A quantitative assay of nuclear import in yeast
To quantitate Kap-mediated import, we developed a nuclear import assay (based, in part, on a previous method; Shulga et al., 1996
) that facilitates rapid, semiautomated cell-by-cell quantitation of import with high spatial and temporal resolution (; for a detailed description of these methods see Leslie et al., 2006
). Our model cargoes were NLSs fused to either GFP or a GFP carrying a C-terminal copy of a single PrA repeat. These fusion proteins were small enough to diffuse rapidly across the NPC; hence, in the absence of active import, they equilibrate between the nucleus and cytoplasm within minutes (Shulga et al., 1996
). Transport was stopped by the addition of metabolic energy poisons, which destroy the RanGTP/GDP gradient (Schwoebel et al., 2002
). Re-import of NLS-GFP was observed seconds after a sample of cells had been washed free of poison and resuspended in glucose-containing media on a microscope slide, allowing the Ran gradient to reform. Import assays were performed in a strain background that contained CFP-tagged Htb2p (a histone) and CFP-tagged Tpi1p (a glycolysis enzyme), which demarked the nucleoplasm and cytoplasm, respectively, whereas Htb2-CFPp also served as an internal calibrant for NLS-GFP concentration. An automated spinning-disk microscopy system was used to acquire confocal images at 15-s intervals over a 10-min time course, by which time the NLS-GFP had fully returned to its steady-state distribution. At each time point, images were taken at three focal points throughout the cells of both the GFP and CFP signals ().
Figure 1. A quantitative measurement of nuclear import rates in vivo. (A) Fluorescence images of a single cell showing NLS-GFP being re-imported over time and of the CFP-tagged marker proteins. (B) Subcellular morphometry obtained from 3D confocal images of the (more ...)
The NLS-GFP concentration in a population of importing cells was calibrated using quantitative Western blotting on a sample from this population, which compared the abundance of NLS-GFP to that of the internal standard, Htb2-CFPp (). Once the quantitative blotting had determined the mean NLS-GFP molar amount for cells in an import assay and correlated this to the mean cellular NLS-GFP fluorescence in that same assay, we could then use a given cell's actual fluorescence to calculate the abundance of NLS-GFP cargo in that cell (Leslie et al., 2006
). We estimated the probable error of these single-cell cargo abundance values to be ±30%.
We needed to measure the size and shape of the assayed cells because a cell with a smaller nucleus would appear to import faster (simply because it has less volume to fill), just as a cell with a greater number of NPCs could potentially import cargo faster. To measure each cell's volumetric statistics, a confocal image series was acquired through the cells, immediately after the time course. From these data, nuclear and cytoplasmic volumes could be directly measured, whereas the area of the nuclear envelope was interpolated from the 3D data using standard isosurface location algorithms (). From similar volumetric measurements of commercially prepared spherical beads (2.5 μm diam), we estimated the uncertainty in our volume measurements to be ±10% and in our surface area measurements to be ±6%. Because it has been shown in yeast that the density of NPCs is a relatively constant 12 NPCs/μm2
throughout the cell cycle (Winey et al., 1997
), we could convert the surface area of the nuclear envelope to an estimate of the number of NPCs in each cell.
These fluorescence microscopy, concentration calibration, and cellular morphometric measurements were combined to give plots of cargo import over time in single cells, which fit single exponential relationships (; average R2 for all single-cell fits, 0.95). The initial rate of cargo import (i.e., import rate at t = 0, where net passive diffusion of naked NLS-GFP across the NPC is negligible) could be estimated in units of cargo molecules/NPC/s for each and every cell assayed. Each time course assayed ~30 cells within a single microscope field, and multiple assays were repeated until the data from ~100 cells had been collected.
Import rates were spread over wide ranges because the NLS-GFP expression constructs were cloned in multiple copy vectors, conferring a random number of copies of the gene to each cell with a commensurate random expression level of the fusion protein. We used this variability to examine the relationship between cargo concentration and import rate (). Each cell's initial import rate measurement and initial cytoplasmic NLS-GFP concentration is displayed as a single point in a scatter plot (gray dots). To better visualize trends in this population dataset, the cargo concentration range was split into several statistical bins, and the mean import rate and cargo concentration within each bin was calculated (blue squares; error bars are the SD of the mean). This moving-average analysis determined that initial import rates of NLS-GFP cargoes followed a simple linear relationship in respect to the available cytoplasmic concentrations of the fluorescent cargo (). We term the slope of this line an effective import rate, being a measure of how quickly a given cargo is imported by its available transport pathways, in units of cargo molecules per NPC per second per micromolar concentration of cargo.
