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Originally published In Press as doi:10.1074/jbc.M608329200 on December 12, 2006

J. Biol. Chem., Vol. 282, Issue 6, 3881-3888, February 9, 2007
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Passive and Facilitated Transport in Nuclear Pore Complexes Is Largely Uncoupled*

Bracha Naim{ddagger}, Vlad Brumfeld§, Ruti Kapon{ddagger}, Vladimir Kiss{ddagger}, Reinat Nevo{ddagger}, and Ziv Reich{ddagger}1

From the Departments of {ddagger}Biological Chemistry and §Plant Sciences, Weizmann Institute of Science, Rehovot 76100, Israel

Received for publication, August 31, 2006 , and in revised form, November 21, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Nuclear pore complexes provide the sole gateway for the exchange of material between nucleus and cytoplasm of interphase eukaryotic cells. They support two modes of transport: passive diffusion of ions, metabolites, and intermediate-sized macromolecules and facilitated, receptor-mediated translocation of proteins, RNA, and ribonucleoprotein complexes. It is generally assumed that both modes of transport occur through a single diffusion channel located within the central pore of the nuclear pore complex. To test this hypothesis, we studied the mutual effects between transporting molecules utilizing either the same or different modes of translocation. We find that the two modes of transport do not interfere with each other, but molecules utilizing a particular mode of transport do hinder motion of others utilizing the same pathway. We therefore conclude that the two modes of transport are largely segregated.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Eukaryotic cell nuclei are separated from the cytoplasm by a double lipid bilayer system known as the nuclear envelope (NE).2 Exchange of material between the two compartments proceeds through nuclear pore complexes (NPCs), large protein assemblies that span the NE and provide the sole medium for exchange. The vertebrate NPC has a molecular mass of ~125 MDa (1) and is made up of ~30 different proteins, called nucleoporins, most of which are present in multiples of eight (2, 3). The core of the NPC consists of a symmetrical framework, measuring ~120 x 90 nm, which is made of two coaxial rings sandwiching a wheel-like array of eight spoke-shaped domains. The spoke-ring assembly encircles the central pore channel, which resembles an hourglass 45–50 nm wide at its waist (4–7). In addition to the central framework, NPCs contain peripheral structures, which are anchored to the ring moieties of the spoke-ring assembly and serve as docking sites for nuclear transport receptors and effectors. These structures include eight short (~50 nm) filaments that protrude toward the cytoplasm and a massive, fish trap-like structure, termed the nuclear basket, which extends into the nucleus (4–11). Yeast NPCs have an overall similar architecture but are smaller (3, 12–14).

Transport across the NPC has been reviewed in detail (15–22) and can be divided into two modes. Small molecules, such as ions, metabolites, and intermediate-sized macromolecules, can pass unassisted by diffusion which becomes increasingly restricted as the particle approaches a size limit of ~10 nm in diameter (23, 24). In contrast, proteins, RNAs, and their complexes are ushered selectively by dedicated soluble transport receptors, which recognize specific import (NLS) or nuclear export signal (NES) peptides displayed by the cargo. In most cases, assembly and disassembly of transport complexes in the appropriate cellular compartment are coordinated by the small GTPase Ran, which performs these activities by binding to transport receptors in its GTP-bound form. Facilitated translocation is often coupled to an input of metabolic energy that permits transport against concentration gradients and can accommodate objects with a diameter of up to ~40 nm (25).

It has been estimated that in proliferating HeLa cells a single NPC supports the facilitated translocation of ≥10–20 MDa of macromolecules and macromolecular complexes/s (26). At the same time, each NPC most likely has to accommodate comparable flows of small molecules that cross it by diffusion. It is generally assumed that both modes of transport share a single diffusion channel located within the central pore of the NPC (see, e.g. Refs. 23 and 27–30). However, up to date, no study had directly tested for concurrent transport of cargoes utilizing different modes. The issue of whether or not a single diffusion channel mediates both modes of transport therefore remains unsettled, despite the fact that it pertains to the very basic function of the NPC. In the present work, we addressed this issue by testing for mutual effects of molecules utilizing either the same or different modes of transport when crossing NPCs. We found that molecules transporting in different modes did not hinder the passage of each other. In contrast, molecules that utilize a given mode of transport for translocation sterically interfered with the passage of other molecules that make use of the same mode. These results indicate that passive and facilitated transport through NPCs proceed along largely segregated routes.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Transport Cargoes—FITC-labeled 9.5- and 19.5-kDa dextrans and RITC-labeled 17.2-kDa dextran were purchased from Sigma. TRITC-labeled bovine serum albumin (BSA) and TRITC-labeled BSA covalently linked to ~10 SV40 large T-antigen NLS peptides (CTPPKKKRKV; the number of peptides attached was determined by mass spectrometry) were a kind gift from Michael Elbaum (Weizmann Institute of Science). A chimera consisting of IgM conjugated to green fluorescent protein (GFP) fused to the naturally NLS-carrying protein, nucleoplasmin, was prepared as follows. Nucleoplasmin-GFP (1 mg in PBS; also a gift from M. Elbaum) was reacted with 92 nmol of m-maleimidobenzoyl-N-hydroxysuccinimide ester (Pierce) for 1 h at room temperature. The IgM (Jackson ImmunoResearch, 0.9 mg dissolved in 200 µl of 0.01 M sodium phosphate buffer (pH 7.6) and 0.5 M NaCl) was incubated with 46 nmol N-succinimidyl 3-(2-pyridyl-dithio)propionate (Pierce) for 30 min (room temperature). Desalting of both mixtures was carried out using Sephadex G-25 spin columns equilibrated with PBS. Dithiothreitol was added to the IgM-SPDP mixture to a final concentration of 1 mM, and the solution was immediately passed through a PD-10 desalting column (Amersham Biosciences), equilibrated with PBS. Fractions containing the IgM-SPDP without dithiothreitol (verified with 5,5'-dithiobis (2-nitrobenzoic acid), Ellman's reagent) were then added to the solution containing Nup-GFP-m-maleimidobenzoyl-N-hydroxysuccinimide ester. Following 30 min of incubation at room temperature, the mixture was concentrated (Vivaspin 6, MWCO 30,000; Vivascience) and separated on Superdex 200 column (Amersham Biosciences). The species used in the experiments had one molecule of nucleoplasmin-GFP per IgM. A plasmid encoding for transportin (human) fused to glutathione S-transferase (GST) was kindly provided by Yuh Min Chook (University of Texas Southwestern Medical Center). The fusion protein was expressed and purified as described (31). The protein was labeled with AlexaFluor 633 succinimidyl ester carboxylic acid by incubation (in PBS, pH 8.4) with 5-fold molar excess of the dye at 4 °C for 14 h. Free dye was removed using PD-10 desalting column (Amersham Biosciences) equilibrated with transport buffer (TB; 30 mM sodium chloride, 90 mM potassium acetate, 5 mM magnesium acetate, 1 mM EDTA, 2 mM dithiothreitol, 250 mM sucrose, and 20 mM Tris-HCl, pH 7.4). Following labeling, the protein was concentrated using Vivaspin 6 (MWCO 30,000; Vivascience) and stored at -70 °C in TB containing 20% (v/v) glycerol. GST sometimes tends to dimerize (24). Mass spectrometry analyses revealed, however, that the fusion is monomeric.

