Kinetic Characterization of the Human Retinoblastoma Protein Bipartite Nuclear Localization Sequence (NLS) in Vivo and in Vitro
A COMPARISON WITH THE SV40 LARGE T-ANTIGEN NLS*

(Received for publication, March 6, 1997, and in revised form, June 20, 1997)

Athina Efthymiadis , Huimin Shao , Stefan Hübner and David A. Jans Dagger

From the Nuclear Signaling Laboratory, Division of Biochemistry and Molecular Biology, John Curtin School of Medical Research, Canberra City, A.C.T. 2601, Australia

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES


ABSTRACT

The retinoblastoma (RB) tumor suppressor is a nuclear phosphoprotein important for cell growth control and able to bind specifically to viral oncoproteins such as the SV40 large tumor antigen (T-ag). Human RB possesses a bipartite nuclear localization sequence (NLS) consisting of two clusters of basic amino acids within amino acids 860-877, also present in mouse and Xenopus homologs, which resembles that of nucleoplasmin. The T-ag NLS represents a different type of NLS, consisting of only one stretch of basic amino acids. To compare the nuclear import kinetics conferred by the bipartite NLS of RB to those conferred by the T-ag NLS, we used beta -galactosidase fusion proteins containing the NLSs of either RB or T-ag. The RB NLS was able to target beta -galactosidase to the nucleus both in vivo (in microinjected cells of the HTC rat hepatoma line) and in vitro (in mechanically perforated HTC cells). Mutational substitution of the proximal basic residues of the NLS abolished nuclear targeting activity, confirming its bipartite character. Nuclear accumulation of the RB fusion protein was half-maximal within about 8 min in vivo, maximal levels being between 3-4-fold those in the cytoplasm, which was less than 50% of the maximal levels attained by the T-ag fusion protein, while the initial rate of nuclear import of the RB protein was also less than half that of T-ag. Nuclear import conferred by both NLSs in vitro was dependent on cytosol and ATP and inhibited by the nonhydrolyzable GTP analog GTPgamma S. Using an ELISA-based binding assay, we determined that the RB bipartite NLS had severely reduced affinity, compared with the T-ag NLS, for the high affinity heterodimeric NLS-binding protein complex importin 58/97, this difference presumably representing the basis of the reduced maximal nuclear accumulation and import rate in vivo. The results support the hypothesis that the affinity of NLS recognition by NLS-binding proteins is critical in determining the kinetics of nuclear protein import.


INTRODUCTION

All passive and active transport into and out of the nucleus occurs through the nuclear pore complex (NPC)1 (1-3). Proteins larger than 45 kDa require a nuclear localization sequence (NLS) (4) to be targeted to the nucleus. NLSs are defined as the sequences sufficient and necessary for nuclear import of their respective proteins (5-7) and are generally functional in targeting heterologous, normally cytoplasmic proteins to the nucleus. NLS-dependent protein transport can be divided into two steps. The first is energy-independent and involves recognition and targeting of the NLS-containing protein to the NPC by a heterodimeric protein complex consisting of importin 58/97 (8, 9) or alpha /beta (10, 11). The second, energy-dependent step is the translocation of the NLS-bearing protein through the NPC into the nucleus (12, 13) and requires GTP hydrolysis mediated by the GTP-binding protein Ran/TC4 (14-16) and the interacting factor p10/NTF2 (17-19).

We were interested in the nuclear import kinetics of tumor suppressor proteins, one of which is the retinoblastoma protein (p110Rb or RB). RB is frequently altered or deleted in a number of tumors and tumor cell lines, including those derived from retinoblastomas, osteosarcomas, breast cancers and prostate, bladder, and small lung carcinomas (20). It is nuclear (21) with a molecular mass of 105-115 kDa, depending on its phosphorylation state (20). Human RB contains a bipartite NLS (amino acids 860-877), conserved in mammalian and amphibian RB proteins (Refs. 22 and 23 and see Table I). Of the two basic types of NLS, bipartite NLSs comprise two clusters of basic amino acids separated by a 10-12-amino acid spacer resembling the NLS of the Xenopus laevis nuclear phosphoprotein nucleoplasmin (Ref. 24 and see Table I), whereas the other type consists of a single short basic amino acid sequence resembling the T-ag NLS (PKKKRKV132; Ref. 5).