Import of the model ribosomal import cargo Rpl25NLS-GFP-PrAp is largely dependent on Kap123p
We began our investigations with one particular model transport pathway, that of Kap123p, chosen for several reasons. First, it is the most abundant yeast Kap (Rout et al., 1997
; Ghaemmaghami et al., 2003
; this study), mediating the import of ribosomal proteins (Rout et al., 1997
; Sydorskyy et al., 2003
), histones (Mosammaparast et al., 2002
; Glowczewski et al., 2004
), and the mRNA export factor Yra1p (Zenklusen et al., 2001
). Second, deletion of Kap123p leads only to a mild growth defect, so we can work with Δkap123
yeast without deleterious effects on cell growth and nuclear transport as a whole. Third, it is partially redundant with other Kaps (Rout et al., 1997
; Zenklusen et al., 2001
; Sydorskyy et al., 2003
), which accounts for its nonessential nature, and is a matter of considerable interest, being typical of the partial redundancy often observed between import pathways.
The NLS of the ribosomal protein Rpl25p (Schaap et al., 1991
) has been used previously as the model cargo for Kap123p (Rout et al., 1997
). To test whether this NLS is largely dependent on Kap123p, we used our import assay to quantitatively compare import of the Rpl25NLS between wild-type and Δkap123
cells (). In wild-type cells, these initial import rates ranged from 5 to 200 cargo molecules/NPC/s/cell, with a mean of 62, whereas these rates varied from 1 to 11 cargo molecules/NPC/s/cell with a significantly lower mean of 3 in Δkap123
cells. By normalizing for the different concentrations of NLS-GFP cargo in each cell (as previously described; ), we determined that Rpl25NLS cargo was imported at a rate of 1.2 ± 0.1 cargo molecules/NPC/s/μM cargo in wild-type cells; a rate that was ~17 times more rapid than in the absence of Kap123p. Thus, in wild-type cells, Rpl25NLS-GFP-PrAp is primarily imported by Kap123p, with only a residual 6% of its import proceeding through alternative pathways. Hence, in cells with Kap123p present, we could reasonably ignore this low residual transport component.
Figure 2. Almost all the import of Rpl25NLS-GFP-PrAp is through Kap123p. (A) Import rate of Rpl25NLS-GFP-PrAp in Δkap123 and wild-type cells was quantitated using the import assay (; see Materials and methods). Import in one cell from each population, (more ...)
To test whether other NLSs that are recognized by Kap123p behave the same as Rpl25p, we compared the import of Rpl25NLS-GFPp with GFP carrying the NLSs from other Kap123p import cargoes. First, we identified numerous Kap123p-binding proteins, all of which were known to be targeted to the nucleus, and thus likely to contain NLSs (Supplemental materials and methods and Figs. S1 and S2, available at http://www.jcb.org/cgi/content/full/jcb.200608141/DC1
); as expected, the majority of these were either ribosomal proteins or proteins involved in the ribosomal assembly process (Table S3; Rout et al., 1997
; Sydorskyy et al., 2003
). We constructed NLS-GFP fusion proteins from five proteins of varied function chosen amongst this Kap123p-binding group (). The distributions of all but one NLS-GFP fusion protein was significantly affected by Kap123p deletion, but most were still strongly localized to the nucleus, indicating that a significant proportion of their import likely goes through pathways mediated by Kaps other than Kap123p (Fig. S3). One of the NLSs identified was that of another ribosomal protein (Rps1b), which was as profoundly mislocalized in the absence of Kap123p as Rpl25NLS-GFPp. Throughout this study, Rps1bNLS-GFPp transport was quantitated in addition to Rpl25NLS-GFPp and demonstrated essentially identical behavior in both wild-type and Δkap123
yeast (). Thus, Rpl25p (and likely other ribosomal proteins) is an appropriate model cargo for our studies.
NLSs cloned as GFP fusion proteins for this study
Figure 3. Rps1bNLS-GFPp is also a cargo of Kap123p and imported similarly to Rpl25NLS-GFPp. (A) Import of the Rps1bNLS-GFPp cargo was compared with that of Rpl25NLS-GFPp at steady-state in wild-type and Δkap123 cells. The average cellular N/C fluorescence (more ...)
