II (63,000 daltons) was only proteolyzed when the luminal face of the microsomal vesicles was made accessible to trypsin by the addition of sublytical detergent concentrations. Only 30--40% of the bound ribosomes were released during trypsinization on intact RM, but ribosome release was almost complete in the presence of low detergent concentrations. Very low glutaraldehyde concentrations (0.005--0.02%) led to the preferential cross-linking of large ribosomal subunits of bound ribosomes to the microsomal membranes. This cross-linking prevented the release of subunits caused by puromycin in media of high ionic strength, but not the incorporation of [3H]puromycin into nascent polypeptide chains. SDS-acrylamide gel electrophoresis of cross-linked samples a preferential reduction in the intensity of the bands representing the ribophorins and the formation of aggregates which did not penetrate into the gels. At low methyl-4-mercaptobutyrimidate (MMB) concentrations (0.26 mg/ml) only 30% of the ribosomes were cross-linked to the microsomal membranes, as shown by the puromycin-KCl test, but membranes could still be solubilized with 1% DOC. This allowed the isolation of the ribophorins together with the sedimentable ribosomes, as was shown by electrophoresis of the sediments after disruption of the cross-links by reduction. Experiments with RM which contained only inactive ribosomes showed that the presence of nascent chains was not necessary for the reversible cross-linking of ribosomes to the membranes. These observations suggest that ribophorins are in close proximity to the bound ribosomes, as may be expected from components of the ribosome-binding sites.

Localization of eukaryotic initiation factor 3 on native small ribosomal subunits.

The localization of eukaryotic initiation factor 3(eIF-3) on native small ribosomal subunits has been established by electron microscopy through a comparison of native small ribosomal subunits with derived subunits and with native subunits stripped of eIF-3. Small subunits derived from reticulocyte ribosomes by the puromycin/KCl method are seen in electron micrographs as elongated particles, divided by a heavily stained partition into approximately one-third and two-third domains. Most particles (60-70%) observed in electron micrographs of native small subunit preparations resemble derived small subunits, but have an additional mass attached to one side, thus producing profiles with a three-lobed appearance. The mass measures approximately 160 x 100 x 60 A, and its particle weight is estimated to be about one-third to one-half that of a 40S subunit. The site of attachment of the additional mass is located on a prominence extending from the central part of the small subunit and is separated by a cleft from the smaller third of the subunit. The remaining particles in preparations of native subunits resemble the profiles seen in electron micrographs of derived subunits. After removal of eIF-3 by treatment with high concentrations of salt, profiles observed in electron micrographs of washed, native subunits were indistinguishable from those of derived subunits. Since removal of eIF-3 coincided with removal of a mass of the correct molecular weight, subunits with the three-lobed appearance are identified as native small subunits carrying eIF-3.

Nonribosomal proteins associated with eukaryotic native small ribosomal subunits.

The native small ribosomal subunit (Sn) from rabbit reticulocytes which is able to initiate translation of globin mRNA in a cell-free system carries additional protein components. The latter can be separated from the subunit in a high salt sucrose gradient yielding a top fraction (T) and a complex fraction (C), sedimenting at about 4 and 15 S, respectively. Polyacrylamide gel electrophoresis in sodium dodecyl sulfate revealed that fraction T contained four dominant polypeptides, while fraction C represents a large protein complex consisting of at least 10 polypeptides. Sn isolated from other sources showed similar patterns of their nonribosomal proteins. Reconstitution experiments revealed that fraction C is absolutely required for protein synthesis, while fraction T enhances protein synthesis only in the presence of C. The adherence of these protein factors to the subunit is not mediated by magnesium ions. Treatment of Sn with EDTA and centrifugation in a magnesium-free sucrose gradient caused unfolding of the subunits and dissociation of several ribosomal proteins, but not of the factors. The unfolded ribosomal subunits sedimented as two distinct peaks. The more slowly sedimenting peak contained proteins of fraction T and the faster sedimenting one contained the 15S complex, indicating heterogeneity of the Sn population with respect to the factors attached to them.

Use of eukaryotic native small ribosomal subunits for the translation of globin messenger RNA.

A highly active in vitro system for the translation of globin mRNA, resulting in more than 10 rounds of translation, is described. The reconstituted system consists of native small ribosomal subunits of rabbit reticulocytes (as a source of initiation factors as well as small ribosomal subunits), large subunits derived from rat liver polysomes by the puromycin-KCl procedure, and a pH 5 fraction obtained from a Krebs ascites cell high speed supernatant. In this system no differences were found between globin messenger ribonucleoprotein and globin mRNA.