Cell Culture—HeLa cells (ATCC CCL-2; up to five passages) were cultured in Dulbecco's modified Eagle's medium with 10% fetal calf serum/antibiotics and were used 1 day after plating, at which time they were 50–70% confluent. Media, antibiotics, and fetal calf serum were purchased from Invitrogen.

Transport Assays and Microinjection—Microinjections were performed using a semi-automatic injection system (Eppendorf 5246 Transjector attached to an Eppendorf 5171 micromanipulator) with a Z-depth limit option, using glass micropipettes (Femtotips; Eppendorf). Air was administrated at 100–250 hPa for 0.2–0.3 s. The cells were kept in Dulbecco's modified Eagle's medium, 25 mM Hepes (pH 7.3) at 37 °C in an open perfusion micro-incubator (PDMI II, Harvard Apparatus) mounted on the stage of the microscope used for the recordings. All of the samples were injected with Cy5-labeled secondary anti-goat antibody (IgG, 150 kDa; Jackson ImmunoResearch), which served as a marker for nuclear envelope integrity. A rough approximation of the initial cytosolic concentration of the injected material was made based on the injected volume, using a spheroid model of the cell. The cells were viewed with an IX70-based Olympus FluoView 500 confocal laser-scanning microscope, using 0.85-numerical aperture 40x (UplanApo) objective; the focal plane was set such that it traversed through the mid-section of the nuclei. The images were collected every 1.6 s before, during, and following injection, until a steady state had been reached. FITC-dextran, GFP, and IgM-nucleoplasmin-GFP were visualized using an argon laser ({lambda}ex = 488 nm) and a 505–525-nm band pass filter. TRITC-BSA and RITC-dextran were excited at 543 nm, using a helium-neon laser, and the emitted fluorescence was collected between 560 and 600 nm. Visualization of the anti-goat Cy5-labeled antibody was made using a helium-neon laser ({lambda}ex = 633 nm) and a >660-nm high-pass filter. The images were acquired sequentially to prevent spectral bleed-through of the fluorescence emission and were processed using the ImagePro Plus (v4.5) software. In all cases, the time-dependent rise in the ratio between (background-corrected) nuclear and cytoplasmic mean fluorescence intensity, F(t), fitted well to a single-exponential curve of the form,

Formula(Eq. 1)
where t is the time post injection, and k is the first order rate constant. Translocation rates (particles/NPC/second) were estimated using the values reported by Ribbeck and Görlich (26) for the volume and NPC density of HeLa cell nuclei.

Fluorescence recovery after photobleaching. HeLa cells stably expressing GFP were obtained by transfection with pCEFL and selection under 700 µg/ml G418 for 3 weeks. The cells were serum-starved (0.15% fetal calf serum) for 14 h. An hour and a half before the measurements, the medium was replaced by a medium containing 10% fetal calf serum, which was used throughout the experiments. The measurements were made at 37 °C with the microscope set-up described above, using a 60x PlanApo oil-immersion objective (NA 1.4). Prior to bleaching, the target cell was imaged using 0.2% of the maximal laser intensity. Photobleaching of nuclear GFP was achieved by repetitive scans of a 72 x 24 pixel area inside the nucleus (covering most of its area) for 3–5 s, using full illumination intensity (~0.3 milliwatt at the focal point). Following bleaching, the laser intensity was brought back to its initial value, and time lapse image sequences were acquired at 2 Hz. The relatively slow acquisition rate and the fact that bleaching often extended to cytosolic domains rendered the rate constants derived from the measurements only approximations. However, we were interested only in relative rates, for which the analysis fully sufficed. Fluorescence recovery inside the nucleus was fitted using the following equation,

Formula 2(Eq. 2)
where F0 and F° denote the mean nuclear fluorescence intensity ratio immediately after bleaching and at the end of recovery.