Table I. Bipartite NLSs in human, mouse, and Xenopus RB, compared to those of nucleoplasmin, N1N2, IL-5, and SWI5


Protein Bipartite NLSa

Nucleoplasmin (24)   KRPAATKKAGQAKKKKLDK174
N1N2 (25) RKKRKTEEESPLKDKAKKSK554
SWI5 (26)   KKYENVVIKRSPRKRGRPRK655
Human IL-5 (27)   KKYIDGQKKKCGEERRRVNQ114
Human RB   KRSAEGSNPPKPLKKLR877
Mouse RB   KRSAEGPNPPKPLKNVR870
Xenopus RB   KRSADTGTTPKLPKKLR840
Consensus   KK- 10-12 aa -KKK
  RR            RRR

a The single-letter amino acid code is used; bold letters indicate the two arms of basic residues of the bipartite NLS. aa, amino acids.

This study examines the nuclear import kinetics of fusion proteins carrying the RB NLS in vivo and in vitro at the single cell level and compares results to those for proteins carrying the T-ag NLS. We assess the dependence of nuclear import on cytosolic factors, ATP, and GTPase activity and use an ELISA-based binding assay to determine the binding affinity for the NLS-binding heterodimeric protein complex importin 58/97. We find that the bipartite NLS of RB is much less efficient than the T-ag NLS in terms of both the maximal level and rate of nuclear import, as well as exhibiting a much lower affinity for importin. The results are consistent with the tenet that the affinity of NLS binding by importin is critical in determining the kinetics of nuclear protein import.


MATERIALS AND METHODS

Chemicals and Reagents

Isopropyl-beta -D-thiogalactopyranoside and the detergent CHAPS were from Boehringer Mannheim, and the sulfhydryl labeling reagent 5-iodoacetamidofluorescein was from Molecular Probes. Other reagents were from the sources described previously (26, 28-30).

Cell Culture

Cells of the HTC rat hepatoma tissue culture (a derivative of Morris hepatoma 7288C) line were cultured as described previously (26, 31, 32).

beta -Galactosidase Fusion Proteins

Plasmids expressing the RB-Bip-beta -galactosidase (RB-Bip-beta -Gal) and RB-BipMut-beta -galactosidase (RB-BipMut-beta -Gal) fusion proteins were derived by oligonucleotide insertion into the SmaI restriction endonuclease site of the plasmid vector pPR2 (28). The resultant fusion proteins express RB amino acids 860-877 fused NH2-terminal to the Escherichia coli beta -galactosidase enzyme sequence (amino acids 9-1023), where RB-Bip-beta -Gal retains the wild type sequence, and the NLS mutated derivative RB-BipMut-beta -Gal possesses Thr-Thr in place of the wild type amino acids Lys-Arg861. The T-ag beta -galactosidase fusion protein (T-ag-CcN-beta -Gal) used in the comparative studies contains T-ag amino acids 111-135, including the CcN motif (comprising protein kinase CK2 and cyclin-dependent kinase phosphorylation sites and the NLS) fused amino-terminal to beta -galactosidase amino acids 9-1023 (31, 32).

1 mM isopropyl-beta -D-thiogalactopyranoside was used to induce expression of beta -galactosidase fusion proteins in E. coli. They were purified by affinity chromatography and labeled with 5-iodoacetamidofluorescein as described previously (31, 32).

Nuclear Import Kinetics

Analysis of nuclear import kinetics at the single cell level was performed using either microinjected (in vivo) or mechanically perforated (in vitro) HTC cells in conjunction with confocal laser scanning microscopy (CLSM) (26-33). In the case of microinjection, HTC cells were fused with polyethylene glycol about 1 h prior to microinjection to produce polykaryons (26, 27, 30). Reticulocyte lysate (Promega) was used as the source of cytosol for the in vitro assay (27, 29, 33). Image analysis of CLSM files using the NIH Image public domain software and curve fitting were performed as described (26, 33).

