1889
Journal of Cell Science 111, 1889-1896 (1998) Printed in Great Britain © The Company of Biologists Limited 1998 JCS7248
The nuclear import factor p10 regulates the functional size of the nuclear pore complex during oogenesis Carl Feldherr1,*, Debra Akin1 and Mary Shannon Moore2 1Dept 2Dept
of Anatomy and Cell Biology, University of Florida, College of Medicine, Gainesville, FL 32610, USA of Cell Biology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA
*Author for correspondence (e-mail:
[email protected])
Accepted 15 April; published on WWW 15 June 1998
SUMMARY Previtellogenic, stage-1 Xenopus oocytes produce mainly 5S and tRNA, whereas vitellogenic oocytes, stages 2-6, synthesize predominantly 18S and 28S rRNA. Using nucleoplasmin-coated gold as a transport substrate, it was determined that the shift in synthesis from small to large RNAs during oogenesis is accompanied by an increase in both the rates of signal-mediated nuclear import and the functional size of nuclear pores. It was observed that, despite the reduction in transport capacity, gold still accumulated at the cytoplasmic surface of the pores in stage-1 oocytes. This suggested that transport in these cells is limited by translocation factors, rather than by
cytoplasmic binding factors. Analysis of extracts prepared from stage-1 and vitellogenic oocytes revealed that the transport factor p10 is more abundant in stage-1 cells. Microinjection of purified p10 into stage-2 oocytes reduced the nuclear import of large gold particles to the level observed in stage-1 cells. It is concluded that p10 can modulate transport through the pores by regulating the functional size of the central transporter element.
INTRODUCTION
of the signal-mediated transport process itself. The first step in transport through the nuclear pores involves binding of an NLS-containing transport substrate to a cytoplasmic receptor. Although there is evidence for different classes of receptors (Pollard et al., 1996; Bonifaci et al., 1997; Michael et al., 1997), the best understood is karyopherin-α/importin-α (Radu et al., 1995; Gorlich et al., 1994), a multigene family encoding proteins of approximately 60 kDa, which bind both the simple (large T antigen-like) and bipartate (nucleoplasmin-like) NLSs. After initially binding the substrate, karyopherin-α complexes with 97 kDa karyopherin-β/importin-β (Radu et al., 1995; Gorlich et al., 1995), which functions in docking the receptorsubstrate complex to one or more nucleoporins at the cytoplasmic surface of the pores (Nigg, 1997). Following the docking step, energy-dependent translocation occurs through the cylindrical transporter element, which occupies the central region of the pore complex. Translocation requires additional soluble factors including, among others, Ran/TC4, a 25 kDa GTPase (Moore and Blobel, 1993; Melchior et al., 1993), and p10/NTF2 (Moore and Blobel, 1994; Paschal and Gerace, 1995), a 14.4 kDa protein that binds to nucleoporins, Ran-GDP and karyopherin-β (Paschal and Gerace, 1995; Nehrbass and Blobel, 1996; Paschal et al., 1996; Wong et al., 1997). The above NLS receptors and translocation factors were originally identified in Xenopus oocytes; subsequently, homologs of these agents have also been found in mammalian cells and yeast (reviewed by Nigg, 1997; Corbett and Silver, 1997).
Regulating macromolecular exchanges across the nuclear envelope is an effective and fundamental method of modulating cellular activity. Basically, there are two general systems for controlling signal-mediated transport between the nucleus and cytoplasm (reviewed by Feldherr and Akin, 1994). One is highly selective, and functions in the exchange of specific cell proteins, such as transcription factors and enzymes. The second involves changes in the transport machinery itself, and would be expected to have a more general effect on cell function. Inhibition of the nuclear import of specific macromolecules containing nuclear localization signals (NLSs) is accomplished either by blocking access to the nuclear pores or by masking the NLS. Specific examples include the catalytic subunit of the cAMP-dependent protein kinase, which is anchored within the cytoplasm in its inactive state, but is released and able to enter the nucleus as the level of cAMP increases (Nigg et al., 1985; Nigg, 1990). The exchange of transcription factors such as NFκB, Rel and dorsal is regulated by reversible binding to inhibitors that mask the NLSs (e.g. Grimm and Baeuerle, 1993; Govind and Steward, 1991). In other instances, such as the cellcycle-dependent nuclear import of SWI5 in yeast (Moll et al., 1991), phosphorylation of sites associated with the NLS serves to mediate transport. The experiments in this report relate to the second method of controlling nucleocytoplasmic exchanges, i.e. to alterations