Permeabilized Cells—HeLa cells were washed with cold TB (see above) and incubated for 5 min with 8 µg/ml digitonin (in TB) on ice, after which they were rinsed five times with cold TB. The measurements were made at 37 °C as described for the microinjection assays, using RITC-labeled 77-kDa dextran as nuclear integrity marker. The images were collected every 1.4 s before, during, and following the addition of the transport cargoes (FITC-labeled 9.5-kDa dextran and AlexaFluor 633-GST-transportin).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Microinjection Experiments—The first set of experiments consisted of competition assays in intact cells. Fluorescently labeled substrates, capable of crossing NPCs either by passive diffusion or in a signal-dependent, receptor-mediated fashion, were injected into the cytoplasm of HeLa cells, either separately or together, and their nuclear import kinetics was followed by confocal microscopy. Passively transported cargoes included 9.5-, 17.2-, and 19.5-kDa dextran molecules (Table 1). Receptor mediated transport was represented by BSA covalently linked to ~10 SV40 large T-antigen NLS peptides, which serve as substrates for importin {alpha}/beta transport receptor complexes. Within the time scale of our measurements, BSA devoid of NLS was virtually excluded from the nuclei and thus can be considered as exclusively dependent on receptor mediation for its transport. To verify that the NE of the injected cells remains intact during the experiments and, hence, that nuclear entry of the probes is the result of authentic translocation events, fluorescently labeled anti-goat secondary antibody (~150 kDa) was co-injected with the transport substrates. Only cells capable of completely excluding the antibody from the nucleus throughout the entire measurement were used in the analysis. Injections and recordings were performed at 37 °C, using an environmental chamber that was mounted on the microscope stage.


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  		TABLE 1
Hydrodynamic radii of molecules used in this study

 
To establish the validity of the experimental system, we injected cells with a mixture containing FITC-labeled 19.5-kDA dextran (green), TRITC-labeled BSA-NLS (red), and the NE integrity marker antibody (Cy5-labeled, magenta). Time series images in Fig. 1A show a homogenous fluorescence of both dextran and BSA inside the cytoplasm within 5 s post-injection. Nuclear import of the two substrates ensued almost immediately after injection and reached a steady state a few minutes later. At this stage, the dextran molecules were distributed more or less evenly between the two cellular compartments, whereas the NLS-carrying BSA markedly accumulated inside the nucleus.

We next set out to obtain the base-line rate constants characteristic of each mode of transport on its own. These rates will serve for comparison in the competition experiment. The inset in Fig. 1B shows single-cell traces of the change in the nuclear/cytoplasmic mean fluorescence intensity ratio, F(t), of either BSA-NLS or 19.5-kDa dextran, plotted against time. Fits to the averaged normalized data sets are shown in the main figure. Both traces fit well to a single exponent F(t) = Fmax(1 - e-kt), akin to first order kinetics. BSA-NLS imported into the nuclei with an apparent rate constant of 7.3 x 10-3 s-1. When the concentration difference between cytoplasm and nucleus is set to 1 µM, this rate leads to a calculated initial influx of ~5000 molecules of BSA-NLS/s or to a translocation rate of approximately two molecules/NPC/s. The rate constants derived for diffusing 9.5- and 19.5-kDa dextran molecules were 27.7 x 10-3 and 18.5 x 10-3 s-1, respectively. These rate constants are 3.8 and 2.5 times higher than that determined for BSA-NLS. Normalized by size, however, the flow rate of the two dextrans through the pores is actually similar to or even slower than that of BSA-NLS bound to its transport receptor(s). This is despite the fact that transport of BSA-NLS involves binding to importin {alpha}/beta, in the cytoplasm, as well as binding of RanGTP to importin beta, at the nucleoplasmic face of the pores. Importin beta-mediated translocation of BSA-NLS across the NPC thus appears to proceed in a facilitated manner. Similar observations have been made before on the passage of other transport receptors (26, 32, 33).


Figure 1
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  		FIGURE 1.
Nuclear import of passively and receptor-mediated transporting cargoes following cytoplasmic injections. FITC-labeled dextran (green) and TRITC-labeled NLS-carrying BSA (red) were injected into the cytoplasm of HeLa cells, separately or together, in the presence of Cy5-labeled secondary antibody (magenta), which served as a marker for NE integrity. Double or triple fluorescence images were acquired in a sequential mode every 1.6 s, before, during, and following injection. A, time series images obtained for a cell injected with a mixture containing 19.5-kDa dextran and BSA-NLS at a molar ratio of 1.5 (dextran per BSA-NLS), as well as the NE integrity marker. B, nuclear accumulation versus time recorded for cells injected with either 19.5-kDa dextran (mixed with BSA lacking NLS) or BSA-NLS. Shown are fits to the averaged normalized data sets. In each case, the data points fit well (solid lines) to a single-exponential of the form F(t) = Fmax (1 - e-kt), suggesting that nuclear entry of both species follows first order kinetics. Inset, single-cell traces. In all the experiments, BSA and BSA-NLS were injected at 52 µM; their cytoplasmic concentrations following injection are estimated to be ~2 µM. Dextran concentration in the cytoplasm was likewise estimated to be 3 µM. Within the time scale of the experiments, BSA lacking NLS and dextran molecules ≥40 kDa failed to enter the nuclei to a detectable level (data not shown).

 


Figure 2
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  		FIGURE 2.
Import rate constants of inert and NLS-carrying cargoes injected alone or together. In all experiments BSA-NLS concentration was kept constant at 2 µM, and dextran concentration was varied from 3 to 100 µM to achieve different molar ratios. A, rate constants of BSA-NLS injected into the cell cytoplasm alone or in the presence of 9.5- or 19.5-kDa dextran. No attenuation in import rate of BSA-NLS is observed up to 50-fold molar excess of dextran. In the latter case, import is actually facilitated, probably because of a crowding effect, which promotes binding of BSA-NLS to its transport receptor. Consistent with this, 40-kDa dextran, which is too big to cross the pores unassisted, also speeded up import of BSA-NLS into the nucleus. B, rate constants of dextran molecules coinjected with BSA or NLS-carrying BSA at molar ratio of 0.7 BSA per dextran or with 10- or 50-fold molar excess of 17.2-kDa dextran. The presence of BSA-NLS in the injected samples did not cause significant changes in the transport rate of the dextran. In contrast, 17.2-kDa dextran (at 30 and 150 µM) effectively attenuated the rate of nuclear entry of the 19.5-kDa dextran probe (kept at 3 µM). The data are presented as the means ± 2 S.E. of determinations involving between 8 and 13 cells for each column. n.s., no significant difference (t test; {alpha} = 0.95).