Kap123p imports cargoes more rapidly than either Kap121p or Kap104p at their wild-type abundances
We compared the import of ribosomal NLS-GFP cargoes with that of similarly sized GFP cargoes carrying other published NLSs (); the NLSs of Nab2p, which is carried predominantly by Kap104p (Lee and Aitchison, 1999
), and of Pho4p, which is a cargo of Kap121p (Kaffman et al., 1998
). Individual cell data from these two other cargoes also produced smooth import curves (), which again gave rise to simple linear relationships between import rate and cargo concentration (). These linear relationships suggested that the import system was never saturated with NLS-GFP cargo, even at cargo concentrations exceeding 100 μM, which is some 20-fold higher than the estimated natural cargo concentrations (Riddick and Macara, 2005
). Import of Rpl25NLS by Kap123p was found to be significantly faster than that of the two other pathways, being ~5-fold faster than Nab2NLS/Kap104p and ~10-fold faster than Pho4NLS/Kap121p. Kap123p, thus, appears to be a significantly more effective importer than the other Kaps tested here.
Figure 4. Quantitative comparison of the import rates of model cargoes for various karyopherins. (A) Import in three representative wild-type cells, each containing similar amounts of three different import cargoes: Rpl25NLS, Nab2NLS, or Pho4NLS. The Kap mainly (more ...)
Kap123p and Kap121p share identical import kinetics and saturation points in respect to Rpl25NLS-YFP
Based on in vitro data, it has been suggested that a large variety of Kaps can replace Kap123p for import of ribosomal proteins (Jakel and Gorlich, 1998
). Indeed, the results in indicate that Kaps other than Kap123p are able to import Rpl25NLS-bearing cargoes, although not nearly as rapidly. Therefore, we tested whether other Kaps could substitute for Kap123p's import of Rpl25NLS cargo in vivo if expressed at comparable levels (). Rpl25NLS-GFPp–expressing Δkap123
cells were transformed with vectors overexpressing HA tagged versions of the four Kaps examined in this study. From quantitative Western blots (Rout et al., 2000
; Cross et al., 2002
) performed on strains containing genomically tagged versions of these Kaps, we measured the natural abundances of Kap95p, Kap104p, Kap121p, and Kap123p to be 60,000, 12,000, 18,000 and 100,000 copies/cell, respectively. These data are consistent with codon bias data indicating that Kap123p is the most highly expressed (i.e., abundant) of all Kaps. Although Western blot analysis demonstrated that all four HA-tagged Kaps were expressed at significantly higher levels than endogenous Kap123p, only overexpression of Kap121-HAp (1.8-fold above the natural Kap123p level) was able to compensate for loss of Kap123p in the import of Rpl25NLS-GFPp (), in agreement with previous studies showing that Kap123p was partially redundant with Kap121p (Rout et al., 1997
; Zenklusen et al., 2001
; Sydorskyy et al., 2003
). Thus, our results confirm that, in vivo, ribosomal proteins are not cargos general to most Kaps, but, instead, are only imported by particular Kaps. This contrasts with results obtained for homologous mammalian Kaps in vitro (Jakel and Gorlich, 1998
Figure 5. Kap121p overexpression specifically compensated for import of Rpl25NLS-GFPp in the absence of Kap123p. Rpl25NLS-GFPp and HA-tagged versions of Kap95p, Kap104p, Kap121p, or Kap123p were expressed from separate overexpression plasmids in Δkap123 (more ...)
As increasing amounts of Kap121p augmented the import rate of the model ribosomal cargo, we tested the hypothesis that Kap123p's effective import rate may be caused by its high cellular abundance. To do this, we manipulated the concentration of either Kap123p or Kap121p and measured how such concentration changes affected Rpl25NLS import. Either Kap123p or Kap121p was expressed as a CFP-tagged fusion protein in Δkap123 cells coexpressing Rpl25NLS-YFPp to allow simultaneous quantitation of both Kap and cargo in individual cells (). Because KAP-CFP and RPL25NLS-YFP expression plasmids were separate 2μ multiple-copy vectors, each cotransformed cell produced random amounts of the fluorescently labeled Kap and cargo, presumably proportionate to the amount of each plasmid they received. The Kap-CFPp and Rpl25NLS-YFPp quantities were found to vary independently of each other, resulting in cells with a wide range of either protein (). With two independent concentration variables, each potentially affecting import-rate, we needed to analyze larger numbers of cells than was practical with the quantitative import assay. Hence, we chose to measure import rate using the nuclear-to-cytoplasmic ratio (N/C) of cargo, as this value could be obtained relatively rapidly. We analyzed dependence of the steady-state N/C ratio of Rpl25NLS-YFPp on the cytoplasmic concentrations of either Kap121-CFPp or Kap123-CFPp.