Key words: Nuclear transport, p10, Oogenesis, Colloidal gold, Xenopus
1890 C. Feldherr, D. Akin and M. S. Moore The translocation process takes place through a central channel located within the pore complexes (Feldherr et al., 1984). The patent diameter of the channel is approximately 90 Å. Passive diffusion of macromolecules can occur through this region, and the rates are inversely related to molecular size. For example, in amphibian oocytes, molecules that have diameters of approximately 45 Å reach diffusion equilibrium between the nucleus and cytoplasm in about 15 hours; however, 70 Å substances are essentially excluded from the nucleus (Paine et al., 1975). In the presence of an NLS-containing substrate, the functional size of the transport channel can increase to approximately 250 Å, allowing the exchange of molecules (or molecular complexes such as RNP particles) that greatly exceed the diffusion limit (Feldherr et al., 1984). Functionally related changes in transport capacity have been reported previously (reviewed by Feldherr and Akin, 1995). For example, as cultured fibroblasts become quiescent, either by growing to confluence or as a result of serum depletion, there is an accompanying size-dependent decrease in nuclear transport (Feldherr and Akin, 1991), which can be attributed to a change in the functional dimensions of the central transporter element. This is indicated by the fact that nucleoplasmin-coated gold particles (NP-gold) as large as 230 Å in diameter are readily transported across the nuclear envelope in proliferating cells, whereas in growth-arrested cells, the upper limit for transport decreases by as much as 40-100 Å, depending on the cell strains. These modifications in functional pore size should be sufficient to restrict the nuclear efflux of large RNP particles, especially ribosomal subunits and, in this way, contribute to the physiological changes that are known to occur during growth arrest, i.e. decreases in both cytoplasmic rRNA content and protein synthesis. However, in this earlier study it was not possible to demonstrate a direct correlation between rRNA synthesis and nuclear transport. There are well-characterized changes in the patterns of RNA synthesis during oogenesis in Xenopus. Stage-1 oocytes synthesize large amounts of 5S and tRNA (approximately 75% of the total RNA synthesized), but relatively little 18S and 28S rRNA (e.g. Ford, 1972; Thomas, 1974; Scheer et al., 1976) or mRNA (Rosbash and Ford, 1974). Lampbrush chromosomes are not present at this stage (Dumont, 1972; Rogers and Browder, 1977). The 5S RNA is stored in the cytoplasm, complexed with TFIIIa and the ribosomal protein L5, in the form of 42S and 7S particles (Picard et al., 1980; Johnson et al., 1984; Rudt et al., 1996). By stage 2 (early vitellogenesis) the oocytes produce predominantly 18S and 28S rRNA (Ford, 1972). At this time the stored 5S RNA is transported back to the nucleus and used in ribosome formation. The variations in nucleic acid production that occur during different stages of oogenesis in Xenopus, plus the available data regarding the factors required for nuclear import, make this an especially useful system for investigating functionally related differences in transport capacity at a molecular level. In this study, it was initially determined, using NP-gold as a transport substrate, that the shift in RNA synthesis from 5S and tRNA to rRNA is accompanied by a significant increase in nuclear import capacity. Evidence was then obtained indicating that nuclear transport, during this period of oogenesis, can be regulated by the amount of translocation factor p10 present in the cells.
MATERIALS AND METHODS Xenopus oocytes Xenopus laevis were purchased from Xenopus I (Ann Arbor, MI). The animals were maintained in filtered artificial pond water and fed beef heart 3 times a week. Postmetamorphic frogs (0.5-1 inch long) were used as the source of stage-1 and stage-2 oocytes; stage-6 oocytes were obtained from mature females (2.5-3 inches long). The oocytes were classified by the criteria established by Dumont (1972). The stage-1 oocytes used in this study ranged from 140-180 µm in diameter; they were transparent (pre-vitellogenic) and had visible mitochondrial masses. The stage-2 oocytes were slightly opaque (early vitellogenic) and had diameters between 260-320 µm. Stage-6 cells had well-delineated animal and vegetal hemispheres, and were approximately 1200 µm in diameter. Ovaries were removed from animals immobilized by hypothermia. Preparation of nucleoplasmin and p10 Nucleoplasmin (NP), a major oocyte nuclear protein that contains a well-characterized bipartite NLS (Dingwall et al., 1988), was employed as a coating agent for colloidal gold particles that were used to assay nuclear transport. NP was isolated from mature Xenopus ovaries by affinity chromatography as described by Dworetzky et al. (1988). All of the following procedures, which were used in the preparation of p10, were performed at 4°C unless otherwise stated. The vector expressing wild-type human p10 (pTacT7L-pp15) was a kind gift of Dr Ulrich Grundmann at Behringwerke (Marburg, Germany) and was used to transform BL21(DE3) cells (Lehmeier and Amann, 1992). 