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Abstract. To study the biochemistry of ribonucleoprotein export from the nucleus, we characterized an in vivo assay in which the cytoplasmic appearance of radiolabeled ribosomal subunits was monitored after their microinjection into Xenopus oocyte nuclei. Denaturing gel electrophoresis and sucrose density gradient sedimentation demonstrated that injected subunits were transported intact. Consistent with the usual subcellular distribution of ribosomes, transport was unidirectional, as subunits injected into the cytoplasm did not enter the nucleus. Transport displayed properties characteristic of a facilitated, energy-dependent process; the rate of export was saturable and transport was completely inhibited either by lowering the temperature or by depleting nuclei of ATP; the effect of lowered temperature was completely reversible. Transport of injected subunits was likely a process associated with the nuclear pore complex, since export was also inhibited by prior or simultaneous injection of wheat germ agglutinin, a lectin known to inhibit active nuclear transport by binding to N-acetyl glucosamine-containing glycoproteins present in the NPC (Hart, G. W., R. S. Haltiwanger, G. D. Holt, and W. G. Kelly. 1989. Annu. Rev. Biochem. 58:841-874). Although GIcNAc modified proteins exist on both the nuclear and cytoplasmic sides of the nuclear pore complex, ribosomal subunit export was inhibited only when wheat germ agglutinin was injected into the nucleus. Finally, we found that ribosomal subunits from yeast and Escherichia coli were efficiently exported from Xenopus oocyte nuclei, suggesting that export of some RNP complexes may be directed by a collective biochemical property rather than by specific macromolecular primary sequences or structures.T HE assembly of ribosomes in eukaryotic cells is a process that requires transfer of macromolecules into and out of the nucleus (33). Ribosomal proteins, synthesized in the cytoplasm, enter the nucleus and assemble with nascent rRNA to form a preribosomal particle. Through a series of maturation steps involving endonucleolytic cleavage of the primary rRNA transcript and further addition of specific ribosomal proteins, the preribosomal particle splits into partially completed 40S and 60S subunits that contain the nearly mature 18S and 28S:5.8S rRNAs, respectively. The pre-60S subunit also acquires a 5S rRNA molecule by addition of a separate 5S-containing RNP (51). Eventually, the 40S and 60S subunits exit the nucleus into the cytoplasm where they assemble with additional ribosomal proteins. While much is known about mechanisms that regulate synthesis of individual ribosomal components (4, 56), we envision equally important mechanisms that promote efficient assembly of ribosomal components. Therefore, we wish to determine the driving forces that bring about exchange of ribosomal components between the nucleus and cytoplasm.Mechanisms of macromolecular transport across the nuclear-cytoplasmic boundary have been the focus of much recent research. A conspicuous structure spanning...

Under specific conditions cycloheximide treatment of Saccharomyces cerevisiae caused the accumulation of a type of polyribosome called "halfmer." Limited ribonuclease digestion of halfmers released particles from the polyribosomes identified as 40S ribosomal subunits. The data demonstrated that halfmers are polyribosomes containing an additional 40S ribosomal subunit attached to the messenger ribonucleic acid. Protein gel electrophoretic analysis of halfmers revealed numerous nonribosomal proteins. Two of these proteins comigrate with subunits of yeast initiation factor eIF2.In current models of the initiation of eucaryotic protein synthesis, the 40S ribosomal subunit binds messenger ribonucleic acid (RNA) before the attachment of the 60S ribosomal subunit. Progress in the study of this process in yeast has been limited by the absence of an in vitro translation system, which was developed only very recently (7). As an alternative approach to in vitro investigation of initiation factors, we have isolated and characterized complexes formed in vivo in the presence of the antibiotic cycloheximide.Cycloheximide, an inhibitor of eucaryotic protein synthesis (22), is known to inhibit elongation over a wide range of concentrations (2,25,27,32). At lower concentrations there is also evidence for inhibition of initiation (2,4,9,24,29).Using a low concentration of cyloheximide, we have detected and characterized a type of polyribosome, termed "halfmer" (9,14,19,20), which has an extra 40S ribosomal subunit. The halfmer is an apparent product of a cycloheximide-induced inhibition of binding of the 60S ribosomal subunit to the 40S ribosomal subunit initiation complex on the polyribosome. Here we report that nonribosomal proteins are associated with the halfmer, and two comigrate with subunits of initiation factor eIF2 in polyacrylamide gels. MATERIALS AND METHODSStrains and media. Saccharomyces cerevisiae haploid strain A364A (ATCC 22244) was grown on "modified synthetic complete" (SCY) medium (13) and YM-1 and YM-5 media (11). Spheroplasts were incubated in YM-5 medium containing 0.5 M MgSO4. Cells grown as above in low-phosphate medium were converted to spheroplasts and diluted into low-phosphate YM-5 medium containing 0.5 M MgSO4 (30). Fifty microcuries of carrier-free [32P]phosphoric acid (New England Nuclear Corp., Boston, Mass.) was added per milliliter of culture, and spheroplasts were allowed to recover for 2.5 h before cycloheximide (10 ,ug/ml of culture) was added. Cells were collected after 10 min at room temperature, and the cell pellets were frozen.Cells were &lso grown in SCY medium, containing 2.5 ,uCi of a '4C-labeled L-amino acid mixture (New England Nuclear Corp.) per ml, to early stationary growth phase (5 x 107 cells per ml). This culture was diluted with 10 volumes of YM-1 medium and grown for one generation before collection, spheroplasting, and recovery as described above.Lysate preparation and analysis. Spheroplast pellets containing about 3 x 107 cells were lysed at 0°C