Figure 6. Measurement of karyopherin-dependent saturation kinetics in vivo. (A) Rpl25NLS-YFPp and either Kap121- or Kap123-CFPp were expressed from separate random-copy plasmids in Δkap123 yeast. Images of cells simultaneously expressing different quantities (more ...)
Around the physiological concentration of Kap123p (~5 μM), we observed an approximately linear relationship between its cytoplasmic concentration and the import of Rpl25NLS-YFPp (). At concentrations of Kap123-CFPp in excess of 15 μM, the N/C ratio of Rpl25NLS-YFPp plateaued with a half-maximal Kap123p concentration of ~7 μM.
Interestingly, Kap121-CFPp displayed a relationship in respect to Rpl25NLS import that was practically indistinguishable from Kap123p-CFPp (). These highly similar import curves were observed despite findings that Kap123p and Kap121p prefer a significantly different subset of Nups to mediate their exchange across the NPC (Rout et al., 1997
; Marelli et al., 1998
; Seedorf et al., 1999
; Denning et al., 2001
A reduction in NLS-Kap affinity proportionally reduces import rate
To further examine the apparent equivalence of Kap121p or Kap123p's import kinetics when normalized for concentration, we examined the strength of binding to their common cargo, Rpl25NLS. If these Kaps recognize their common cargo with similar affinities, this would result in equivalent cytoplasmic concentrations of Kap–cargo import complex, which would be indicative of comparable import rates across the NPC.
Hence, we measured the dissociation binding constants (Kd) between Kaps and NLSs, using recombinant purified proteins and an in vitro binding assay ( and ; see Materials and methods). Binding of Kap121p and Kap123p to Rpl25NLS was found to be essentially indistinguishable, with Kd values of 82 and 94 nM, respectively; this is consistent with both Kaps having similar import kinetics. We also measured binding between Pho4NLS and Kap121p to be similar to Rpl25NLS binding, at 93 nM, whereas Nab2NLS binding to Kap104p was significantly tighter at 17 nM. Cleavage of the GST from the Kaps had no significant effect on the measured Kd values (unpublished data). Binding controls found no quantifiable binding between nonspecific Kap–cargo pairs, such as Pho4NLS-Kap123p (unpublished data).
Figure 7. Measurement and manipulation of NLS-Kap dissociation constants. (A) Kds between NLSs and their Kap partners were determined by binding assays using purified, recombinantly expressed proteins (see Materials and methods). Results of four typical experiments (more ...)
Summary of measured import statistics
To test whether these binding affinities were important in determining import rates, we manipulated the Kd of the Rpl25NLS interaction with Kap123p and Kap121p. We mutagenized lysine residues 21 and 22 in the Rpl25NLS sequence to alanines, which reduced the binding strength of the NLS–Kap interaction with both Kap121p and Kap123 ~2.8-fold, and consequently reduced the steady-state NLS-GFP N/C distributions by ~3.5-fold (). This indicates that an increase in Kd between Kap and cargo gives a proportionate decrease in cargo import.
Nonspecific binding effectively competes with NLSs for Kap binding
Certain observations from our studies led us to propose that binding between NLS-GFP cargo and Kaps is highly competitive with its cytoplasmic environment (see Discussion). To test this hypothesis, we examined the effect of adding bacterial cytosol, absent of any natural Kap cargoes, to the NLS–Kap interactions. Increasing concentrations of bacterial cytosol were found to effectively compete with the binding of Pho4NLS to Kap121p (). 1 mg/ml cytosol disrupted ~50% of the specific Kap–NLS binding, and 10 mg/ml cytosol was sufficient to disrupt >90% of the import complexes. This result is unlikely to be a consequence of specific competition, because there are no NLSs in Escherichia coli, which is a prokaryote.
Import is fitted well by a pump–leak model
We showed that import fits well to single exponential relationships, the initial rates of which are linearly related to cytoplasmic cargo concentration (). Around physiological levels, the Kap concentration is linearly related to the import rates. However, at still higher concentrations of Kap the import rates ultimately saturate, following Michaelis–Menten–like curves (). In addition, we find that reducing the affinity of a cargo for its Kap proportionally reduced its import (). Considering these observations, nuclear import of our small NLS-GFP cargoes appears to be well represented by a pump–leak model, such as those used to describe a variety of biological pumps (e.g., ion channels). These pumps actively transport their cargo across a membrane, against a tendency for that cargo to leak back across the membrane through channels (Hodgkin and Keynes, 1955
). We therefore fitted our import data to a simple pump–leak model (). From this model, we obtained quantitative estimates of the kinetic parameters of nuclear import in living yeast cells.
