Importin alpha can migrate into the nucleus in an importin beta- and Ran-independent manner
2002; Springer Nature; Volume: 21; Issue: 21 Linguagem: Inglês
10.1093/emboj/cdf569
ISSN1460-2075
Autores Tópico(s)Genomics and Chromatin Dynamics
ResumoArticle1 November 2002free access Importin α can migrate into the nucleus in an importin β- and Ran-independent manner Yoichi Miyamoto Yoichi Miyamoto Department of Frontier Biosciences, Graduate School of Frontier Biosciences, Osaka University, 2-2 Yamada-oka, Suita, Osaka, 565-0871 Japan Search for more papers by this author Miki Hieda Miki Hieda Department of Frontier Biosciences, Graduate School of Frontier Biosciences, Osaka University, 2-2 Yamada-oka, Suita, Osaka, 565-0871 Japan Search for more papers by this author Michelle T. Harreman Michelle T. Harreman Department of Biochemistry,Emory University School of Medicine, 1510 Clifton Road, NE, Atlanta, GA, 30322 USA Search for more papers by this author Masahiro Fukumoto Masahiro Fukumoto Department of Frontier Biosciences, Graduate School of Frontier Biosciences, Osaka University, 2-2 Yamada-oka, Suita, Osaka, 565-0871 Japan Search for more papers by this author Takuya Saiwaki Takuya Saiwaki Department of Frontier Biosciences, Graduate School of Frontier Biosciences, Osaka University, 2-2 Yamada-oka, Suita, Osaka, 565-0871 Japan Search for more papers by this author Alec E. Hodel Alec E. Hodel Department of Biochemistry,Emory University School of Medicine, 1510 Clifton Road, NE, Atlanta, GA, 30322 USA Search for more papers by this author Anita H. Corbett Anita H. Corbett Department of Biochemistry,Emory University School of Medicine, 1510 Clifton Road, NE, Atlanta, GA, 30322 USA Search for more papers by this author Yoshihiro Yoneda Corresponding Author Yoshihiro Yoneda Department of Frontier Biosciences, Graduate School of Frontier Biosciences, Osaka University, 2-2 Yamada-oka, Suita, Osaka, 565-0871 Japan Search for more papers by this author Yoichi Miyamoto Yoichi Miyamoto Department of Frontier Biosciences, Graduate School of Frontier Biosciences, Osaka University, 2-2 Yamada-oka, Suita, Osaka, 565-0871 Japan Search for more papers by this author Miki Hieda Miki Hieda Department of Frontier Biosciences, Graduate School of Frontier Biosciences, Osaka University, 2-2 Yamada-oka, Suita, Osaka, 565-0871 Japan Search for more papers by this author Michelle T. Harreman Michelle T. Harreman Department of Biochemistry,Emory University School of Medicine, 1510 Clifton Road, NE, Atlanta, GA, 30322 USA Search for more papers by this author Masahiro Fukumoto Masahiro Fukumoto Department of Frontier Biosciences, Graduate School of Frontier Biosciences, Osaka University, 2-2 Yamada-oka, Suita, Osaka, 565-0871 Japan Search for more papers by this author Takuya Saiwaki Takuya Saiwaki Department of Frontier Biosciences, Graduate School of Frontier Biosciences, Osaka University, 2-2 Yamada-oka, Suita, Osaka, 565-0871 Japan Search for more papers by this author Alec E. Hodel Alec E. Hodel Department of Biochemistry,Emory University School of Medicine, 1510 Clifton Road, NE, Atlanta, GA, 30322 USA Search for more papers by this author Anita H. Corbett Anita H. Corbett Department of Biochemistry,Emory University School of Medicine, 1510 Clifton Road, NE, Atlanta, GA, 30322 USA Search for more papers by this author Yoshihiro Yoneda Corresponding Author Yoshihiro Yoneda Department of Frontier Biosciences, Graduate School of Frontier Biosciences, Osaka University, 2-2 Yamada-oka, Suita, Osaka, 565-0871 Japan Search for more papers by this author Author Information Yoichi Miyamoto1, Miki Hieda1, Michelle T. Harreman2, Masahiro Fukumoto1, Takuya Saiwaki1, Alec E. Hodel2, Anita H. Corbett2 and Yoshihiro Yoneda 1 1Department of Frontier Biosciences, Graduate School of Frontier Biosciences, Osaka University, 2-2 Yamada-oka, Suita, Osaka, 565-0871 Japan 2Department of Biochemistry,Emory University School of Medicine, 1510 Clifton Road, NE, Atlanta, GA, 30322 USA ‡Y.Miyamoto and M.Hieda contributed equally to this work *Corresponding author. E-mail: [email protected] The EMBO Journal (2002)21:5833-5842https://doi.org/10.1093/emboj/cdf569 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info A classical nuclear localization signal (NLS)-containing protein is transported into the nucleus via the formation of a NLS-substrate/importin α/β complex. In this study, we found that importin α migrated into the nucleus without the addition of importin β, Ran or any other soluble factors in an in vitro transport assay. A mutant importin α lacking the importin β-binding domain efficiently entered the nucleus. Competition experiments showed that this import pathway for importin α is distinct from that of importin β. These results indicate that importin α alone can enter the nucleus via a novel pathway in an importin β- and Ran-independent manner. Furthermore, this process is evolutionarily conserved as similar results were obtained in Saccharomyces cerevisiae. Moreover, the import rate of importin α differed among individual nuclei of permeabilized cells, as demonstrated by time-lapse experiments. This heterogeneous nuclear accumulation of importin α was affected by the addition of ATP, but not ATPγS. These results suggest that the nuclear import machinery for importin α at individual nuclear pore complexes may be regulated by reaction(s) that require ATP hydrolysis. Introduction Molecular migration between the nucleus and cytoplasm occurs through the nuclear pore complex (NPC) present in the double membrane of the nuclear envelope. The NPC, a huge proteinaceous structure composed of 50–100 different