1 Department of Biochemistry and Biophysics, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA
2 Zentrum für Molekulare Biologie der Universität Heidelberg, Im Neuenheimer Feld 282, D-69120 Heidelberg, Federal Republic of Germany
* Present address: Department of Genetics, Duke University Medical Center, Box 3657, Durham, NC 27710, USA
Author for correspondence (e-mail: refried{at}email.unc.edu)
Accepted June 18, 2001
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SUMMARY |
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Key words: Importin, Nuclear transport, Nucleolus, Signal recognition particle, SRP19
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INTRODUCTION |
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Nuclear-cytoplasmic transport is largely mediated by the importin ß family of nuclear transport receptors (Görlich and Kutay, 1999; Nakielny and Dreyfuss, 1999). These transport receptors bind cargo molecules (proteins, RNAs and RNA-protein complexes) in either the nucleus or cytoplasm, translocate with their cargo through nuclear pore complexes (NPC), release their cargo in the opposite compartment and finally return to the original compartment. All of the importin ß family members possess an N-terminal domain that binds RanGTP (Görlich et al., 1997). It is the receptors interaction with Ran, a Ras-related GTPase, that controls the binding and release of cargo. Receptors that carry cargo into the nucleus are termed importins. Importins bind their cargo in the absence of RanGTP in the cytoplasm and dissociate from cargo in the presence of RanGTP in the nucleus. Exportins such as Xpo1p/CRM1 carry cargo out of the nucleus; they have weak affinity for their cargo in the absence of RanGTP but simultaneous binding of RanGTP in the nucleus dramatically increases the affinity between exportins and their respective cargoes. The different responses to RanGTP exhibited by importins and exportins are controlled by a RanGTP gradient established across the nuclear envelope (Görlich and Kutay, 1999).
In this report, we examined the question of how mammalian SRP proteins enter the nucleus, using SRP19 as an example. SRP19 directly bound and was efficiently imported into nuclei in vitro by two members of the importin ß family of transport receptors, importin 8 and transportin. SRP19 also bound to several other members of the importin ß family of transport receptors. However, compared to transportin and importin 8, these other receptors were less efficient in promoting in vitro nuclear import of SRP19. Transportin is a well-characterized importin ß-like transport factor whose cargoes include hnRNP proteins (Pollard et al., 1996; Fridell et al., 1997; Siomi et al., 1997) and ribosomal proteins (Jäkel and Görlich, 1998). Prior to this study, no cargo had been identified for importin 8 (previously known as RanBP8) (Görlich et al., 1997). Furthermore, we found that SRP19 localizes to the nucleoli during in vitro import assays and an anti-SRP19 antibody revealed a considerable amount of endogenous SRP19 in the nucleus and the nucleolus in vivo. These findings show that for at least one mammalian SRP protein, nuclear import is an authentic process, mediated by members of the importin ß family of transport receptors. Our results add additional evidence for nuclear assembly of SRP.
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MATERIALS AND METHODS |
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Immunocytochemistry
Endogenous SRP19 in HeLa cells was detected by indirect immunofluorescence followed by confocal microscopy. HeLa cells grown on coverslips were washed briefly in phosphate-buffered saline (PBS) and fixed with 100% acetone or 4% paraformaldehyde. Paraformaldehyde-fixed cells were permeabilized with 0.2% Triton X-100 in PBS. After a blocking step, primary antibodies were applied and incubated with the fixed cells for 1 hour in a humid chamber. Following extensive washing, primary antibodies were detected using either donkey anti-rabbit (Amersham) or sheep anti-mouse (Sigma) fluorescently labeled secondary antibodies. All incubations were at room temperature.