Figure 8. Import through the NPC was modeled as a saturable first-order process balanced against a passive leak. (A) Diagram of the seven basic processes considered in our pump–leak model of import: (i) the passive leak of naked NLS-GFP cargo across the (more ...)
In building our model, we assumed a constant Kap concentration in the cytoplasm, consistent with our observations that Kap distributions were not altered by even the highest obtainable Kap or cargo concentrations, and that Kap distributions did not change during our import assays (unpublished data). This assumption was further supported by the reported rapid translocation of naked Kaps (Ribbeck and Gorlich, 2001
). Thus, the recycling system for Ran and Kaps, as previously modeled (Smith et al., 2002
; Riddick and Macara, 2005
), was regarded as an essential but imperturbable and very rapid background process. We also assumed instantaneous mixing in the nucleus and cytoplasm, justified by the small size of yeast cells and the estimated 100-ms intracellular diffusion rates (Wachsmuth et al., 2000
; Smith et al., 2002
), as compared with the several minutes each cargo required to return to steady-state ().
illustrates the components of our pump–leak model. In this model, NLS-GFP cargo binds its cognate Kap (), competing with other unlabeled or “bulk” cargoes (iii) and nonspecific cytosolic proteins (iv and v). Because it was not possible to quantitate the degree of nonspecific competition (see Discussion), we assumed a value for this nonspecific competition that was common to all Kap–cargo pairs; namely, an interaction of 500 nM Kd
with 1-mM competitors. The magnitude of this competition is the minimum capable of simulating the linearity of the observed relationships (), but is physiologically reasonable, given the high concentration of total protein in the cytoplasm ( Zimmerman and Minton, 1993
). Also, we cannot state whether this competition is indicative of binding to the NLS-GFP, to the Kap, or to both, but the resulting import complex concentrations will be the same in either case (unpublished data); hence, we ignore nonspecific protein binding to NLS-GFP (). We adopted an estimate for unlabeled NLS-bearing cargo (B
) of 5 μM for each Kap (Riddick and Macara, 2005
) and assumed a mean Kd
of 20 nM for these cargoes (Catimel et al., 2001
), except for Kap104p, for which we assumed a competitor concentration of 0 μM, as it was observed to bind very few cargos (Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200608141/DC1
). All three binding processes () were assumed to be at equilibrium. The three equilibrium binding expressions, of the general form Kd
= [A][B]/[AB], were solved for [AB] in terms of total Kap and binding protein concentrations (denoted by subscript “T
” in their variable names), resulting in the quadratic equations shown in (bottom). Thus, the concentration of NLS-GFP–Kap import complexes (KC
) was calculated by simultaneous solution of these equations, using the measured and estimated values for the dissociation constants and the total cytoplasmic abundances of each reactant, as listed in .
Next, we considered the import processes. Import through NPCs and subsequent nuclear dissociation of import complexes was modeled as a saturable first-order process () because we had observed saturation of import by Kap that followed a Michaelis–Menten–like relationship (). Here, the kinetic constant k is analogous to the Michaelis–Menten's VMAX, and the saturation constant KS is analogous to KM. Because nonspecific proteins likely interact only transiently with Kaps, we assumed such complexes (KU) were not involved in import. Saturation of import was assumed to be caused by the total concentration of import-competent complexes (i.e., KB + KC), although at nonsaturating conditions (i.e., low Kap concentrations) we modeled import of complexes as a pseudolinear system. Pseudolinear rate constants (k') were calculated for each cell importing each model import cargo in and from their initial import rates and their expected intracellular NLS-GFP–Kap concentration at that time point (see values in ).
Lastly, we considered the relevant passive diffusion processes. Naked cargo is free to leak through NPCs via diffusion, where the rate of passive flux is proportional to the difference between the nuclear and cytoplasmic concentrations of cargo, with an NPC permeability constant of p
(). At steady-state, cargo maintains a constant N/C ratio, which can be shown to be proportional to the ratio k'/p
(Shulga et al., 2000
). Having estimated k'
as described in the previous paragraph, we calculated p
, simply using the final N/C ratio for each cell in these import assays. Estimated k'
values were combined with the measurements and estimates of the other cellular and biochemical parameters listed in , creating a parameter set that was used to simulate import of NLS-GFP cargoes in single cells over time. These simulated curves closely fit the measured import data ().