2 l of cells were induced with 1 mM IPTG for 4 hours at 30°C. After washing once in Buffer A (20 mM Hepes-KOH, pH 7.3, 1 mM DTT) containing 50 mM NaCl, the cells were French-pressed in 40 ml of the same buffer containing 1 mM PMSF followed by centrifugation at 60,000 rpm (264,902 g) in a Ti70 rotor for 90 minutes. The supernatant was diluted with Buffer A to a NaCl concentration of 10 mM and loaded on a 40 ml Q-Sepharose (Pharmacia, Piscataway, NJ) column (3.8×10.5 cm) equilibrated in Buffer A containing 10 mM NaCl. The sample was loaded overnight at a flow rate of 10 ml/hour and then washed at 50 ml/hour with 200 ml of column buffer. The column was eluted with a 200 ml gradient of 0-300 mM NaCl in Buffer A and 4 ml fractions were collected. The column fractions containing p10 (which eluted at approximately 150 mM NaCl) were determined by SDS-PAGE and pooled. The p10 was precipitated by solid ammonium sulfate at 50% saturation (29.1 g/100 ml) and the pellet was resuspended to a final volume of 10 ml in Buffer A containing 100 mM NaCl. One-tenth volume of 50% glycerol was added and the sample was microfuged for 10 minutes. The sample was loaded on a 564 ml Sephacryl S-200 (Pharmacia, Piscataway, NJ) column (2.5×115 cm) equilibrated in Buffer A containing 100 mM NaCl and run at 30 ml/hour. 4 ml fractions were collected and the elution position of p10, which was always consistent with the dimeric molecular mass of p10 of approximately 29 kDa, was determined by SDS-PAGE. The pooled fractions were dialyzed against Buffer A containing 100 mM potassium acetate, divided into single-use portions, snap-frozen in liquid nitrogen, and stored at −80°C. The purified, recombinant human protein exhibited a similar specific activity in the in vitro nuclear import assay to the purified, native, Xenopus protein (Moore and Blobel, 1994). The average yield was approximately 12 mg from 2 l of culture. Fig. 1 is an SDS-PAGE gel of a final p10 preparation. Prior to microinjection, p10 was dialyzed against intracellular medium (100 mM KCl, 10 mM NaCl, 6 mM K2HPO4, 4 mM KH2PO4, pH 7.0). Cytoplasmic extracts The cytosolic extracts to be analyzed for translocation factors were prepared from stage-1 and vitellogenic oocytes. Stage-1 oocytes were
Nuclear transport during oogenesis 1891 blocking solution. Next, they were incubated for 1 hour with goat antimouse IgG:horseradish peroxidase (Bio-Rad; Hercules, CA) diluted 1:4000 in blocking solution. After washing 3× 10 minutes in TBST, the blots were developed with the ECL kit (Amersham; Arlington Heights, IL).
Fig. 1. Purified recombinant p10. 1 µg of p10 was run on a 15% gel and stained with Coomassie Blue. Molecular mass markers (Std) are the broad-range markers from Bio-Rad (Hercules, CA).
obtained from postmetamorphic frogs. The ovaries were removed, cut into small pieces, and shaken for 1 hour at room temperature in calcium-free OR-2 extracellular medium (Wallace et al., 1973) containing 0.2% collagenase (Sigma; St Louis, MO). The dissociated oocytes were then rinsed in complete OR-2 medium with 0.1% BSA (Sigma; St Louis, MO) and filtered, sequentially, through 149 µm and 105 µm Spectra/Mesh screens (Fisher Scientific; Atlanta, GA). The cells that passed through the first filter, but were retained by the second were collected and used for extraction. Mature females served as the source of vitellogenic oocytes. The ovaries from these animals were treated with collagenase and rinsed as described above; however, the filtration step was omitted since it is estimated that vitellogenic cells make up over 90% of the tissue mass. The cells were not separated into different developmental stages (i.e. stages 2-6) since it was found that after the initiation of vitellogenesis the oocytes have similar nuclear transport properties. The subsequent extraction steps were the same for both stage-1 and vitellogenic oocytes. The procedures were modeled from those used by Adam et al. (1992) to prepare fractions that support nuclear import in permeabilized cells, and thus contain a full component of transport factors. All procedures were performed at 4°C. The oocytes were first pelleted by centrifuging at 45 g for 30 seconds, and resuspended in an equal volume of buffer containing 20 mM Hepes, pH 7.3, 110 mM potassium acetate, 5 mM sodium acetate, 2 mM magnesium acetate, 1 mM EGTA, 2 mM DTT, 1 mM PMSF and 1 µl/ml each of aprotinin, leupeptin and pepstatin. Next, the cells were lysed in a Dounce homogenizer, and centrifuged at 12,000 g for 10 