Recombinant protein expression and purification
The following proteins were expressed in E. coli BLR/Rep4 and purified as described previously: Xenopus importin (Görlich et al., 1994); human importin ß (Görlich et al., 1996); Ran, NTF2, RanBP1 and Rna1p (Kutay et al., 1997); RanQ69L[GTP] (Görlich et al., 1997) and transportin (Izaurralde et al., 1997); importin 5 (RanBP5) (Jäkel and Görlich, 1998) and importin 7 (RanBP7) (Jäkel and Görlich, 1998). Importin 8 was expressed with an N-terminal 2z-tag (IgG-binding domain from Staphylococus aureas protein A) and a C-terminal his-tag (Görlich et al., 1997) and purified on nickel-NTA agarose (Qiagen) followed by precipitation with ammonium sulfate (33% saturation) and chromatography on Superdex 200 (Pharmacia). Human ribosomal protein L23a was expressed as a fusion with N-terminal z tags as previously described (Jäkel and Görlich, 1998); 2z-rpL23a was used in binding assays, whereas 4z-rpL23a was used as a substrate in import assays. Human SRP19 was expressed in E. coli BL21(DE3) with an N-terminal his-tag (Henry et al., 1997) or as an N-terminal his-tagged fusion to gluathione S tranferase (GST); expression vector was obtained from Young and Gautel (Young and Gautel, 2000). Human SRP19 was also expressed in E. coli BLR/Rep4 with N-terminal 2z tags and a C-terminal his-tag. Disruption of cells and purification of HIS6-SRP19, GST-SRP19, and 2z-SRP19 on nickel agarose was in the presence of 1 M lithium chloride.
Binding assays
HeLa cell extract: 2z-rpL23a and 2z-SRP19 were immobilized on IgG-sepharose 4B (Pharmacia) at approximately 2 mg/ml in 50 mM Tris-HCl, pH 7.5, 200 mM NaCl and 5 mM MgCl2. For each binding reaction, 20 µl of the affinity matrix with either pre-bound rpL23 or SRP19 was rotated for 4 hours at 4°C with 500 µl of HeLa cell extract. The beads were recovered by gentle centrifugation and washed extensively with binding buffer. Bound proteins were eluted with 1.5 M MgCl2, 50 mM Tris-HCl, pH 7.5, precipitated with 90% isopropanol (final concentration), resuspended in SDS sample buffer and analyzed by SDS PAGE.
Recombinant transport receptors: a GST-SRP19 fusion protein was bound to glutathione-sepharose (Pharmacia) for 1 hour at 4°C in binding buffer (50 mM Tris-HCl, pH 7.5, 0.1 M NaCl, 5 mM MgCl2), washed twice in the same buffer containing 1.0 M NaCl, and then equilibrated with binding buffer. Transport receptors at 1.5 µM concentration were preincubated for 30 minutes at 4°C in binding buffer containing 0.5 mM GTP, 0.5 mM ATP, 10 mM creatine phosphate and 50 µg/ml creatine kinase, with or without 10 µM RanQ69L. Transport receptor samples were then added to 20 µl of GST-SRP19 matrix and incubated for 4 hours at 4°C in binding buffer. The final concentration of GST-SRP19 per binding reaction was approximately 3 µM. The resin was washed four times with binding buffer and bound material eluted with SDS sample buffer and analyzed by SDS-PAGE.
Preparation of labeled recombinant import substrates
The preparation of fluorescent 4z-rpL23a has been described (Jäkel and Görlich, 1998). Fluorescent labeling of HIS6-SRP19 was with Alexa 594 C5 malemide (Molecular Probes) in 50 mM potassium phosphate, pH 7.2, 250 mM NaCl. Protein was separated from free label on a Sephadex G25 column (Pharmacia) equilibrated in 50 mM potassium phosphate, pH 7.2, 250 mM NaCl.
Nuclear import assays
Permeabilized HeLa cells, prepared essentially as described by Adam et al. (Adam et al., 1990), were used for in vitro nuclear import assays according to Jäkel and Görlich (Jäkel and Görlich, 1998). Import mixtures contained an energy-regenerating system consisting of the following components: 0.5 mM ATP, 0.5 mM GTP, 10 mM creatine phosphate and 50 µg/ml creatine kinase. The Ran mix constituents were 3 µM RanGDP, 0.3 µM RanBP1, 0.2 µM Schizosaccharomyces pombe Rna1p (Ran GTPase-activating protein) and 0.4 µM NTF2 (each final concentrations). Import buffers for HIS6-SRP19 and 4z-rpL23a contained 20 mM potassium phosphate, pH 7.2, 5 mM magnesium acetate, 0.5 mM EGTA and 250 mM sucrose with 140 mM potassium acetate for HIS6-SRP19 or 200 mM potassium acetate for 4z-rpL23a. Import assays were performed without addition of a cytosol extract, so that import was dependent on addition of specific transport factors and Ran.
