Signal-mediated nuclear transport in the amoeba

Transcription

1 Journal of Cell Science 112, (1999) Printed in Great Britain The Company of Biologists Limited 1999 JCS Signal-mediated nuclear transport in the amoeba Carl M. Feldherr* and Debra Akin Dept of Anatomy and Cell Biology, University of Florida, College of Medicine, Gainesville, FL 32610, USA *Author for correspondence ( Accepted 20 April; published on WWW 26 May 1999 SUMMARY The evolutionary changes that occur in signal-mediated nuclear transport would be expected to reflect an increasing need to regulate nucleocytoplasmic exchanges as the complexity of organisms increases. This could involve changes in both the composition and structure of the pore complex, as well as the cytosolic factors that mediate transport. In this regard, we investigated the transport process in amoebae (Amoeba proteus and Chaos carolinensis), primitive cells that would be expected to have less stringent regulatory requirements than more complex organisms. Colloidal gold particles, coated with bovine serum albumin (BSA) conjugated with simple (large T) nuclear localization signals (NLSs), bipartite (nucleoplasmin) NLSs or mutant NLSs, were used to assay nuclear import. It was found that in amoebae (1) the diameter of the particles that are able to enter the nucleoplasm is significantly less than in vertebrate cells, (2) the simple NLS is more effective in mediating nuclear import than the bipartite NLS, and (3) the nucleoporins do not appear to be glycosylated. Evidence was also obtained suggesting that, in amoebae, the simple NLS can mediate nuclear export. Key words: Nuclear transport, Pore complex, Amoeba INTRODUCTION The signal-mediated import of macromolecules into the nucleoplasm in vertebrate cells occurs through the nuclear pore complex, a supramolecular structure that has a molecular mass of approximately 125 MDa, and is made up of different proteins, the nucleoporins. Many, but not all of the nucleoporins contain FG repeat motifs, and/or O-linked GlcNAc moieties. Import is a multistep process that is initiated by a nuclear localization signal (NLS). The most thoroughly studied signals are the simple NLS, a single short basic amino acid domain, and the bipartite NLS, two basic regions separated by an amino acid spacer. These NLSs were first identified in SV40 large T antigen (Kalderon and Smith, 1984) and nucleoplasmin (Robbins et al., 1991), respectively, and are commonly found in nuclear proteins (Richter and Standiford, 1992). Both signals utilize the same nuclear import pathway. They initially bind to the cytoplasmic adapter importin α (also referred to as karyopherin α), and importin β (or karyopherin β), the receptor responsible for docking the transport complex to the cytoplasmic face of the pores (Gorlich, 1998). There are also alternative pathways for nuclear import; for example, RNP proteins (Siomi et al., 1997) and ribosomal proteins (Rout et al., 1997; Jakel and Gorlich, 1998) not only contain different NLSs, but utilize different receptors for docking. The existence of different pathways could allow the transport of specific functional classes of proteins to be controlled independently. Following the docking step, translocation occurs through a gated, central transporter element, which allows the passage of particles up to 240 Å in diameter. By varying the degree of dilation of the central transport channel, the size of particles entering and leaving the nucleus can be controlled (Feldherr and Akin, 1995b). This could be especially important in regulating the exchange of large RNP particles (Feldherr and Akin, 1991). The translocation step involves at least two factors: Ran, a 24 kda GTPase, and p10/ntf2, a 14 kda polypeptide (Gorlich, 1997; Moore, 1998). Ran, which is mainly in the GDP form in the cytoplasm and the GTP form in the nucleoplasm, provides directionality to the transport process. There is evidence that p10/ntf2 mediates the nuclear uptake of Ran (Ribbeck et al., 1998), and might also regulate dilation of the central transport channel (Feldherr et al., 1998). In addition to studies on vertebrate cells, there is a considerable amount of data regarding the transport apparatus in yeast. The nuclear pore complex in these cells has an estimated mass of 66 MDa (Yang et al., 1998), approximately half that of vertebrate cells. Technically, it has not yet been possible to measure the functional size of the pores in yeast, although morphological studies indicate the presence of a central transporter that has a diameter of approximately 350 Å, which is comparable to that in vertebrate cells (Yang et al., 1998). The yeast nucleoporins also have FG repeat motifs, but they are not glycosylated, and show limited homology with vertebrate nucleoporins (Fabre and Hurt, 1997). The NLS receptors in yeast and vertebrate cells show a greater degree of homology and, in addition, yeast contain homologs of Ran and p10/ntf2 (Corbett and Silver, 1997). The similarities in signal receptors and translocation factors suggest that the transport pathways are highly conserved. The differences in the pore complexes, on the other hand, could