In in vitro experiments where the ATP dependence of transport was tested, apyrase pretreatment was used to hydrolyze endogenous ATP in cytosolic extracts (10 min at room temperature with 800 units/ml) and perforated cells (15 min at 37 °C with 0.2 units/ml) (12, 33), and transport assays were then performed in the absence of the ATP-regenerating system (31, 33), which was otherwise used. In experiments where the dependence of transport on the GTP-binding protein Ran/TC4 (34) was tested, cytosolic extract was treated with 850 µM GTPgamma S (nonhydrolyzable GTP analog) for 5 min at room temperature, prior to use in the in vitro assay (final GTPgamma S concentration of 300 µM).

Nuclear accumulation was also examined in vitro in the presence of a <FR><NU>1</NU><DE>10</DE></FR> volume of 20 mM Tris (pH 7.0) containing 10% glycerol and 0.25% CHAPS, which results in permeabilization of the nuclear envelope; accumulation under these conditions only results from binding to nuclear components such as lamins, chromatin, etc.

ELISA-based Binding Assay

An ELISA-based binding assay was used to examine the binding affinity between importin subunits (mouse importin 58 and 97 glutathione S-transferase (GST) fusion proteins expressed as described (8, 9, 30)) and RB or T-ag fusion proteins (30). Briefly, 96-well microtiter plates were coated with beta -galactosidase fusion proteins, blocked with bovine serum albumin, hybridized with increasing concentrations of importin 58-GST or precomplexed importin 58/97-GST and then successive incubations carried out with goat anti-GST primary, and alkaline phosphatase-coupled rabbit anti-goat secondary, antibodies. After the addition of the substrate p-nitrophenyl phosphate, A405 was measured at 5-min intervals for 90 min using a plate reader (Molecular Devices). Values were corrected by subtracting both A405 at 0 min and A405 in wells incubated without importin 58/97-GST complex. To assess importin binding specifically to the NLSs, quantitation was performed in identical fashion for beta -galactosidase itself and the values subtracted from those for the respective fusion proteins (30).

To correct for differences in coating, the RB and T-ag fusion proteins were subjected to a parallel beta -galactosidase ELISA assay using a beta -galactosidase-specific monoclonal antibody together with an anti-mouse alkaline phosphatase-conjugated secondary antibody and p-nitrophenyl phosphate (30). As above, values were corrected by subtracting the absorbance at 0 min. Measurements for importin binding were ultimately corrected for any differences in coating efficiencies quantified in the beta -galactosidase ELISA, to enable a true estimate of bound importin to be made (30). Fusion proteins denatured through preincubation for 10 min at 65 °C were found to exhibit greatly reduced reactivity in the beta -galactosidase ELISA, as well as an over 100-fold increase in the value of the KD (apparent affinity constant).


RESULTS

The Bipartite NLS of Human RB Is Capable of Targeting a Heterologous Protein to the Nucleus

Human RB contains a consensus bipartite NLS that is conserved in X. laevis and mouse RB (see Table I and Refs. 22 and 23). To test whether this sequence in human RB is functional, beta -galactosidase fusion proteins were derived containing either the wild type human RB NLS (amino acids 860-877, RB-Bip-beta -Gal) or a mutated version (RB-BipMut-beta -Gal) in which lysine 860 and arginine 861 were substituted by threonine residues. This mutation abolishes the bipartite character of the NLS; similar mutations abolish the nuclear targeting activity of bipartite NLSs such as those of nucleoplasmin (24), and the yeast transcription factor SWI5 (26) (see Table I). The nuclear import kinetics of RB-Bip-beta -Gal and RB-BipMut-beta -Gal were measured using in vivo (microinjected cells of the HTC rat hepatoma line) (26, 32) and in vitro (mechanically perforated HTC cells) (29, 33) nuclear transport assay systems. The human RB NLS was capable of targeting the heterologous E. coli protein beta -galactosidase (476 kDa) to the nucleus in both assay systems (Figs. 1 and 2). RB-Bip-beta -Gal accumulated in nuclei maximally to levels 3-4 fold those in the cytoplasm (Fig. 1B and 2B; Table II). Results were similar to those for beta -galactosidase fusion proteins containing the bipartite NLSs from SWI5 (26) or human interleukin-5 (hIL-5; Ref. 27) in that RB-Bip-beta -Gal accumulated in the nucleus to a significantly lower extent than T-ag-CcN-beta -Gal; the T-ag NLS is clearly a more potent targeting signal than the bipartite NLS of RB. The initial nuclear import rate of the RB-Bip-beta -Gal in vivo was also significantly less (p < 0.0005) than that of T-ag-CcN-beta -Gal (rates of 0.49 and 1.12 Fn/c/min, respectively, (where Fn/c is defined as the ratio of nuclear to cytoplasmic fluorescence after the subtraction of fluorescence due to autofluorescence; see Table II), supporting the idea that the T-ag NLS is more efficient than the bipartite NLS of RB. The RB-BipMut-beta -Gal protein was completely excluded from the nucleus both in vivo and in vitro (Fn/cmax of about 0.5; Figs. 1 and 2B, left panel; Table II), even up to 12 h in vivo (not shown), where fusion protein localization was scored using a histochemical stain for beta -galactosidase activity in situ (32). This confirmed the bipartite nature of the RB NLS.


