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Nucleocytoplasmic Transportof Ribosomes in a Eukaryotic System: Is Proc. Natl. Acad. Sci. USA Vol. 86, pp. 1791-1795, March 1989 Biochemistry Nucleocytoplasmic transport of ribosomes in a eukaryotic system: Is there a facilitated transport process? (Tetrahymena ribosomal subunits/ribosomal RNA/Xenopus laevis oocyte) ARATI KHANNA-GUPTA AND VASSIE C. WARE* Department of Biology and Center for Molecular Bioscience and Biotechnology, Mountaintop Campus, Building A, Lehigh University, Bethlehem, PA 18015 Communicated by Clement L. Markert, December 5, 1988 ABSTRACT We have examined the kinetics of the process Studies involving the transport of tRNAs from microin- by which ribosomes are exported from the nucleus to the jected Xenopus oocyte nuclei have revealed that a carrier- cytoplasm using Xenopus laevis oocytes microinjected into the mediated tRNA nuclear transport mechanism exists and that germinal vesicle with radiolabeled ribosomes or ribosomal the rate of tRNA movement is dramatically affected by subunits from X. laevis, Tetrahymena thermophila, or Esche- nucleotide changes in a conserved domain of the tRNA richia coli. Microinjected eukaryotic mature ribosomes are structure itself (6, 7). Therefore, tRNA may interact directly redistributed into the oocyte cytoplasm by an apparent carrier- with nuclear pore constituents to promote its transport. mediated transport process that exhibits saturation kinetics as To gain an understanding of the process for translocating ribosomal subunits from the nuclei of eukaryotic cells, we increasing amounts of ribosomes are injected. T. thermophila have introduced radiolabeled homologous (Xenopus laevis) ribosomes are competent to traverse the Xenopus nuclear and heterologous (Tetrahymena thermophila and Esche- envelope, suggesting that the basic mechanism underlying richia coli) ribosomes or ribosomal subunits into the nucleus ribosome transport is evolutionarily conserved. Microinjected of individual Xenopus oocytes by microinjection and have E. coli ribosomes are not transported in this system, indicating followed the partitioning of the injected ribosomes between that prokaryotic ribosomes lack the "signals" required for the nucleus and cytoplasm as a function of time. We report transport. Surprisingly, coinjected small (40 S) and large (60 S) that the nuclear export of ribosomal subunits, like that of subunits from T. thermophila are transported significantly tRNA species (6), is saturable, indicative of a carrier- faster than individual subunits. These observations support a facilitated transport process. Our data suggest that the basic facilitated transport model for the translocation of ribosomal ribosomal subunit transport mechanism in eukaryotes has subunits as separate units across the nuclear envelope whereby been conserved through evolution since ribosomes from a the transport rate of 60S or 40S subunits is enhanced by the diverse species such as Tetrahymena can be transported in presence of the partner subunit. Although the basic features of Xenopus oocytes. Translocation of subunits across the nu- the transport mechanism have been preserved through evolu- clear envelope is, however, a property specifically limited to tion, other aspects of the process may be mediated through eukaryotic subunits, as prokaryotic ribosomes are not trans- species-specific interactions. We hypothesize that a species- ported over the time interval studied. The kinetics of heter- specific nuclear 40S-60S subunit association may expedite the ologous eukaryotic ribosomal subunit transport suggest a transport of individual subunits across the nuclear envelope. model in which the transport rate of individual subunits may be facilitated through nuclear subunit associations. The assembly ofribosomal subunits and their eventual export into the cytoplasm is a complex process. Our knowledge is MATERIALS AND METHODS more advanced for earlier events in this process, such as rRNA gene transcription (1) and the maturation pathways for Synthesis of 3H/32P-Labeled Ribosomes. E. coli cells (strain rRNA ref. than for later steps in ribosome HB101) in early logarithmic phase were labeled with 2 mCi of precursor (e.g., 2), 32P-labeled H3PO4 in water (ICN; 1 Ci = 37 GBq) or 0.5 mCi maturation and nucleocytoplasmic transport. of [3H]uridine (ICN) in low-phosphate M9 medium. Cells Several investigators have hypothesized that nuclear ribo- were harvested after 16 hr at 37°C. T. thermophila cells grown nucleoproteins (RNPs) are associated with the nuclear matrix at 25°C for 16 hr were transferred to medium 357 (American and that the matrix may play a role in processing and in the Type Culture Collection) containing 32P-labeled H3PO4 as movement ofRNPs toward the nuclear pore complexes (3, 4). above and allowed to grow with gentle shaking for 16 hr at Although electron microscopy studies (5) have implicated 25°C. X. laevis stage VI oocytes were excised from the ovary