 
Having established the validity of the experimental system and having obtained the base-line rate constants for the substances used, we proceeded with the competition experiments. First, we examined the effect of passive transport of dextran, on receptor-mediated traffic of BSA-NLS (Fig. 2). Fig. 2A shows the import rate constants derived for BSA-NLS in the presence of 9.5- or 19.5-kDa dextran. The molar ratio between the two species was 1.5 dextran molecules/BSA-NLS. Under these conditions, neither the 9.5-kDa nor the 19.5-kDa dextran had a significant effect on the rate of BSA-NLS import into the nucleus. In principle, the lack of BSA-NLS sensitivity to the application of dextran can be simply the result of too low a concentration of dextran competitors. To ascertain that this is not the case, we conducted two more experiments: one in which differently labeled BSA-BLS substrates competed against each other at a low molar ratio and one at which the ratio of dextran to BSA-NLS was raised dramatically. For the first experiment TRITC-BSA-NLS at a concentration of 2 µM was introduced alone or together with FITC-BSA-NLS at the same concentration. Even at this low molar ratio of 1:1, a significant reduction of ~1.6 in the transport rate of TRITC-BSA-NLS was observed.

Next, we increased the molar ratio between dextran (19.5 kDa) and BSA-NLS to 50 (by increasing the concentration of the former). At this concentration (~100 µM), the dextran molecules imposed an additional mass flow of roughly 6 MDa (~300 molecules)/s on each NPC, ~25–50% of the estimated flow of material crossing in a facilitated manner. Notably, this additional load not only failed to slow down the transport of BSA-NLS but, in fact, exerted an opposite effect, increasing the rate constant of the latter by ~2.5-fold. We hypothesized that this latter effect might be due to depletion interactions (molecular crowding), affected by the high concentration of dextran in the cytoplasm, which facilitated association of BSA-NLS with its transport receptors. To test this, we substituted the 19.5-kDa dextran with 40-kDa dextran, which is too big to diffuse through the pores, thus separating possible effects in the cytoplasm and within the pores. As shown, the 40-kDa dextran also speeded the transport of BSA-NLS into the nucleus. The smaller effect produced by this dextran as compared with that exerted by the 20-kDa dextran is expected because it is larger and, because of solubility limits, was applied at lower molar excess (30-fold versus 50-fold).

Next, we conducted the reverse experiment, where the effect of BSA-NLS on the nuclear import of dextran was studied (Fig. 2B). Here too, no statistically significant change could be observed in the rate constant of the dextran probes when BSA-NLS was co-injected at a molar ratio of 0.7 BSA-NLS/dextran. Substituting dextran with GFP led to similar results.3 In this set of experiments, however, we could not significantly increase the ratio between the two species (in favor of BSA-NLS) because it was impossible to concentrate the protein much further or, alternatively, to sufficiently dilute the dextran (or GFP) solution and still retain a reliable signal.

We therefore took another approach where the pores were plugged by chimeric molecules consisting of IgM molecules conjugated to NLS (5x)-carrying nucleoplasmin fused to GFP. This construct has an estimated diameter of 35–40 nm and thus should occupy a very large fraction of the central channel of the pores, at least at its waist. To ensure that potential effects on facilitated transport are not due to competition over available transport receptors, the IgM-nucleoplasmin-GFP chimeras were injected into the cells (cytoplasm) at very low concentrations such that their intracellular concentration (1–10 nM) was well below that of their transport receptors ~5–10 µM; (34). Because of its size, the chimera transports into the nucleus very slowly with a half-life of 2 h (data not shown). The cells were therefore let to incubate for ~2 h (37 °C), during which the chimeras reached the NE and began translocating through the pores (Fig. 3A). Following incubation, the cells containing the IgM chimeras were injected again with either BSA-NLS or 17.2-kDa dextran, and the nuclear import kinetics of either species was monitored. The presence of the IgM conjugates inside the cells had no effect on the transport of the 17.2-kDa dextran (Fig. 3B). On the other hand, the presence of the chimera molecules led to a 2-fold decrease in the transport rate constant of BSA-NLS (Fig. 3C). The relatively small inhibitory effect probably reflects that only a small fraction of pores contained IgM-nucleoplasmin-GFP molecules at the time of the second injection (Fig. 3A).


Figure 3
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  		FIGURE 3.
Effect of translocating IgM-nucleoplasmin-GFP chimeras on nuclear import of inert and NLS-carrying cargo. A, IgM conjugated to NLS-carrying nucleoplasmin-GFP fusion (green) was injected into the cell cytoplasm together with Cy5-labeled secondary antibody (magenta). ~2 h later, the cells were injected again with either 17.2-kDa dextran (B) or BSA-NLS (C), and nuclear import was followed by confocal imaging. The IgM conjugates noticeably interfered with the import of BSA-NLS, leading to a ~2-fold decrease in its apparent transport rate constant but had no effect on the passage of the dextran molecules. The data are presented as the means ± 2 S.E. of determinations involving between 7 and 15 cells for each column. n.s., no significant difference (t test, {alpha} = 0.95).

 
The above set of experiments demonstrates that passively diffusing molecules do not slow down the translocation of receptor-cargo complexes through NPCs and vice versa. In principle, the observed lack of inhibition may be attributed to the use of insufficiently high loads. However, in the case of dextran, very high intracellular concentrations could be achieved. Moreover, the results obtained from the last experiment show that IgM chimeras, although not affecting the transport of passively diffusing dextran, significantly attenuated the passage of BSA-NLS molecules through the pores.

