Cytoplasmic transport of ribosomal subunits microinjected into the Xenopus laevis oocyte nucleus: a generalized, facilitated process.

Abstract

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...

A Viral Movement Protein as a Nuclear Shuttle (The Geminivirus BR1 Movement Protein Contains Domains Essential for Interaction with BL1 and Nuclear Localization)

“…In addition, the partitioning of BRlN219A between the cytoplasm and nuclei of Xanthi protoplasts, combined with the absence of NLSs in this C-terminal region of BRl and our previous studies (Sanderfoot and Lazarowitz, 1995), argues that shuttling of BRl may be essential for its proper function as well. Nuclear shuttle proteins are important components of intercompartmental communication in all eukaryotic cells and have historically been identified based on their nuclear redistribution in heterokaryons or microinjection studies (Bataille et al, 1990;Guichon-Mantel et al, 1994). The only other reported virus-encoded nuclear shuttle protein, the matrix protein M1 of influenza A virus, was deduced based on immunogold localization of M1 to the cytoplasm and nucleus at different times following infection of animal cells (Martin and Helenius, 1991b).…”

“…Such proteins include the major nucleolar proteins nucleolin and No38 (Borer et al, 1989;Laskey and Dingwall, 1993;SchmidtZachmann et al, 1993), members of the 70-kD family of heat-shock proteins (Mande1 and Feldherr, 1990), the heterogeneous nuclear RNA-packaging protein A1 (Pinol-Roma and Dreyfuss, 1992), splicing factor UlA (Kambach and Mattaj, 1992), the nucleolar protein Noppl40 (Meier and Blobel, 1992), the progesterone receptor (Guichon-Mantel et al, 1991), and the influenza M1 matrix protein (Martin and Helenius, 1991b). Because the rate of nuclear import far exceeds that of nuclear export, these proteins characteristically accumulate in nuclei, and shuttling is usually demonstrated by the redistribution of nuclear proteins in heterokaryons or in microinjection studies (Bataille et al, 1990;Schmidt-Zachmann et al, 1993;Guichon-Mantel et al, 1994). Studies demonstrate that masking of nuclear retention signals, as well as the presente of an intact NLS, is required for a karyophilic protein to exit to the cytoplasm and thus function as a shuttle protein, and they have further suggested that most nuclearlocalized proteins may in fact potentially be shuttle proteins (Schmidt-Zachmann et al, 1993;Guichon-Mantel et al, 1994).…”

Nuclear and nucleolar targeting of human ribosomal protein S6.

“…Newly synthesized ribosomal proteins are imported into the nucleus and subsequently accumulate in the nucleoli at the site of ribosome biogenesis (Lastick, 1980;Sommerville, 1986;Warner, 1990). Assembled ribosomal subunits are exported through the nuclear pores into the cytoplasm (Khanna-Gupta and Ware, 1989;Bataille et al, 1990) in which they become engaged in protein synthesis.…”

A conditional yeast mutant deficient in mRNA transport from nucleus to cytoplasm.

“…mRNA export is facilitated by its 5' m7G cap (10, 11). Exit of tRNAs and ribosomal subunits appears to be receptor mediated (9,12).…”

“…mRNA export is facilitated by its 5' m7G cap (10, 11). Exit of tRNAs and ribosomal subunits appears to be receptor mediated (9,12).Because colloidal gold coated with poly(A), tRNA, or 5S RNA microinjected into the nucleus exits via nuclear pores (13) and because exit of tRNA and 5S RNA is inhibited by antibodies that react with pore complexes (14), it is likely that mRNA also exits via pores. Maximal mRNA release from isolated animal cell nuclei requires ATP (15-17).…”