nuclear pore complexes as the sites from which ribosomal of a mature female and transferred to Holtfreter's medium particles as well as other RNPs emerge into the cytoplasm, (8). Radiolabeling and harvesting were as above. the mechanism for transport remains unknown. According to Isolation of 3H/32P-Labeled Ribosomes and RNA. 32p- a general "gating" mechanism proposed by Wunderlich and labeled E. coli cells were disrupted by the NaDodSO4 lysis Speth (5), nuclear RNPs bind to pore complex constituents method (9). The crude lysate was centrifuged at 30,000 rpm until a threshold of bound RNP particles is reached. Above (SW 41 rotor) for 2 hr at 4°C. The transparent pellet was a critical concentration the RNPs are translocated through resuspended in 10 mM Tris (pH 8). Ribosome and RNA nuclear pores and released into the cytoplasm. The possible preparations from all sources were treated with RNase-free contribution of other nuclear factors to the transport process DNase (Promega Biotec) for 30 min at 25°C. 32P-labeled T. is unclear. Presumably specific molecular interactions must thermophila cells were pelleted and disrupted by freezing and occur involving both the RNP and pore complex constitu- thawing three times at -70°C. Cells were resuspended in ents. ice-cold 10 mM Tris (pH 8) and centrifuged at 10,000 rpm (Ti 60 rotor) for 15 min. The supernatant was recentrifuged at The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" Abbreviation: RNP, ribonucleoprotein. in accordance with 18 U.S.C. §1734 solely to indicate this fact. *To whom reprint requests should be addressed. 1791 Downloaded by guest on September 26, 2021 1792 Biochemistry: Khanna-Gupta and Ware Proc. Natl. Acad Sci. USA 86 (1989) 30,000 rpm (SW 41 rotor) for 2 hr. The pellet was resuspended either in 10 mM Tris (pH 8) or in dissociation buffer (20 mM 100_ Tris HCl, pH 7.4/16 mM MgCl2/1.0 M KCl/12 mM 2- mercaptoethanol/0.2 mM EDTA). Radiolabeled X. laevis stage VI oocytes were washed in Holtfreter's solution (8) and homogenized at 40C. Centrifugation was as described. RNA was prepared by phenol/chloroform extraction of crude ribosomes followed by ethanol precipitation. 32P-labeled I-C RNA was resuspended in 10 mM Tris (pH 8). Isolation of Ribosomal Subunits. Ribosomes in dissociation '0 buffer were loaded onto a 15-30% (wt/vol) linear sucrose gradient in dissociation buffer and centrifuged at 10'C for 17 hr at 20,000 rpm (SW 41 rotor). The radioactivity in each 0 15 30 45 60 0.5-ml fraction was assayed by liquid scintillation counting. Incubation time, min or small ribosomal subunits were Fractions containing large FIG. 1. Kinetics ofXenopus ribosome transport as a function of pooled and pelleted at 30,000 rpm (SW 41 rotor) for 16 hr at the concentration of ribosomes injected into the nuclei of X. laevis 10'C. Pellets were resuspended in 10 mM Tris (pH 8). oocytes. 32P-labeled X. laevis ribosomes were injected in a vol of 20 Microinjection and Microdissection of X. laevis Oocytes. nl into the nuclei of X. laevis oocytes. After incubation of injected Microinjections into the germinal vesicle of stage V and VI oocytes for 0, 5, 15, 30, 45, and 60 min in Holtfreter's medium at oocytes and manual dissections of oocytes at various times 250C, the oocytes (average number 10 per time point per experiment) after injection were carried out as described (6). An average were fixed in ice-cold 1% CC13COOH and manually dissected, and of 10 oocytes was injected for each time point. In some the radioactivity present in either pooled or individual nuclear and experiments nuclear and cytoplasmic fractions for each time cytoplasmic fractions was determined by liquid scintillation count- was In other ing. A range of 0.2-2.8 ng of rRNA per nucleus (0.4-6 x 103 cpm) point were pooled and radioactivity quantitated. was used in this experiment. Several concentrations are not shown. experiments the radioactivity in nuclear and cytoplasmic For clarity, SDs between 2 and 10%6 have been omitted from the fractions of each individual oocyte was determined. Meth- figure. The best-fit first-order exponential plots were generated. ylene blue (0.1%) was added to the injected material in most Statistically significant differences (at a minimum 95% confidence experiments as an indicator of the success of nuclear injec- level) are noted as follows: 0.2 ng/0.4 ng; 0.2 ng/0.6 ng; 0.2 ng/0.8 tion. Only those oocytes with dye remaining in the nucleus ng; and 0.4 ng/0.6 ng. v, 0.6 ng; 0, 0.8 ng; A, 0.4 ng; o, 0.2 ng. were analyzed. The dye has been shown to have no measur- able effect on the transport of ribosomes or ribosomal In a parallel set of oocytes, radiolabeled Xenopus ribosomes subunits (unpublished observations). On the average only 1 were injected. After incubation of the oocytes for periods up nucleus in 20-30 oocyte nuclei was not targeted. to 30 min, no detectable transport of the labeled DNA was Measurement of Ribosome Specific Activity.
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