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RESULTS |
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DISCUSSION |
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Signal recognition particle protein 19 also bound, in a RanGTP-sensitive manner, to importin ß, 5 and 7 present in a HeLa cell lysate and as purified recombinant proteins; however, these other receptors appeared to promote less nuclear import of SRP19 compared with importin 8 and transportin, as only weak nucleolar fluorescence was observed in the presence of these other receptors. Similarly, ribosomal protein L23a recovered importin 8 from the cell lysate, in a RanGTP-sensitive manner, yet importin 8 was considerably less efficient in importing rpL23a compared with other transport receptors. In yeast some ribosomal proteins are preferentially transported by one receptor but will associate with a second receptor of lower affinity when the primary receptor is absent (Rout et al., 1997). Clearly our results do not establish which transport receptor or receptors represent the principal import pathway of SRP19 in vivo. Although importin 8 and/or transportin would appear to be the most likely candidates based on efficiency, by analogy to yeast ribosomal proteins, importin ß, 5 and/or 7 may constitute backup pathways for SRP19 import. Grosshans et al. (Grosshans et al., 2001) reported that nuclear localization of Sec65p, the yeast homolog of SRP19, was greatly reduced in a mutant defective in importin , an adapter that bridges importin ß and cargo having so-called classical nuclear localization sequences. However, inclusion of importin
with importin ß in our in vitro import assays did not produce any increased nuclear import of SRP19 beyond the minimal import seen with importin ß alone. Grosshans et al. (Grosshans et al., 2001) also reported that nuclear localization of Sec65p was affected to some degree in mutants defective in several other import receptors, including Pse1p and Kap123p/Yrb4p, which are related to mammalian importin 5.
Signal recognition particle protein 19 is one of many proteins imported into the nucleus by more than one member of the importin receptor family. Most yeast SRP proteins, as well as ribosomal protein L25, are imported by Pse1p and Kap123p/Yrb4p (Grosshans et al., 2001; Rout et al., 1997; Schlenstedt et al., 1997). Kap114p, Kap121p and Kap123p are involved in import of the yeast TATA-binding protein (Pemberton et al., 1999) and these same three importins, along with Kap95p (yeast importin ß), are involved in histone H2A and H2B nuclear import (Mosammaparast et al., 2001; Mühlhäusser et al., 2001). Similarly, human rpL23a, the homologue of yeast L25, as well as human rpS7, can be imported by importin ß, transportin, importin 7 and importin 5; human rpL5 is also imported by several receptors (Jäkel and Görlich, 1998). Having multiple receptors, it will be interesting to determine which portion of SRP19 interacts with importin 8 and transportin. Although importin 8 and transportin do not share any significant similarity other than their N-terminal RanGTP-binding domains, this lack of homology does not necessarily mean that the two receptors bind different regions of SRP19. The four receptors that bind rpL23a share <15% sequence identity yet all four interact with the same segment of rpL23a, the BIB domain (residues 32-74) (Jäkel and Görlich, 1998). Also, transportin has at least two distinct, nonoverlapping cargo binding sites, one for M9-containing substrates and another for those that contain the BIB domain (Pollard et al., 1996; Jäkel and Görlich, 1998). Sequence comparisons have not revealed any significant similarity between SRP19 and either M9 or BIB although, given that SRP19 and BIB are highly basic, it seems more likely that SRP19 interacts with the BIB-binding site of transportin.
Finally, immunostaining of fixed HeLa cells revealed endogenous SRP19 not only in the cytoplasm, where mature SRP resides, but also in the nucleoplasm and the nucleolus. A previous study using another polyclonal SRP19 antibody detected SRP19 only in the cytoplasm (Bovia et al., 1995). The basis for this difference is not known, although a different fixative was used in the previous study. Our results clearly demonstrate that SRP19 is a nuclear protein in animal cells and support a model in which mammalian SRP assembles in the nucleus, as is the case for yeast (Ciufo and Brown, 2000; Grosshans et al., 2001). Politz et al. (Politz et al., 2000) also provided evidence for nuclear assembly of SRP, as they observed both nucleoplasmic and nucleolar localization of GFP fusions of SRP19, SRP68 and SRP72 in transiently transfected cells. Importantly, our immunolocalization results confirm the observations of Politz et al. in a way that is not subject to potential mislocalization arising from overexpression of proteins with high affinities for RNA. The nucleolar localization of SRP proteins is especially interesting. Using in situ hybridization, Politz et al. have reported that SRP RNA is found in the nucleolus. This observation, coupled with the nucleolar localization of SRP proteins, suggests that at least one stage in SRP assembly occurs in the nucleolus, a structure previously assumed to be host to only ribosome assembly. Precursor tRNAs, the RNA and protein subunits of RNase P, and tRNA base modification enzymes have also been recently localized in the nucleolus (Carmo-Fonseca et al., 2000; Pederson and Politz, 2000). This, in combination with our results, suggests that the nucleolus is not only the assembly site for ribosomes but is involved in assembly and processing of other RNAs and ribonucleoproteins.