minutes. The supernatents were collected, centrifuged again at 100,000 g for 1 hour, clarified with an equal volume of 1,1,2-trichlorotrifluoroethane and stored at −80°C. SDS-PAGE and immunoblotting SDS-PAGE of purified, recombinant p10 was carried out on a 15% mini-gel. 1 µg of protein was loaded, and the gel was stained with Coomassie Blue. For the oocyte extracts, 30 µg samples of each extract were analyzed on a 7.5%-15% gel, which was stained with Coomassie Blue. For western blotting, 15 µg of protein from each oocyte extract were run on a 15% SDS-PAGE mini-gel and transferred to nitrocellulose (Schleicher and Schuell; Keene, NH). After transfer, the blot was blocked with 5% dry milk (Carnation; Los Angeles, CA) in TBST (20 mM Tris, pH 7.4, 137 mM NaCl, 0.3% Tween-20) at room temperature for 1 hour. The blots were then incubated with a 1:1000 dilution of either an anti-Ran or an anti-p10 monoclonal antibody (Transduction Laboratories; Lexington, KY) in blocking solution for 2 hours at room temperature, followed by 3× 10 minute washes in
Preparation of colloidal gold Small and large gold fractions, containing particles that ranged from 20-50 and 80-280 Å in diameter, respectively, were prepared and coated with nucleoplasmin, BSA or p10, as described previously (Feldherr et al., 1984; Dworetzky et al., 1988; Frens, 1973). It is estimated that the addition of the protein coat increased the overall particle size by approximately 30 Å. All of the coated gold preparations were dialyzed against intracellular medium prior to microinjection. Microinjection, EM procedures and data analysis For microinjection experiments, stage-1 and 2 oocytes were dissected in OR-2 medium, along with their follicles, into groups of 2-5 cells. They were then treated with 0.2% collagenase in complete OR-2 medium for 30-45 minutes. (This procedure facilitated microinjection by partially digesting the follicles.) After rinsing in 3 changes of OR2, the oocytes were covered with mineral oil, and sufficient medium was removed to immobilize the cells for injection. Due to the color of the colloidal gold, the extent of the injections could be visualized, and it was estimated that the amount of material introduced into stage1 and 2 oocytes represented approximately 5% of the cell volume. The injections were performed with a Narishige micromanipulator using micropipettes with a 1-2 µm tip diameter. The stage-6 oocytes were manually defolliculated, placed in depressions in a paraffin chamber, and injected with approximately 45 nl of solution. The micropipettes used for these injections had a 10-15 µm tip diameter. At the indicated times after injection of colloidal gold, the oocytes were prepared for TEM. The procedures employed for fixation (glutaraldehyde followed by OsO4), dehydration and embedding have been described by Dworetzky et al. (1988). Sections were examined with a JEOL 100CX electron microscope. The nuclear to cytoplasmic gold ratios (N/C ratios), which are a measure of relative rates of nuclear import, were obtained by counting particles in equal and adjacent areas of nucleoplasm and cytoplasm. The functional dimensions of the transport channels, located in the centers of the pores, were determined by measuring the diameters of the NP-gold particles that entered the nucleus. The size distribution of the particles that were injected into the cytoplasm, i.e. particles available for transport, were also determined. To compensate for variations that normally occur among oocytes obtained from different animals, separate controls were performed for each experiment. The relative density of the nuclear pores, a factor that could affect nuclear transport rates, was determined by counting the pores in perpendicular sections through the nuclear envelope in stage-1 and 2 oocytes, and calculating the number of pores per unit length of envelope.
RESULTS Nuclear import of NP-gold The nuclear uptake results obtained for large (110-310 Å, total diameter) NP-gold particles in stage-1, 2 and 6 oocytes, fixed 30 minutes after injection, are shown in Table 1. Both the N/C gold ratios (relative nuclear import) and the dimensions of the particles present in the nucleoplasm (functional pore size) were significantly less in stage-1 oocytes, as compared to either stage-2 or stage-6 cells. N/C ratios were also obtained for small (50-80 Å) NP-gold in stage-1 and 2 oocytes. At 30 minutes,
1892 C. Feldherr, D. Akin and M. S. Moore Table 1. Nuclear import of large NP-gold Experiment Stage 1 (7)* Stage 2 (7) Stage 6 (7) Cytoplasmic gold size
Size distribution (%)‡
N/C ratio
P values§
No. particles measured
80-120 Å
120-160 Å
160-200 Å
200-240 Å
240+ Å
Mean ± s.e.m. (counts)
Size
N/C
2739 2906 520 428
7.1 4.8 1.1 0.9
57.3 35.7 34.5 26.6
35.0 56.8 58.5 53.0
0.7 2.7 5.9 15.0
0 0 0.1 4.4
0.77±0.15 (2244) 2.23±0.19 (2876) 1.51±0.16 (1689)