Fig. 1. Nuclear uptake of RB-Bip-beta -Gal and RB-BipMut-beta -Gal fusion proteins in vivo as measured by quantitative CLSM. A, CLSM images are shown for cells 35-40 min after microinjection (see "Materials and Methods"). B, nuclear import kinetic measurements were performed as described under "Materials and Methods" (26, 31) and represent a single typical experiment, where each point represents the average of 10-11 separate measurements for each of nuclear, cytoplasmic, and background (autofluorescence) fluorescence. Data were fitted for the function Fn/c(t) = Fn/cmax(1 - e-kt) (26, 29, 31); collated data are presented in Table II. Results are compared with those for T-ag-CcN-beta -Gal.
[View Larger Version of this Image (36K GIF file)]


Fig. 2. Dependence on cytosol and ATP of the nuclear transport kinetics of RB-Bip-beta -Gal and T-ag-CcN-beta -Gal in mechanically perforated HTC cells. A, CLSM images are shown for 5-iodoacetamidofluorescein-labeled T-ag-CcN-beta -Gal (left panels) and RB-Bip-beta -Gal (right panels) in the presence and absence of either exogenously added cytosol or an ATP-regenerating system as indicated after 30-40 min at room temperature (see "Materials and Methods"). B, experiments were carried out in the absence and presence of exogenous cytosol and an ATP-regenerating system as indicated. Results are compared with those for T-ag-CcN-beta -Gal and RB-BipMut-beta -Gal (left panel). Measurements and curve fitting were performed as described in the legend to Fig. 1B and represent the average of at least two separate experiments, where each point represents the average of up to 10 separate measurements for each of Fn and Fc, respectively, with autofluorescence subtracted. Collated data are presented in Table II.
[View Larger Version of this Image (46K GIF file)]

Table II. Nuclear import kinetics of RB-Bip-beta -galactosidase fusion protein derivatives compared to those of T-ag-CcN-beta -Gal


Protein Nuclear import parametera
Fn/cmax Initial rate (Fn/c/min) n

A. In vivo (microinjected cells)
  RB-Bip-beta -Gal 3.16  ± 0.31b 0.49  ± 0.04b 3
  RB-BipMut-beta -Gal 0.54  ± 0.04 0.13  ± 0.01 3
  T-ag-CcN-beta -Galc 7.47  ± 1.10b 1.12  ± 0.06b 4
B. In vitro (mechanically perforated cells)d
  RB-Bip-beta -Gal 3.76  ± 0.46b 0.43  ± 0.07 7
  RB-Bip-beta -Gal (-cytosol) 1.21  ± 0.04 0.24  ± 0.04 3
  RB-Bip-beta -Gal (-ATP) e 1.21  ± 0.14 0.33  ± 0.06 2
  RB-Bip-beta -Gal (-cytosol/-ATP) g 1.18  ± 0.14 0.23  ± 0.01 3
  RB-Bip-beta -Gal (+GTPgamma S) 1.92  ± 0.10f 0.32  ± 0.03f 1
  RB-Bip-beta -Gal (+CHAPS) 1.23  ± 0.06 NDh 2
  RB-BipMut-beta -Gal 1.04  ± 0.24 0.16  ± 0.06 2
  T-ag-CcN-beta -Galc 5.35  ± 0.24b 0.48  ± 0.06 5
  T-ag-CcN-beta -Gal (-cytosol) 1.43  ± 0.35 0.25  ± 0.02 3
  T-ag-CcN-beta -Gal (-ATP) e 1.30  ± 0.10 0.25  ± 0.04 3
  T-ag-CcN-beta -Gal (-cytosol/-ATP) g 0.97  ± 0.04 0.24  ± 0.02 3
  T-ag-CcN-beta -Gal (+GTPgamma S) 2.23  ± 0.27f 0.30  ± 0.03f 1
  T-ag-CcN-beta -Gal (+CHAPS) 1.26  ± 0.06 NDh 2
  70-kDa dextran 0.23  ± 0.03 NDh 4
  70-kDa dextran (+CHAPS) 0.98  ± 0.06 NDh 2