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ACKNOWLEDGMENTS |
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REFERENCES |
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Adam, S. A., Sterne Marr, R. and Gerace, L. (1990). Nuclear protein import in permeabilized mammalian cells requires cytoplasmic factors. J. Cell Biol. 111, 807-816.[Abstract]
Batey, R. T., Rambo, R. P., Lucast, L., Rha, B. and Doudna, J. A. (2000). Crystal structure of the ribonucleoprotein core of the signal recognition particle. Science 287, 1232-1239.
Bischoff, F. R., Klebe, C., Kretschmer, J., Wittinghofer, A. and Ponstingl, H. (1994). RanGAP1 induces GTPase activity of nuclear ras-related Ran. Proc. Natl. Acad. Sci. USA 91, 2587-2591.[Abstract]
Bovia, F., Fornallaz, M., Lefters, H. and Strub, K. (1995). The SRP9/14 subunit of the signal recognition particle (SRP) is present in more than 20-fold excess over SRP in primate cells and exists primarily free but also in complex with small cytoplasmic Alu RNAs. Mol. Cell Biol. 6, 471-484.
Carmo-Fonseca, M., Mendes-Soares, L. and Campos, I. (2000). To be or not to be in the nucleolus. Nat. Cell Biol. 2, E107-E112.[Medline]
Ciufo, L. F. and Brown, J. D. (2000). Nuclear export of yeast signal recognition particle lacking Srp54p by the Xpo1p/Crm1p NES-dependent pathway. Curr. Biol. 10, 1256-1264.[Medline]
Fridell, R. A., Truant, R., Thorne, L., Benson, R. E. and Cullen, B. R. (1997). Nuclear import of hnRNP A1 is mediated by a novel cellular cofactor related to karyopherin-beta. J. Cell Sci. 110, 1325-1331.
Görlich, D. and Kutay, U. (1999). Transport between the cell nucleus and the cytoplasm. Annu. Rev. Cell Dev. Biol. 15, 607-660.[Medline]
Görlich, D., Prehn, S., Hartmann, E., Herz, J., Otto, A., Kraft, R., Wiedmann, M., Knespel, S., Dobberstein, B. and Rapoport, T. A. (1990). The signal sequence receptor has a second subunit and is part of a translocation complex in the endoplasmic reticulum as probed by bifunctional reagents. J. Cell Biol. 111, 2283-2294.[Abstract]
Görlich, D., Prehn, S., Laskey, R. A. and Hartmann, E. (1994). Isolation of a protein that is essential for the first step of nuclear protein import. Cell 79, 767-778.[Medline]
Görlich, D., Vogel, F., Mills, A. D., Hartmann, E. and Laskey, R. A. (1995). Distinct functions for the two importin subunits in nuclear protein import. Nature 377, 246-248.[Medline]
Görlich, D., Pante, N., Kutay, U., Aebi, U. and Bischoff, F. R. (1996). Identification of different roles for RanGDP and RanGTP in nuclear protein import. EMBO J. 15, 5584-5594.[Abstract]
Görlich, D., Dabrowski, M., Bischoff, F. R., Kutay, U., Bork, P., Hartmann, E., Prehn, S. and Izaurralde, E. (1997). A novel class of RanGTP binding proteins. J. Cell Biol. 138, 65-80.
Grosshans, H., Deinert, K., Hurt, E. and Simos, G. (2001). Biogenesis of the signal recognition particle (SRP) involves import of SRP proteins into the nucleolus, assembly with SRP-RNA, and Xpo1p-mediated export. J. Cell Biol. 153, 745-761.
He, X. P., Bataillé, N. and Fried, H. M. (1994). Nuclear export of signal recognition particle RNA is a facilitated process that involves the Alu sequence domain. J. Cell Sci. 107, 903-912.
Henry, K. A., Zwieb, C. and Fried, H. M. (1997). Purification and biochemical characterization of the 19-kDa signal recognition particle RNA-binding protein expressed as a hexahistidine-tagged polypeptide in Escherichia coli. Protein Expres. Purif. 9, 15-26.[Medline]
Izaurralde, E., Kutay, U., von Kobbe, C., Mattaj, I. W. and Görlich, D. (1997). The asymmetric distribution of the constituents of the Ran system is essential for transport into and out of the nucleus. EMBO J. 16, 6535-6547.