2 2044 C. M. Feldherr and D. Akin mean that vertebrate cells have additional regulatory requirements that necessitate modifications in organization and composition of these structures. In this study, signal-mediated nuclear transport was investigated in amoebae. Assuming that the complexity of the nuclear transport machinery is related to the level of regulation required for cell function, it would be expected that amoebae would have a more primitive transport system than that found in higher organisms. If this assumption is correct, comparative studies should not only provide a better understanding of the evolution of the nucleocytoplasmic exchange process, but might also provide clues regarding the functional role of the different elements of the transport apparatus. Previous studies relating to nuclear transport in amoebae focused on passive diffusion across the nuclear envelope. A comparison of the data obtained for amoebae, in which polyvinylpyrrolidone-coated gold particles were used as a transport substrate (Feldherr, 1965), with diffusion studies performed on other cell types (Peters, 1986; Paine, 1992), clearly indicate that both the rate of diffusion and the dimensions of the diffusion channel are greater in amoebae than in vertebrate cells. The present data show that there are also significant differences in signal-mediated transport across the envelope. In amoebae, the transport channels have a smaller diameter, bipartite NLS is less effective in facilitating nuclear import, and the nucleoporins do not appear to be glycosylated. MATERIALS AND METHODS Cell cultures Amoeba proteus and the multinucleated amoeba Chaos carolinensis were obtained from Carolina Biological Supply (Burlington, NC). The cells were cultured at room temperature (21 C) in a medium containing 0.5 mm CaCl 2, 0.05 mm MgCl 2, 0.16 mm K 2HPO 4, 0.12 mm KH 2PO 4 (ph ), and were fed Tetrahymena pyriformis (Prescott and James, 1955). Preparation of BSA conjugates The following peptides were synthesized by the University of Florida protein core facility: CGGGPKKKRKVGG; CGGGAVKRPAATKKAGQAKKKKLNGG; CGGGPKNKRKVGG; CGGGVKRKKKPGG. They contain, respectively, the SV40 large T NLS (a simple signal), the nucleoplasmin NLS (a bipartite signal), an inactive mutant form of the large T NLS (ct mutant; Lanford and Butel, 1984) and the reverse large T NLS. The signal domains are underlined. The synthetic peptides were conjugated to bovine serum albumin (BSA; Sigma, St Louis, MO) using the cross-linker m- maleimidobenzoyl-n-hydroxysuccinimide ester (Pierce, Rockford, IL), as outlined by Lanford et al. (1986). Based on SDS-PAGE analysis, it was estimated that an average of 8 peptides were conjugated to each BSA molecule. Fluorescent conjugates were prepared as described above, except that the BSA was labeled with fluorescein before it was conjugated with the signal peptides. Fluorescent labeling was accomplished by treating 10 mg/ml BSA in 100 mm carbonate buffer (ph 9.0) for 1 hour with 2.25 mm fluorescein isothiocyanate (Pierce; Rockford, IL) dissolved in 60 µl dimethylformamide (Pierce; Rockford, IL). The labeled protein was purified by gel filtration. Preparation of colloidal gold Colloidal particles, ranging in diameter from 20 to 180 Å, were prepared by reducing chloroauric acid with a saturated solution of phosphorous in ether (Feldherr, 1965). A second fraction, containing particles up to 290 Å in diameter, was prepared by using sodium citrate as a reducing agent (Frens, 1973). These fractions, designated intermediate and large, respectively, were coated with BSA conjugates as described by Dworetzky et al. (1988). It is estimated that the protein coat increases the diameter of the particles by approximately 30 Å. Prior to microinjection, the coated gold preparations were concentrated about 200-fold using Centriprep and Microcon concentrators (Amicon, Inc., Beverly, MA), and dialyzed against amoebae medium. Microinjection, EM, and fluorescent analysis Microinjections were performed at room temperature using an inverted microscope and a hydraulic micromanipulator (Narishige USA, Inc., Greenvale, NY). The amoebae were immobilized in an oil chamber and injected with pipettes that had tip diameters of about 2 µm. Approximately 5-10% of the cell volume was injected. For EM analysis, the amoebae were fixed in 4% glutaraldehyde for 30 minutes, postfixed in 2% osmium tetroxide for 30 minutes, embedded in Spurr s resin, sectioned and