a Raw data (see Figs. 1B and 2B and data not shown) were fitted for the function Fn/c (t) = Fn/cmax(1 - e-kt) (26, 29, 31), where Fn/cmax is the maximal level of accumulation at steady state in the nucleus, and t is time in minutes. An Fn/cmax of 1.0 indicates nuclear entry and equilibration between nucleus and cytoplasm, with values below 1 indicating exclusion from the nucleus. The S.E. is indicated.
b Denotes significant differences between the respective parameters for RB-Bip-beta -Gal and T-ag-CcN-beta -Gal; p values were <0.02, <0.0005, and <0.01, for Fn/cmax and initial rate in vivo and Fn/cmax in vitro, respectively.
c The T-ag beta -galactosidase fusion protein (described in Ref. 32) contains the NLS together with the regulating phosphorylation sites, known as the CcN motif (31).
d Results are shown for transport in the presence of exogenously added cytosol and an ATP-regenerating system, unless otherwise indicated.
e Apyrase pretreatment was used and the ATP-regenerating system omitted (Ref. 33; see "Materials and Methods").
f S.E. for curve fit.
g Cytosol and the ATP-regenerating system were omitted (no apyrase pretreatment).
h ND, not able to be determined.

Dependence of Nuclear Uptake Conferred by the Human RB Bipartite NLS on Cellular Factors in Vitro

To compare the NLSs of RB and T-ag in more detail, we examined the dependence of nuclear transport on cellular factors. NLS-dependent nuclear protein import in vitro is known to be dependent on energy (12) and on the addition of exogenous cytosol (33-35). The latter supplies the NLS-binding/NPC-docking dimer of importin 58/97 (11) as well as the monomeric GTP-binding protein/GTPase Ran/TC4 (34) and interacting proteins (see Ref. 36), all of which are essential for nuclear accumulation. In vitro experiments were performed to investigate the dependence of RB-Bip-beta -Gal nuclear import on ATP and cytosolic factors (Fig. 2; Table II). In identical fashion to that of T-ag-CcN-beta -Gal, nuclear accumulation of RB-Bip-beta -Gal was found to be dependent on both ATP and exogenous cytosol. In the absence of either, the Fn/cmax was about 1, indicating no accumulation in the nucleus (Fig. 2; Table II). Similarly, the nonhydrolyzable GTP analog GTPgamma S inhibited transport (Table II). The results thus indicated that, like T-ag-CcN-beta -Gal, RB-Bip-beta -Gal localizes in the nucleus through an energy and cytosolic factor-dependent pathway inhibitable by GTP analogs, accumulation thus being an NLS-dependent, active process, most likely dependent on the importin subunits and the GTPase activity of Ran/TC4.

The nuclear envelope can be perforated by detergents like CHAPS, under which conditions molecules can diffuse freely between cytoplasm and nucleoplasm and accumulation in the nucleus is exclusively due to binding to nuclear components. No accumulation of either RB-Bip-beta -Gal or T-ag-CcN-beta -Gal was observed in the presence of CHAPS (Table II and data not shown), indicating that binding in the nucleus is unlikely to be part of the mechanism of nuclear accumulation conferred by either NLS (see also Refs. 5-7), although it should be remembered that some soluble nuclear components may be lost through the CHAPS permeabilization. That the RB bipartite NLS itself is unlikely to have a role in binding to nuclear components is supported by other studies (Ref. 23; see also "Discussion").