Jäkel, S. and Görlich, D. (1998). Importin beta, transportin, RanBP5 and RanBP7 mediate nuclear import of ribosomal proteins in mammalian cells. EMBO J. 17, 4491-4502.
Kutay, U., Izaurralde, E., Bischoff, F. R., Mattaj, I. W. and Görlich, D. (1997). Dominant-negative mutants of importin-beta block multiple pathways of import and export through the nuclear pore complex. EMBO J. 16, 1153-1163.
Lingelbach, K., Zwieb, C., Webb, J. R., Marshallsay, C., Hoben, P. J., Walter, P. and Dobberstein, B. (1988). Isolation and characterization of a cDNA clone encoding the 19 kDa protein of signal recognition particle (SRP): expression and binding to 7SL RNA. Nucleic Acids Res. 16, 9431-9442.[Abstract]
Luirink, J. and Dobberstein, B. (1994). Mammalian and Escherichia coli signal recognition particles. Mol. Microbiol. 11, 9-13.[Medline]
Lütcke, H. 1995. Signal recognition particle (SRP), a ubiquitous initiator of protein translocation. Eur. J. Biochem. 228, 531-550.[Abstract]
Maxwell, E. S. and Fournier, M. J. (1995). The small nucleolar RNAs. Annu. Rev. Biochem. 35, 897-934.
Mosammaparast, N., Jackson, K. R., Guo, Y., Brame, C. J., Shabanowitz, J., Hunt, D. F. and Pemberton, L. F. (2001). Nuclear import of histone H2A and H2B is mediated by a network of karyopherins. J. Cell Biol. 153, 251-262.
Mühlhäusser, P., Müller, E.-C., Otto, A. and Kutay, U. (2001). Multiple pathways contribute to nuclear import of core histones. EMBO Rep. 2, 690-696.
Nakielny, S. and Dreyfuss, G. (1999). Transport of proteins and RNAs in and out of the nucleus. Cell 99, 677-690.[Medline]
Pederson, T. and Politz, J. C. (2000). The nucleolus and the four ribonucleoproteins of translation. J. Cell Biol. 148, 1091-1095.
Peluso, P., Herschlag, D., Nock, S., Freymann, D. M., Johnson, A. E. and Walter, P. (2000). Role of 4.5S RNA in assembly of the bacterial signal recognition particle with its receptor. Science 288, 1640-1643.
Pemberton, L. F., Rosenblum, J. S. and Blobel, G. (1999). Nuclear import of the TATA-binding protein: mediation by the karyopherin Kap114p and a possible mechanism for intranuclear targeting. J. Cell Biol. 145, 1407-1417.
Politz, J. C., Yarovoi, S., Kilroy, S. M., Gowda, K., Zwieb, C. and Pederson, T. (2000). Signal recognition particle components in the nucleolus. Proc. Natl. Acad. Sci. USA 97, 55-60.
Pollard, V. W., Michael, W. M., Nakielny, S., Siomi, M. C., Wang, F. and Dreyfuss, G. (1996). A novel receptor-mediated nuclear protein import pathway. Cell 86, 985-994.[Medline]
Rout, M. P., Blobel, G. and Aitchison, J. D. (1997). A distinct nuclear import pathway used by ribosomal proteins. Cell 89, 715-725.[Medline]
Schlenstedt, G., Smirnova, E., Deane, R., Solsbacher, J., Kutay, U., Görlich, D., Ponstingl, H. and Bischoff, F. R. (1997). Yrb4p, a yeast Ran-GTP-binding protein involved in import of ribosomal protein L25 into the nucleus. EMBO J. 16, 6237-6249.
Siomi, M. C., Eder, P. S., Kataoka, N., Wan, L., Liu, Q. and Dreyfuss, G. (1997). Transportin-mediated nuclear import of heterogeneous nuclear RNP proteins. J. Cell Biol. 138, 1181-1192.
Yoneda, Y., Hieda, M., Nagoshi, E. and Miyamoto, Y. (1999). Nucleocytoplasmic protein transport and recycling of Ran. Cell Struct. Funct. 24, 425-433.[Medline]
Young, P. and Gautel, M. (2000). The interaction of titin and -actinin is controlled by a phospholipid-regulated intramolecular pseudoligand mechanism. EMBO J. 19, 6331-6340.