examined with a JEOL 100CX electron microscope. Kinetic analysis of the nuclear import of the fluorescein-labeled conjugates was performed using an intensified Hamamatsu CCD camera and a MetaMorph imaging system (Universal Imaging Corporation, West Chester, PA). At the specified times after injection, images were acquired and N/C fluorescent ratios were determined by analyzing equal areas of nucleoplasm and cytoplasm. To minimize the effects of UV on the cells, each exposure was limited to less than 1 second, and a number 16 neutral density filter was used. RESULTS To monitor nuclear transport, different size gold particles coated with BSA conjugated to either simple, bipartite or mutant NLSs were microinjected into the cytoplasm of amoebae, and their subsequent nucleocytoplasmic distribution was analyzed by electron microscopy. The relative rates of nuclear import are expressed as nuclear/cytoplasmic (N/C) gold ratios, which were determined by counting particles in equal, randomly selected areas of nucleoplasm and cytoplasm. The functional size of the transport channel was established by measuring the diameters of the particles that entered the nucleus. Table 1 shows the nucleocytoplasmic distribution obtained in A. proteus for Å gold particles coated with BSA conjugates. The large T antigen NLS (Table 1A) facilitated the transport of gold into the nucleoplasm. The reached a value of 3.37 at 20 hours; however, there was restricted import of particles larger than 100 Å in diameter (not including the protein coat) and, as a consequence, the overall size distribution of the particles present in the nucleoplasm was significantly less than in the cytoplasm (Table 1E; P< ; chi square test). Table 1B shows that conjugates containing the bipartite, nucleoplasmin NLS had only a limited capacity to facilitate nuclear import. No uptake was detected at 30 minutes, and the s at 90 minutes and 20 hours were approximately 47 and 5.5 times lower, respectively, than those obtained for the large T NLS conjugates. In addition, the size distribution of the particles in the nucleoplasm was significantly less than that observed for the simple NLS (P< ). Gold particles coated with the ct mutant (Table 1C) or reverse large T NLS conjugates (Table 1D) were excluded from the nucleus. To verify that there was limited nuclear uptake of particles over 100 Å in diameter, experiments were performed using a gold fraction in which the majority of the particles (>96%) had diameters exceeding 120 Å. Fractions of this size (approximately Å in diameter) have been used previously to investigate

3 Nuclear transport in the amoeba 2045 Table 1. Nuclear import in Amoeba proteus A BSA-large T NLS 30 min (6)* (496) 90 min (6) (285) 20 hours (7) (491) B BSA-nucleoplasmin NLS 30 min (6) 0.00 (233) 90 min (7) 0.06 (222) 20 hours (6) (232) C BSA-cT mutant NLS 30 min (6) 0.00 (260) 90 min (7) 0.00 (231) 20 hours (7) (195) D BSA-reverse large T NLS 30 min (4) 0.00 (460) 90 min (2) 0.00 (202) 20 hours (4) 0.00 (427) E Cytoplasmic gold the properties of the nuclear pores (Feldherr and Akin, 1995b). In vertebrate cells, the N/C gold ratios obtained for these fractions varied from about 1-3, and the largest particles able to enter the nuclei were approximately Å in diameter (not including the protein coat). Furthermore, under normal growth conditions, the size distribution of particles smaller than 200 Å was essentially the same in both the nucleus and cytoplasm, demonstrating that particles in this size range readily enter the nucleoplasm. When large gold particles coated with BSA-large T NLSs were microinjected into A. proteus, a very different nucleocytoplasmic distribution pattern was observed (see Table 2). The was 0.05, demonstrating that the large particles were essentially excluded from the nucleus. Furthermore, measurements of the few particles that did enter the nucleoplasm showed that only 40% were larger than 100 Å, whereas 98% of the cytoplasmic particles (gold available for transport) were in this size category. These results provide convincing evidence that the functional size of the nuclear transport channel in amoebae is significantly smaller than in vertebrate cells. The nuclear import data obtained for C. carolinensis, with regard to both s and size distribution, were similar to those described for A. proteus. In short-term experiments (30 minutes), there was facilitated uptake of gold particles coated with BSA-large T NLSs (Table 3A). The nucleoplasmin signal was much less effective in mediating transport (Table 3B), and both the ct mutant (Table 3C) and reverse signals (Table 3D) were excluded from the nucleus. The size distribution of particles in the cytoplasm are shown in Table 3E. The main difference in the C. carolinensis results was the finding that essentially