Affinity of RB-Bip-beta -Gal for the NLS-binding Importin 58/97 Dimer

The reduced nuclear import of RB-Bip-beta -Gal relative to the wild type T-ag-CcN-beta -Gal fusion protein both in vivo and in vitro (Figs. 1 and 2) could result from reduced efficiency of recognition of the NLS by the importin 58/97 complex. To test this hypothesis, an ELISA-based binding assay (Ref. 30; see "Materials and Methods") was employed. RB-Bip-beta -Gal, RB-BipMut-beta -Gal, and T-ag-CcN-beta -Gal fusion proteins were coated onto microtiter plates and incubated with increasing amounts of importin 58-GST or importin 58/97-GST complex. Binding was then quantitated using antibodies specific to GST and an alkaline-phosphatase-labeled secondary antibody.

For importin 58 alone the apparent dissociation constant (KD) for both T-ag-CcN-beta -Gal and RB-Bip-beta -Gal was about four times higher than that for the importin 58/97 complex, confirming that the latter is the high affinity NLS binding component for both types of NLS (16, 30, 37). The KD of T-ag-CcN-beta -Gal for importin 58/97 (10.3 ± 2.1 nM, mean ± S.D. for two separate experiments) was about 6.5 times lower than that for RB-Bip-beta -Gal (67.5 ± 16.9 nM) (Fig. 3). The KD for importin 58/97 for RB-BipMut-beta -Gal could not be determined due to the lack of binding (<1% the maximal amount of importin 58/97 bound by RB-Bip-beta -Gal), confirming the specificity of the NLS binding assay (see also Ref. 30). Although the basis was not entirely clear, the maximal amount of importin bound (the Bmax) also varied between T-ag-CcN-beta -Gal and RB-Bip-beta -Gal, whereby the T-ag NLS maximally bound about three times more importin 58/97 complex than the bipartite NLS of RB. These results were not attributable to partial denaturation of the RB-Bip-beta -Gal preparation as indicated by the results for the beta -galactosidase ELISA (see also "Materials and Methods"), which is routinely used to standardize results for equivalent amounts of native protein (30). We have obtained very similar results for an IL-5 bipartite NLS containing beta -galactosidase fusion protein (27), so that it seems reasonable to conclude that the results for RB-Bip-beta -Gal do not represent an artifact of either the RB-Bip-beta -Gal protein preparation or the ELISA-based binding assay. NLS masking within the beta -galactosidase tetramer can also not be the basis of the differences in Bmax, since the four subunits of beta -galactosidase are structurally identical (see Ref. 38); that is, a reduced Bmax is not interpretable in terms of differences in the number of importin 58/97 binding sites (i.e. number of NLSs) (30). The basis of the differences in Bmax may simply be that beta -galactosidase itself binds significant amounts of importin 58/97 at concentrations of the latter above 0.1 µM (see Ref. 30 and data not shown), and since the values for beta -galactosidase are always subtracted from those for the respective fusion proteins, lower affinity NLSs such as that of RB thereby exhibit ostensibly lower Bmax values than the high affinity T-ag NLS as a result.


Fig. 3. Binding of the importin 58/97 complex to T-ag and RB fusion proteins. Binding of RB-Bip-beta -Gal and T-ag-CcN-beta -Gal to mouse importin 58/97 complex was quantitated using an ELISA-based binding assay. Microtiter plates coated with beta -galactosidase fusion proteins (0.5 µg/well) were hybridized with increasing amounts of importin complex (30; see "Materials and Methods"). Binding curves, where the values for importin 58/97 binding to beta -galactosidase alone have already been subtracted, were fitted for the function B(x) = Bmax (1 - e-kx), where x is the concentration of importin complex. The KD represents the concentration of importin complex yielding half-maximal binding. No KD could be calculated for RB-BipMut-beta -Gal due to the fact that, after subtraction of the binding results for beta -galactosidase itself, importin 58/97 binding was negligible (see "Results"). The correlation coefficient for the curve fit in both cases was greater than 0.98. Collated data are presented in the text.
[View Larger Version of this Image (17K GIF file)]

Based on the results for importin 58/97 binding, we conclude that the relatively low efficiency of the RB bipartite NLS compared with the T-ag NLS in terms of nuclear import (Figs. 1 and 2) is likely to be the direct result of its lower affinity for and binding to the importin 58/97 complex. This is consistent with our recent study demonstrating that the rate of nuclear import in vivo correlates directly with the respective binding affinity for importin 58/97 of T-ag-CcN-beta -Gal variants (30).


