Nonribosomal proteins associated with eukaryotic native small ribosomal subunits.

Abstract

The native small ribosomal subunit (S9) 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 (7) 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. SI' 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 SO 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 9' population with respect to the factors attached to them.Initiation factors for protein synthesis in pro-and eukaryotes are in general found to be bound to ribosomes, from which they can be extracted by high concentrations of monovalent ions (1, 2). In eukaryotes it was recently established that the so-called native small ribosomal subunits (Sn), which comprise only a few percent of the total ribosomes, are able to initiate translation of mRNA in a reconstituted system containing derived large ribosomal subunits and pH 5 enzymes as the only other components (3, 4). Furthermore, it was reported that Sn contained several nonribosomal proteins (5) and that a salt extract from sn could stimulate globin mRNA translation in a cell-free protein synthesis system (6).These findings prompted us to identify the nonribosomal proteins found in Sn and to investigate their possible localization in other components of the ribosomal fraction, such as monosomes and polysomes, their mode of linkage to the small ribosomal subunit, their occurrence in a variety of cells, and their role in a reconstituted system. METHODSPreparation of Native Small Ribosomal Subunits (9'). Procedures were essentially as described (3). In brief: blood from anemic rabbits (7) was collected in the presence of cycloheximide. Further operations were performed in the cold (1-4°). Washed reticulocytes were lysed in 2 volumes of a solution of 10 mM Tris-HCl (pH 7.5)-10 mM KC1-1.5 mM MgCl2-2 mM dithiothreitol and homogenized with three strokes in a Potter-Elvehjem homogenizer. The postmitochondrial supernatant (10 min, 20,000 X gav) was centrifuged first for 30 min and the resulting supernatant was subsequently centrifuged again for 5 hr at 105,0...

Irreversible aggregation of protein synthesis machinery after focal brain ischemia

“…The optical density ratio of S6 versus L28 was 3.87 ± 0.28 (mean ± SD) in the sedimentation peaks from sham‐operated non‐ischemic brains, but changed to 0.91 ± 0.26 and 0.64 ± 0.04 in the sedimentation peaks from brains subjected to ischemia followed by 4 and 24 h of reperfusion, respectively. The presence of eIF3 in the control sedimentation peaks is likely because native 40S subunits in the cytoplasm are stably associated with eIF3, which prevents association of 40S with 60S subunits (Freienstein and Blobel 1975; Thompson et al . 1977; Kolupaeva et al .…”

Binding of eukaryotic initiation factor 3 to ribosomal 40S subunits and its role in ribosomal dissociation and anti-association

“…Although the mechanism of post-termination recycling of ribosomes in eukaryotes is not understood (Janosi et al 1996;Kisselev and Buckingham 2000), eIF3 and, to some extent, eIF1A have been implicated in preventing reassociation of free 40S and 60S subunits and in dissociation of empty 80S ribosomes (Thompson et al 1977;Trachsel and Staehelin 1979;Thomas et al 1980a). Native 40S subunits in the cytoplasm are stably associated with eIF3 (Freienstein and Blobel 1975) and purified eIF3 can bind directly to 40S subunits in the absence of other initiation factors (Benne and Hershey 1976;Peterson et al 1979;Trachsel and Staehelin 1979;Nygard and Westermann 1982). The ability of eIF3 to bind to 40S subunits in the absence of other initiation factors depends strictly on the presence of its loosely associated eIF3j subunit (Fraser et al 2004).…”

Interaction of a limited set of proteins with different mRNAs and protection of 5′-caps against pyrophosphatase digestion in initiation complexes

“…This model predicts that eIF-3 or some of its subunits would be present in natural 80S initiation complexes, either on the ribosome or on the mRNA 5'-terminus. Although studies with reconstituted initiation systems have demonstrated the release of eIF-3 from 80S complexes (30,35 (36,37). Further characterization of the proteins that can be cross-linked to the cap of mRNA in initiation complexes should indicate whether they correspond to eIF-3 subunits and if cap-binding proteins promote translation by the pathway suggested.…”