If this differential effect of receptor-mediated transport cargoes on other complexes is due to a steric segregation of paths, then the same should hold true for passively diffusing cargoes. We have already shown that these do not interfere with their receptor-mediated counterparts. It thus remains to be shown that they do interfere with other passively diffusing cargoes. To this end, we studied the effect of 17.2-kDa dextran on the nuclear import of 19.5-kDa dextran, which served as the test probe. The concentrations and molar ratios between the two dextrans were almost identical to those used in the experiments involving dextran and BSA-NLS, ensuring that similar loads are applied. Applied at a molar excess of 10, the 17.2-kDa dextran molecules led to an almost 2-fold decrease in the import rate constant of the 19.5-kDa dextran probe. Increasing the ratio between the two species to 50 led to a further diminution, decreasing the rate by a factor of ~3 (Fig. 2B). Thus, both inert and signal-carrying molecules can selectively interfere with the passage of other molecules that utilize the same pathway but have no significant effect on the passage of molecules transporting in a different mode.

FRAP Experiments—Another strategy we employed to study the effects produced by receptor-bound translocating material on the passive diffusion of inert molecules relied on the fact that nuclear export of RNA molecules and their protein complexes constitutes a major fraction of the total mass flow through NPCs. In addition, ribonucleoprotein complexes can be very large and thus can plug the pores for relatively long times during their translocation. As inert cargo, capable of freely diffusing through the pores, we used GFP that was stably expressed in the (HeLa) cells. In the experiments, the nuclear import kinetics of GFP was followed by semi-quantitative FRAP (see "Experimental Procedures"; Fig. 4A) in control cells and in cells treated with actinomycin D, which blocks synthesis of all RNA types and inhibits nuclear import of pre-mRNA-binding proteins (35).

An averaged trace of the time-dependent recovery of GFP fluorescence inside the nuclei of untreated cells is shown in Fig. 4B (black dots). Consistent with the results obtained from the microinjection experiments, nuclear import of GFP followed apparent first order kinetics with a half-time of 34 s. The transport rate constant derived from the fit (red, using Equation 2 under "Experimental Procedures") is 22.6 x 10-3 s-1, close to the 27.7 x 10-3 s-1 value we measured for the similarly sized 9.5-kDa dextran.

To study the effect of RNA synthesis blockage and, consequently, lack of its nuclear export on GFP transport through the pores, we subjected cells to 5 µg/ml actinomycin D. To cover most of the activation time range of the drug (36), we took multiple measurements at 10-min intervals, starting 70 min and ending 180 min after addition of the reagent. Because no significant change in import kinetics was observed throughout the entire time range, the data were clustered and analyzed collectively. The global fit of the data, shown in Fig. 4B (green line), is statistically similar to the curve obtained for the untreated cells. The rate constant derived from the fit is 25 x 10-3 s-1, statistically indistinguishable from the value determined in the untreated cells (Fig. 2B, inset).


Figure 4
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  		FIGURE 4.
Effect of RNA export on nuclear import of endogenously expressed GFP studied by FRAP. GFP was stably expressed in HeLa cells, and its nuclear import was followed by FRAP in control cells or in cells treated with 5 µg/ml actinomycin D. Treated cells were incubated with the drug for different times before measurements (70–180 min). Recordings were taken at 10-min intervals. However, because no significant changes were observed throughout the entire time range, the data were clustered and analyzed collectively. A, images of an untreated cell before and after photobleaching of nuclear GFP. B, fluorescence recovery recorded in control cells and in cells treated with actinomycin D. For clarity, standard errors are shown only for control cells; similar values of S.E. were observed for treated cells. Inset, corresponding transport rate constants. Blocking RNA synthesis (and, consequently, its export) had no statistically significant effect on the nuclear import rate of GFP. The data are presented as the means ± 2 S.E. of determinations involving between 9 and 28 cells for each column. n.s., no significant difference (one-way analysis of variance; {alpha} = 0.95).

 
Competition Assays in Permeabilized Cells—A problem with the above set of experiments is that the load exerted on the pores by the transporting RNA molecules, although probably substantial, is essentially unknown. To overcome this limitation, we turned to permeabilized cells (37). Such cells allow the introduction of very large amounts of transporting substrates and are largely devoid of endogenous nucleocytoplasmic exchange, leaving the majority of NPCs in an "empty" state. As a substrate for facilitated transport, we chose the protein import receptor transportin, which can be applied at high concentrations (i.e. >50 µM) and imports efficiently and irreversibly into the nucleus in the absence of RanGTP (26). The combination of permeabilized cells with exogenously applied transportin thus provides an efficient, simple translocation system where the competition between substrates transporting in a passive or facilitated manner can be directly assessed. In addition, transportin translocates through NPCs by a pathway that is distinct from that utilized by importin beta (which mediates the passage of BSA-NLS used in the microinjection assays), extending the scope of potentially competing pathways.


Figure 5
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  		FIGURE 5.
Effect of increasing concentrations of transportin on the nuclear entry of passively diffusing dextran studied in permeabilized cells. Nuclear import of 9.5-kDa dextran ({Delta}C = 2 µM) was studied in permeabilized HeLa cells in the absence and presence of increasing concentrations of recombinant (human) transportin fused to GST. With the exception of 60 µM, no significant change in the rate of import of dextran into the nuclei was observed when the concentration of the protein was varied between 2 and 100 µM. The data are presented as the means ± 2 S.E. of determinations involving between 12 and 50 cells for each column. n.s., no significant difference (one-way analysis of variance; {alpha} = 0.95). Inset, averaged mean fluorescence intensity versus time measured for GST-transportin ({Delta}C = 60 µM) in permeabilized HeLa cells. The fusion protein entered the nuclei with a (first order) rate constant of 8.6 x 10-3 s-1.