no gold particles coated with BSA-large T NLS conjugates were present in either the nucleus or cytoplasm in 20 hour experiments (Table 3A). Electron microscopic analysis of the amoebae revealed that particles coated with BSA-large T NLSs, but not other signals, are sequestered from the cytoplasm into food vacuoles (Fig. 1A-D). The fact that the gold count in the nucleoplasm also drops, demonstrates that particles are able to reenter the cytoplasm. Since the effectiveness of a specific NLS could be a function of the molecular size of the transport substrate, differences in NLS activity were also investigated using fluorescein-labeled BSA conjugates. The nuclear import kinetics of conjugates containing either large T antigen, nucleoplasmin or ct mutant NLSs, as well as labeled BSA alone, were determined in A. proteus by measuring the N/C fluorescence ratio at intervals of 5, 10, 15, 30 and 60 minutes after injection. The results are given in Fig. 2. Tracers containing the large T antigen or the nucleoplasmin NLS concentrated in the nuclei during the course of the experiments; however, the large T conjugates were transported at a significantly greater rate (P<0.0001, ANOVA). Interestingly, the nucleoplasmin signal was more effective in Table 2. Nuclear import of large gold particles in Amoeba proteus Experiment measured < mean (counts) BSA-large T NLS 90 min (4)* (1017) Cytoplasmic gold

4 2046 C. M. Feldherr and D. Akin Fig. 1. Electron micrographs showing the fate of gold particles coated with BSA conjugates following injection into the cytoplasm of C. carolinensis. At 30 minutes, gold particles (arrowheads) coated with BSA-large T NLSs were present in both the cytoplasm and the nucleoplasm (A). By 20 hours, the BSA-large T NLS-gold (arrowhead) was found in food vacuoles (B), but was no longer present in either the cytoplasm or nucleoplasm (C). Gold particles coated with BSA-cT mutant signals were still located in the cytoplasm after 20 hours (D). c, cytoplasm; n, nucleus; fv, food vacuole. mediating the transport of fluorescent conjugates than the larger colloidal gold particles. The BSA-cT mutant NLS, and BSA alone showed low levels of nuclear associated fluorescence. In both instances, the s were less than 1, and were not significantly different. Since these ratios did not increase with time, it is most likely that the nuclear fluorescence observed for the two substrates that lacked an active NLS actually represents background from overlapping and underlying cytoplasm. The effect of wheat germ agglutinin (WGA) on nuclear import in A. proteus is shown in Table 4. WGA, which binds GlcNAc, blocks signal-mediated transport in vertebrate cells when injected at a concentration of 3 mg/ml (Daubauvalle et al., 1988). In amoebae, the same concentration of WGA had no effect on the nuclear uptake of gold particles coated with BSA-large T NLS conjugates. Both the s and the size of the particles that entered the nucleus were not significantly different from controls. In a positive control, performed on 3T3-BALB/c cells, 3 mg/ml WGA completely blocked the nuclear import of gold (data not shown). As is the case in vertebrate cells, nuclear transport in Table 3. Nuclear import in Chaos carolinensis A BSA-large T NLS 30 min (5)* (359) 20 hours (6) 0.00 (0) B BSA-nucleoplasm NLS 30 min (6) 0.01 (126) 20 hours (5) (150) C BSA-cT mutant NLS 30 min (6) 0.00 (165) 20 hours (5) 0.00 (148) D BSA-reverse large T NLS 30 min (4) 0.00 (324) 20 hours (4) (958) E Cytoplasmic gold

5 Nuclear transport in the amoeba 2047 Fig. 2. The nuclear import rates of fluorescein-labeled BSA alone ( ), and labeled BSA conjugated with either large T antigen ( ), nucleoplasmin ( ) or ct mutant ( ) NLSs. Respectively, 5, 6, 7 and 5 cells were analyzed for the different transport substrates. Bars indicate s.e.m. amoebae is temperature-dependent (Table 5). A decrease in temperature from 21 C to 4 C prevented transport of gold particles coated with the BSA-large T NLS conjugate. DISCUSSION It was found that (1) the size limit for signal-mediated transport into the nucleus is significantly lower in amoebae than in vertebrate cells; (2) both the simple and bipartite NLSs mediated nuclear transport, but the simple signal was significantly more effective; (3) WGA had no effect on nuclear import, indicating that amoeba nucleoporins are not glycosylated; and (4) signalmediated import is temperature-dependent. Results were also obtained suggesting that, in amoebae, the simple NLS can also function in mediating nuclear export. Differences in functional pore size could be related to the way in which specific cells process and transport ribosomal subunits (presumably the largest particles that cross the nuclear envelope). There is evidence that nascent ribosomal subunits in amoebae are