DISCUSSION

This study presents the first kinetic analysis of nuclear import mediated by the RB bipartite NLS. We demonstrate that amino acids 860-877 of human RB (p110Rb) are capable of targeting a large heterologous protein into the nucleus, both in vivo and in vitro, and therefore constitute a functional NLS. Mutation of KR861 to TT abolishes the bipartite character of the RB NLS, abrogating its nuclear targeting ability both in vivo and in vitro in analogous fashion to the effect of similar mutations on other bipartite NLSs (24-27). Nuclear import of RB-Bip-beta -Gal in vitro requires ATP and the addition of exogenous cytosol and is inhibited by the nonhydrolyzable GTP analog GTPgamma S (Fig. 2B, Table II). RB-Bip-beta -Gal thus appears to be accumulated in the nucleus by a classical NLS-dependent, active pathway, dependent on importin, ATP, and the monomeric GTP-binding protein/GTPase Ran/TC4 (14, 15, 17, 39; see also Ref. 40). In vitro measurements (Table II) in the absence of an intact nuclear envelope due to the action of the nuclear membrane permeabilizing detergent CHAPS, demonstrate that nuclear accumulation is unlikely to be due to binding of the RB fusion protein to nuclear components (Ref. 23; see also below).

This study embodies the first definitive kinetic comparison of two basic types of NLS, namely the T-ag NLS and the bipartite NLS of RB, both of which, as shown in this study, confer ATP-, cytosolic factor- and GTP-binding protein/GTPase-dependent nuclear accumulation, which appears to be independent of binding to nuclear components. The RB NLS confers significantly lower maximal nuclear accumulation both in vivo and in vitro compared with the T-ag NLS, while the initial import rate of the fusion protein carrying the RB NLS is reduced over 2-fold in vivo. The T-ag NLS is clearly a more efficient NLS than the RB bipartite NLS. Since the results for the RB NLS with respect to in vivo nuclear transport and importin binding properties (see also below) are similar to those for the bipartite NLSs of SWI5 (26) and IL-5 (27), the conclusions reported here with respect to the properties of the RB NLS may be valid for bipartite NLSs in general.

An ELISA-based binding assay (30) was employed to examine the interaction of the NLS-binding importin 58/97 complex with the T-ag NLS and the RB bipartite NLS. The specificity and sensitivity of the binding assay allows the measurement of high affinity (nanomolar) interactions between importin subunits and different NLSs. The dimeric importin 58/97 complex, rather than the NLS-binding importin 58 subunit alone, was confirmed through direct affinity determinations to be the high affinity NLS receptor for both types of NLS (see Refs. 16, 30, and 37). Apart from our previous study (30), measurements of NLS-binding protein/NLS binding affinity have only been performed for the low affinity NLS receptor (100 nM and higher; see Ref. 41). Our assay's specificity is demonstrated by the fact that NLS mutant derivatives are severely impaired in importin binding as shown in this and our previous study (30). We found that the T-ag beta -galactosidase fusion protein has a 6.5 times higher binding affinity for the importin 58/97 complex than the beta -galactosidase fusion protein containing the RB NLS; a similarly reduced binding affinity for importin 58/97 on the part of the bipartite NLS of IL-5 supports the idea that the results for the RB NLS are representative of bipartite NLSs in general and that bipartite NLSs may exhibit lower importin binding affinity than those of the T-ag NLS type. The lower affinity of the RB bipartite NLS for the importin 58/97 complex presumably constitutes the basis of the observed reduced maximal nuclear accumulation and import rate in vivo conferred by the RB NLS relative to the T-ag NLS. This implies, as shown previously (Ref. 30 and see Refs. 36 and 42), that the initial events of nuclear transport, and NLS-binding by importin in particular, are critical in determining overall nuclear protein import kinetics. That different members of the NLS-binding protein family, such as the importins, may have distinct NLS binding affinities (41) is consistent with this idea.