 
For the experiments, we used recombinant (human) transportin fused to GST. The fused protein entered the nuclei of permeabilized HeLa cells with a rate constant of 8.6 x 10-3 s-1 (Fig. 5, inset). At the highest concentration we used (100 µM), this corresponds to an initial translocation rate of ~200 molecules·NPC-1·s-1; we note that it has previously been shown that nuclear entry of transportin deviates from ideal Michaelis-Menten kinetics at concentrations above 5–10 µM (26), indicating that above this limit, the passage of transporting molecules is influenced by the presence of others.

Fig. 5 summarizes the effect produced by increasing concentrations of GST-transportin on the nuclear import of 9.5-kDa dextran applied at a constant concentration of 2 µM. No significant changes in the nuclear entry rate constant of the dextran molecules were observed when GST-transportin was added up to a final concentration of 50 µM (Fig. 5). When present at a concentration of 60 µM, GST-transportin led to a small but statistically significant drop (~15%) in the transport rate constant of the dextran, but a further increase of the concentration of the protein to 100 µM had, again, no significant effect on this constant. Overall, there is no apparent trend in the dependence of the rate constant of nuclear import of the dextran probe. Thus, even when competed by heavy loads of cargoes translocating in a facilitated manner, diffusion of molecules through the pores remains unperturbed.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Traffic through NPCs can be considered as consisting of two components. The first is comprised of a population of small, passively diffusing molecules, and the second includes protein, RNA, and ribonucleoprotein complexes requiring interaction with receptors to be transported. Maintaining these two components unperturbed is essential for cell viability and homeostasis, requiring the NPCs to accommodate huge fluxes of both populations at the same time. Mechanistically, as well physiologically, it makes sense that the two components should traffic along spatially separated routes. Such a separation would minimize the risks of steric hindrances that may compromise flows and should simplify control over transport occurring in either mode.

Performing single-particle three-dimensional reconstructions on NPCs released from Xenopus oocytes NEs by detergent treatment, Hinshaw et al. (4) showed that the central pore channel is surrounded by eight 10-nm-wide channels that perforate the central framework of the NPC. Morphologically similar channels (although positioned at smaller radii) were observed in three-dimensional reconstructions of detergent-released frozen-hydrated NPCs from Xenopus (5) and yeast (14). A recent cryo-electron tomography study (6) of native, NE-associated NPCs, also revealed the presence of eight channels roughly similar in shape, size, and radial position to those identified by Hinshaw et al. These channels have been implicated in trafficking of integral membrane proteins to the inner nuclear membrane (38) and in maintenance of NE electrical conductance (39–42). However, they may also serve as an additional conduit for passively diffusing molecules (4, 43). In a meticulous assay, Feldherr and Akin (43) studied the distribution of gold colloids following microinjection into cytoplasm or nucleoplasm of Xenopus oocytes. They found that particles smaller than 10 nm could be found at sites that correspond to peripheral channels in addition to those found in the central pore channel. Larger particles were not observed at these sites. These studies indicate that facilitated transport occurs solely in the central channel, whereas passive diffusion of small particles may occur in both the central and the peripheral channels.

On the other hand, a transport assay on isolated nuclear patches (24) suggested that passive transport occurs only through the central channel of the NPC. Using optical single transporter recording and statistical analysis of the transport of 4–20-kDa dextran molecules, the authors concluded that NPCs contain a single diffusion conduit, located within the central pore channel, which mediates both passive and receptor-mediated transport. However, a recent model published by this group suggests that transport of small molecules occurs within a dedicated tube located in the center of the central pore channel (44), whereas macromolecules traffic along the walls of the NPC.

In the present study, we tackled the question of how the two components of traffic interact by presenting the NPC with cargoes of both types, at various ratios, concomitantly. If both components utilize the same conduit exclusively, one would expect that, at high enough loads, some inhibitory effect would be observed. However, in our experiments, such an inhibitory effect was observed only for species belonging to the same component. At all molar ratios used in our experiments, up to 50, and with applied loads greater than 100 µM, no inhibitory effect was observed for passive transport on facilitated transport, or vice versa. One could argue that the molar ratios used in our experiments are not sufficient to observe hindrance. However, because the same ratios were sufficient to impede members of the same group, the reason for lack of mutual effects of the two traffic components must lay elsewhere. The most parsimonious explanation for this indifference is that they proceed, at least partly, along physically segregated routes.

Our data do not preclude that passive diffusion can also take place across the central pore of the NPC; indeed there is no reason to believe that small molecules do not take advantage of this massive conduit as observed by Feldherr (43). However, our results indicate that a significant fraction of the molecules that cross NPCs by diffusion utilize alternative routes that are sterically not overlapping with the central pore channel. This conclusion is in accordance with observations that passive diffusion of small inorganic ions is largely uncoupled from receptor-mediated, facilitated transport (39–42). It also rationalizes early results reported on the effect of the lectin wheat germ agglutinin on passive and signal-dependent transport. This substance, which binds to O-linked N-acetyl-glucosamine moieties present on certain nucleoporins (45–47), effectively blocks receptor-mediated translocation (48–52) but has little effect on the diffusion of intermediate-sized polymers and proteins (49, 50, 53). Electron microscopy studies indicate that wheat germ agglutinin accumulates mostly, although not exclusively, in the central region of the pores (52, 54, 55) and does not prevent binding of NLS-carrying cargoes to the surface of the pores (48, 52, 56). The different effects exerted by wheat germ agglutinin on passive and facilitated transport through NPCs are naturally explained by the existence of separate routes of transport for each mode. Based on the above, we conclude that passive and facilitated transport across NPCs proceed, to a large extent, through routes that are sterically not overlapping with each other. Segregation of the two modes of transport may follow the configuration proposed by Peters (44), in which facilitated transport occurs along the walls of the central pore channel and passive diffusion occurs through a narrow diffusion tube located at the pore center. More probable to our opinion, the routes mediating passive diffusion of small molecules are provided by the peripheral channels that surround the central pore, as was originally proposed by Hinshaw et al. (4). In contrast to the central pore that possesses a high degree of hydrophobicity, which is believed to promote passage of nuclear transport receptors (34, 44, 57, 58), the peripheral channels (or the central diffusion channel in the model proposed by Peters) are hydrophilic in nature. The difference in local dielectric may then act to sort incoming transport substrates into the different translocation paths according to their surface hydrophobicity/hydrophilicity.