contained in extended helical structures. These structures can range from Å in width, and up to 7000 Å in length; however, they are formed by the supercoiling of filaments that are only Å in diameter (Stevens, 1967). Furthermore, there is morphological data suggesting that the filaments exit the nucleus through the pores and dissociate once in the cytoplasm (Stevens and Prescott, 1965). Thus, it is likely that a functional pore diameter, of the order of 130 Å, is sufficient to satisfy the transport requirements in amoebae, but would not be adequate in vertebrate cells, where the ribosomal subunits that exit the nucleus are much larger. Although the simple and bipartite NLSs are both recognized by the transport machinery in amoebae, the bipartite signal was significantly less effective in the transport of both fluoresceinlabeled BSA conjugates and colloidal gold particles; furthermore, its relative activity was found to vary inversely in relation to the size of the transport substrate. The fact that the bipartite NLS is more active in vertebrate cells could be a response to more complex regulatory requirements in higher organisms, and might be related to evolutionary changes in importin α. Recent analysis of the crystal structure of importin α (Conti et al., 1998) demonstrated that it contains two NLS binding sites. One site can accommodate up to five lysine and/or arginine residues, whereas the second, smaller site is able to bind only two basic residues. Furthermore, the positioning of the two binding sites is ideally suited for interactions with bipartite signals. If amoebae contain a homolog of importin α that lacks the smaller NLS binding domain, it would still be effective in transporting simple signals but, due to lower binding affinity, would be less active in mediating the transport of bipartite signals. A number of vertebrate nucleoporins contain O-linked GlcNAc (Pante and Aebi, 1996). Consistent with this finding, WGA, or antibodies against epitopes that include O-linked GlcNAc, bind to the pore complex and block signal-mediated Table 4. The effect of 3 mg/ml WGA on nuclear transport in Amoeba proteus BSA-large T NLS+WGA (9)* (422) BSA-large T NLS (9) (314) Cytoplasmic gold Table 5. The effect of temperature on nuclear transport in Amoeba proteus BSA-large T NLS+4 C (6)* (517) BSA+4 C (5) (357) BSA-large T NLS+21 C (8) (1134) BSA+21 C (8) (423) Cytoplasmic gold

6 2048 C. M. Feldherr and D. Akin transport at the translocation step. Based on the apparent absence of glycoproteins in yeast and amoeba nucleoporins, it is unlikely that GlcNAc is essential for translocation. Evidence favoring a regulatory role for GlcNAc in higher organisms has recently been reviewed by Snow and Hart (1998). GlcNAc moieties can be readily added or removed from proteins, a dynamic feature that would facilitate regulatory activities. Furthermore, there are indications that O-linked glycosylation and phosphorylation can occur at the same sites on proteins. Since there is evidence that phosphorylation is involved in nuclear import (Vandromme et al., 1994; Feldherr and Akin, 1995a; Mishra and Parnaik, 1995), GlcNAc could affect transport by modulating the levels of nucleoporin phosphorylation. The sequestration of gold particles coated with active, but not mutated NLSs into the food vacuoles of C. carolinensis not only suggests a possible method of regulating the intracellular levels of nuclear proteins in these primitive organisms, but also represents a fortuitous experimental system for selectively reducing the cytoplasmic concentration of nuclear import substrates. In this study, when gold particles coated with BSAlarge T NLS conjugates were sequestered from the cytoplasm, there was an accompanying loss of particles from the nucleus, demonstrating that transport is reversible. Since the gold particles used in these experiments are too large to diffuse through the pores, as indicated by the fact that particles of the same size coated with mutant signals are unable to enter the nucleus (see Table 1C,D), the possibility exists that, in amoebae, the simple NLS can also function as an export signal. This dual role for the NLS is consistent with a facilitated transport mechanism (Paine, 1993). The data obtained in this report demonstrate that there are significant differences in nuclear transport capacity in organisms that presumably have different regulatory requirements for nucleocytolasmic exchanges. In future experiments it will be important to correlate these changes with specific cellular activities; for example, the relationship between functional pore size and ribosome production. From an evolutionary standpoint, it is also important to understand the differences that exist at the molecular level. 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