One aspect that arose from this study in terms of the comparison between the T-ag and RB NLSs was the fact that the results for nuclear import kinetics in vitro did not concur completely with those obtained in vivo. The wild type RB fusion protein exhibited over 50% reduced maximal nuclear accumulation and initial import rates compared with the T-ag fusion protein in vivo, but the differences between the two proteins were not as great in vitro and not significant in the case of the import rate. Clearly, the in vivo and in vitro nuclear transport assays are complementary rather than being identical, the most important differences being the source of the exogenously added cytosol (rabbit reticulocyte lysate), the ATP-regenerating system, and the temperature of incubation (37 °C in vivo as opposed to room temperature in vitro). Theoretically, at least, no transport component/ATP etc. is limiting in vitro, which may explain why the affinity of NLS recognition does not appear to be a significant factor in determining the rate of accumulation in vitro, i.e. why there is no significant difference in the initial rates of fusion proteins containing low affinity (RB) or high affinity (T-ag) NLSs, as opposed to in vivo. The in vivo system is clearly more physiologically relevant with respect to transport kinetics in living cells, while the in vitro system enables the dependence on particular transport components, ATP, etc. to be tested. The level and type of the NLS-binding proteins expressed has recently been shown to vary between cell types (41), so that it seems reasonable to surmise that differences in the nature and amount of NLS-binding proteins and other cytosolic factors compared with in vivo are the basis of the fact that the import rate of RB and T-ag fusion proteins is not significantly different in vitro (see also Ref. 27).

While the conclusions of this study are quite clear with respect to the functional activity of the RB NLS, it should be mentioned that additional sequences may be involved in the nuclear accumulation of the complete RB protein. Cell cycle/phosphorylation-dependent regulation of nuclear association ("tethering") of RB has been reported (43, 44), but our in vitro results here using CHAPS support the idea that the RB bipartite NLS is not likely to be involved in binding to nuclear components. We previously identified a putative CcN motif in RB (36), but the results of Zacksenhaus et al. (23) with respect to mouse p110RB1 imply that this sequence is not essential for RB nuclear localization. The latter study indeed implicates the role of sequences other than the mouse RB bipartite NLS (in exon 25) in nuclear localization, since deletion of exon 22 (within the RB T-ag/E1A binding domain), additional to mutation of the RB bipartite NLS, was found to be necessary to effect an exclusively cytoplasmic location of an NLS mutant. The authors suggest a role for these additional sequences in RB nuclear localization through conferring association with nuclear proteins (23). In the light of our results and those of others (23, 43, 44), it seems reasonable to propose that the bipartite NLS constitutes the primary nuclear entry signal of RB, while other sequences may mediate interactions with nuclear components (23).

In conclusion, this study shows that archetypal representatives of the two basic types of NLS are both accumulated in the nucleus by a classical NLS-dependent, active pathway, but that the RB bipartite NLS is not as efficient in nuclear targeting as the T-ag NLS. The difference is probably due to a lower affinity for the NLS-binding importin 58/97 complex, consistent with our findings for T-ag derivatives (30), indicating a direct correlation between importin binding affinity and the initial nuclear import rate. Reduced affinity for the importin 58/97 complex and reduced in vivo nuclear import conferred by the NLS, compared with the properties conferred by the T-ag NLS, may be a common feature of bipartite NLSs.


FOOTNOTES

*   The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Dagger    To whom correspondence should be addressed: Nuclear Signaling Laboratory, Division for Biochemistry and Molecular Biology, John Curtin School of Medical Research, Australian National University, P. O. Box 334, Canberra City, A.C.T. 2601, Australia. Tel.: 616-2494188; Fax: 616-2490415; E-mail: daj224{at}leonard.anu.edu.au; Telex: curtmed 62033.
1   The abbreviations used are: NPC, nuclear pore complex; NLS, nuclear localization sequence; T-ag, SV40 large tumor-antigen; RB, retinoblastoma protein (p110Rb); CHAPS, 3[(3-cholamidopropyl)dimethylammonio]-1-propane-sulfonate; GST, glutathione S-transferase; ELISA, enzyme-linked immunosorbent assay; CLSM, confocal laser scanning microscopy; GTPgamma S, guanosine 5'-O-(3-thiotriphosphate); IL, interleukin.

ACKNOWLEDGEMENTS

We thank Joanna Reid for helpful discussions and Lyndall J. Briggs and Patricia Jans for skilled technical assistance.


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