   FOOTNOTES
 
* This work was supported by grants from the Human Frontier Science Program and from the Minerva Foundation with funding from the federal German ministry for education and research. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 To whom correspondence should be addressed. Tel.: 972-8-934-2982; Fax: 972-8-934-6010; E-mail: ziv.reich{at}weizmann.ac.il.

2 The abbreviations used are: NE, nuclear envelope; NLS, nuclear localization signal; BSA, bovine serum albumin; GFP, green fluorescent protein; GST, glutathione S-transferase; NPC, nuclear pore complex; FITC, fluorescein isothiocyanate; RITC, rhodamine isothiocyanate; TRITC, tetramethylrhodamine isothiocyanate; PBS, phosphate-buffered saline; TB, transport buffer. Back

3 B. Naim, V. Brumfeld, R. Kapon, V. Kiss, R. Nevo, and Z. Reich, unpublished data. Back


   ACKNOWLEDGMENTS
 
We thank Michael Elbaum for providing us NLS-conjugated BSA and nucleoplasmin-GFP and Yuh Min Chook for providing us with the plasmid encoding for GST-transportin.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Reichelt, R., Holzenburg, A., Buhle, E. L., Jr., Jarnik, M., Engel, A., and Aebi, U. (1990) J. Cell Biol. 110, 883-894[Abstract/Free Full Text]
  2. Cronshaw, J. M., Krutchinsky, A. N., Zhang, W., Chait, B. T., and Matunis, M. J. (2002) J. Cell Biol. 158, 915-927[Abstract/Free Full Text]
  3. Rout, M. P., Aitchison, J. D., Suprapto, A., Hjertaas, K., Zhao, Y., and Chait, B. T. (2000) J. Cell Biol. 148, 635-651[Abstract/Free Full Text]
  4. Hinshaw, J. E., Carragher, B. O., and Milligan, R. A. (1992) Cell 69, 1133-1141[CrossRef][Medline] [Order article via Infotrieve]
  5. Akey, C. W., and Radermacher, M. (1993) J. Cell Biol. 122, 1-19[Abstract/Free Full Text]
  6. Stoffler, D., Feja, B., Fahrenkrog, B., Walz, J., Typke, D., and Aebi, U. (2003) J. Mol. Biol. 328, 119-130[CrossRef][Medline] [Order article via Infotrieve]
  7. Beck, M., Forster, F., Ecke, M., Plitzko, J. M., Melchior, F., Gerisch, G., Baumeister, W., and Medalia, O. (2004) Science 306, 1387-1390[Abstract/Free Full Text]
  8. Goldberg, M. W., and Allen, T. D. (1993) J. Cell Sci. 106, 261-274[Abstract]
  9. Ris, H. (1991) EMSA Bull. 21, 54-56
  10. Nevo, R., Markiewicz, P., Kapon, R., Elbaum, M., and Reich, Z. (2000) Single Molecules 1, 109-114
  11. Kapon, R., Nevo, R., Shuhmaher, N., Shahar, D., and Reich, Z. (2001) Single Molecules 2, 86-89
  12. Rout, M. P., and Blobel, G. (1993) J. Cell Biol. 123, 771-783[Abstract/Free Full Text]
  13. Fahrenkrog, B., Hurt, E. C., Aebi, U., and Pante, N. (1998) J. Cell Biol. 143, 577-588[Abstract/Free Full Text]
  14. Yang, Q., Rout, M. P., and Akey, C. W. (1998) Mol. Cell 1, 223-234[CrossRef][Medline] [Order article via Infotrieve]
  15. Corbett, A. H., and Silver, P. A. (1997) Microbiol. Mol. Biol. Rev. 61, 193-211[Abstract]
  16. Mattaj, I. W., and Englmeier, L. (1998) Annu. Rev. Biochem. 67, 265-306[CrossRef][Medline] [Order article via Infotrieve]
  17. Gorlich, D., and Kutay, U. (1999) Annu. Rev. Cell Dev. Biol. 15, 607-660[CrossRef][Medline] [Order article via Infotrieve]
  18. Yoneda, Y. (2000) Genes Cells 5, 777-787[Abstract]
  19. Fried, H., and Kutay, U. (2003) Cell Mol. Life Sci. 60, 1659-1688[CrossRef][Medline] [Order article via Infotrieve]
  20. Weis, K. (2003) Cell 112, 441-451[CrossRef][Medline] [Order article via Infotrieve]
  21. Suntharalingam, M., and Wente, S. R. (2003) Dev. Cell 4, 775-789[CrossRef][Medline] [Order article via Infotrieve]
  22. Pemberton, L. F., and Paschal, B. M. (2005) Traffic 6, 187-198[CrossRef][Medline] [Order article via Infotrieve]
  23. Paine, P. L., Moore, L. C., and Horowitz, S. B. (1975) Nature 254, 109-114[CrossRef][Medline] [Order article via Infotrieve]
  24. Keminer, O., and Peters, R. (1999) Biophys. J. 77, 217-228
  25. Pante, N., and Kann, M. (2002) Mol. Biol. Cell 13, 425-434[Abstract/Free Full Text]
  26. Ribbeck, K., and Gorlich, D. (2001) EMBO J. 20, 1320-1330[CrossRef][Medline] [Order article via Infotrieve]
  27. Stevens, B. J., and Swift, H. (1966) J. Cell Biol. 31, 55-77[Abstract/Free Full Text]
  28. Peters, R. (1984) EMBO J. 3, 1831-1836[Medline] [Order article via Infotrieve]
  29. Dworetzky, S. I., Lanford, R. E., and Feldherr, C. M. (1988) J. Cell Biol. 107, 1279-1287[Abstract/Free Full Text]
  30. Fahrenkrog, B., and Aebi, U. (2003) Nat. Rev. Mol. Cell. Biol. 4, 757-766[Medline] [Order article via Infotrieve]
  31. Chook, Y. M., and Blobel, G. (1999) Nature 399, 230-237[CrossRef][Medline] [Order article via Infotrieve]
  32. Siebrasse, J. P., and Peters, R. (2002) EMBO Rep. 3, 887-892[CrossRef][Medline] [Order article via Infotrieve]
  33. Kubitscheck, U., Grunwald, D., Hoekstra, A., Rohleder, D., Kues, T., Siebrasse, J. P., and Peters, R. (2005) J. Cell Biol. 168, 233-243[Abstract/Free Full Text]
  34. Ribbeck, K., Lipowsky, G., Kent, H. M., Stewart, M., and Gorlich, D. (1998) EMBO J. 17, 6587-6598[CrossRef][Medline] [Order article via Infotrieve]
  35. Pinol-Roma, S., and Dreyfuss, G. (1992) Nature 355, 730-732[CrossRef][Medline] [Order article via Infotrieve]
  36. Andersen, J. S., Lam, Y. W., Leung, A. K., Ong, S. E., Lyon, C. E., Lamond, A. I., and Mann, M. (2005) Nature 433, 77-83[CrossRef][Medline] [Order article via Infotrieve]
  37. Adam, S. A., Marr, R. S., and Gerace, L. (1990) J. Cell Biol. 111, 807-816[Abstract/Free Full Text]
  38. Soullam, B., and Worman, H. J. (1995) J. Cell Biol. 130, 15-27[Abstract/Free Full Text]
  39. Shahin, V., Danker, T., Enss, K., Ossig, R., and Oberleithner, H. (2001) FASEB J. 15, 1895-1901[Abstract/Free Full Text]
  40. Danker, T., Schillers, H., Storck, J., Shahin, V., Kramer, B., Wilhelmi, M., and Oberleithner, H. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 13530-13535[Abstract/Free Full Text]
  41. Enss, K., Danker, T., Schlune, A., Buchholz, I., and Oberleithner, H. (2003) J. Membr. Biol. 196, 147-155[CrossRef][Medline] [Order article via Infotrieve]
  42. Mazzanti, M., Bustamante, J. O., and Oberleithner, H. (2001) Physiol. Rev. 81, 1-19[Abstract/Free Full Text]
  43. Feldherr, C. M., and Akin, D. (1997) J. Cell Sci. 110, 3065-3070[Abstract]
  44. Peters, R. (2005) Traffic 6, 421-427[CrossRef][Medline] [Order article via Infotrieve]
  45. Davis, L. I., and Blobel, G. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 7552-7556[Abstract/Free Full Text]
  46. Holt, G. D., Snow, C. M., Senior, A., Haltiwanger, R. S., Gerace, L., and Hart, G. W. (1987) J. Cell Biol. 104, 1157-1164[Abstract/Free Full Text]
  47. Snow, C. M., Senior, A., and Gerace, L. (1987) J. Cell Biol. 104, 1143-1156[Abstract/Free Full Text]
  48. Newmeyer, D. D., and Forbes, D. J. (1988) Cell 52, 641-653[CrossRef][Medline] [Order article via Infotrieve]
  49. Finlay, D. R., Newmeyer, D. D., Price, T. M., and Forbes, D. J. (1987) J. Cell Biol. 104, 189-200[Abstract/Free Full Text]
  50. Yoneda, Y., Imamoto-Sonobe, N., Yamaizumi, M., and Uchida, T. (1987) Exp. Cell Res. 173, 586-595[CrossRef][Medline] [Order article via Infotrieve]
  51. Adam, E. J., and Adam, S. A. (1994) J. Cell Biol. 125, 547-555[Abstract/Free Full Text]
  52. Rutherford, S. A., Goldberg, M. W., and Allen, T. D. (1997) Exp. Cell Res. 232, 146-160[CrossRef][Medline] [Order article via Infotrieve]
  53. Adachi, M., Fukuda, M., and Nishida, E. (1999) EMBO J. 18, 5347-5358[CrossRef][Medline] [Order article via Infotrieve]
  54. Akey, C. W. (1989) J. Cell Biol. 109, 955-970[Abstract/Free Full Text]
  55. Wilken, N., Kossner, U., Senecal, J. L., Scheer, U., and Dabauvalle, M. C. (1993) J. Cell Biol. 123, 1345-1354[Abstract/Free Full Text]
  56. Dabauvalle, M. C., Schulz, B., Scheer, U., and Peters, R. (1988) Exp. Cell Res. 174, 291-296[CrossRef][Medline] [Order article via Infotrieve]
  57. Ribbeck, K., Kutay, U., Paraskeva, E., and Gorlich, D. (1999) Curr. Biol. 9, 47-50[CrossRef][Medline] [Order article via Infotrieve]
  58. Macara, I. G. (2001) Microbiol. Mol. Biol. Rev. 65, 570-594[Abstract/Free Full Text]
  59. Peters, R. (1986) Biochim. Biophys. Acta. 864, 305-359[Medline] [Order article via Infotrieve]

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