species of proteins called nucleoporins, is estimated to have a total mass of ∼125 MDa in higher eukaryotes and 66 MDa in Saccharomyces cerevisiae. Whereas molecules <20–40 kDa are able to passively diffuse through the NPC into the nucleus, the nuclear import of larger molecules is generally dependent on the presence of a specific signal sequence, the nuclear localization signal (NLS), and is often associated with a requirement for metabolic energy (Görlich and Kutay, 1999). The first identified NLS was that of the SV40 large T-antigen, which consists of a short stretch of basic amino acids, designated as the basic type NLS. This type of NLS is divided into two groups, monopartite and bipartite, based on the number of basic amino acid clusters (Dingwall and Laskey, 1991). The nuclear import of basic type NLS-containing proteins is mediated by specific soluble factors that form a stable complex, the nuclear pore-targeting complex, in the cytoplasm (Imamoto et al., 1995a). The complex is composed of two essential components that are referred to as importin α and β (Görlich and Mattaj, 1996). In addition to these molecules, several factors participate in this transport system, including a small GTPase Ran (Moore and Blobel, 1993) and Ran-binding proteins (Paschal and Gerace, 1995). Ran has a low intrinsic activity with respect to GDP/GTP exchange and GTP hydrolysis. RCC1, a guanine nucleotide exchange factor of Ran, accelerates the dissociation of the guanine nucleotide from Ran, thereby converting RanGDP to RanGTP in the cells (Bischoff and Ponstingl, 1991). Since RCC1 is a chromatin protein and is located in the nucleus, it is generally thought that the generation of the GTP-bound form of Ran occurs in the nucleus. NTF2 facilitates the uptake of RanGDP from the cytoplasm into the nucleus in order to maintain the RanGTP gradient. In the cytoplasm, importin α interacts with a classical basic type NLS-bearing karyophile and importin β. The resultant importin α/β/NLS-substrate ternary complex migrates into the nucleoplasm through the NPC in an importin β-dependent manner. Once the importin α/β/NLS-substrate complex reaches the nucleoplasm, importin β interacts directly with RanGTP, resulting in the release of importin α and the NLS-bearing karyophile. Structural analysis of importin α has revealed three functional domains. One is an importin β binding (IBB) domain, which is located in the region comprised of residues 10–55 in the N-terminus (Görlich et al., 1996; Weis et al., 1996). The second is a hydrophobic central domain known as the armadillo (arm) repeat domain (Yano et al., 1994). The crystal structure of yeast importin α (KAP60) or mouse importin α2 (importin αP1) showed that this central domain is composed of a tandem array of 10 arm repeats, organized in a right-handed superhelix of helices. Each arm repeat consists of three α-helices that are connected by loops. Moreover, it has been shown that a classical NLS binds to two sites within a helical surface groove of the arm repeat domain in importin α (Conti et al., 1998; Kobe, 1999; Fontes et al., 2000). The third is a short acidic domain in the C-terminus. This region binds to the cellular apoptosis susceptibility gene product (CAS), the function of which is to export importin α from the nucleoplasm (Kutay et al., 1997; Herold et al., 1998). Saccharomyces cerevisiae contains a single importin α, referred to as Srp1p, which is an essential gene originally identified as a suppressor of the temperature-sensitive RNA polymerase I mutation (Yano et al., 1992). In contrast to yeast, mammals, such as the human and mouse, possess at least six importin α isoforms [importin α1, αS1, NPI1; importin α2, αP1, PTAC58, Rch1; importin α3, αQ1, Qip1; importin α4, αQ2; importin α6 (identified only in human); and importin αS2, NPI2] (Cortes et al., 1994; Cuomo et al., 1994; Imamoto et al., 1995b; Köhler et al., 1997; Tsuji et al., 1997). These importin α proteins can be classified into three subfamilies. One subfamily consists of importin α1, α6 and αS2; the second, importin α2; and the third includes importin α3 and α4. The amino acid sequence identity among these three subfamilies is ∼50%, and the identity between members in a subfamily is ∼80–85%. These known importin α molecules interact specifically with importin β, resulting in high-affinity binding to classical NLSs. They are also exported by CAS from the nucleoplasm. The results of several investigations suggest that these importin α molecules show different expression patterns in adult tissues or cell lines, and display specific recognition for distinct classical NLSs or karyophiles (Prieve et al., 1996; Miyamoto et al., 1997; Nadler et al., 1997; Sekimoto et al., 1997; Tsuji et al., 1997). The existence of distinct importin α isoforms in mammals implies that different isoforms could recognize distinct target proteins. The dynamic movement of importin β has been extensively analyzed and a large body of data has accumulated to show that importin β by itself translocates through the NPC bi-directionally by binding directly to nucleoporins. However, the intracellular behavior of importin α has not been extensively examined. The goal of this study was to develop a better understanding of the intracellular dynamics of importin α. As a result, we found that importin α alone can migrate into the nucleus in an importin β- and Ran-independent manner. In addition, our findings show that the pathway of importin α import is distinct from that of importin β import. Furthermore, this novel import is conserved from mammals to yeast, suggesting that the importin α import may have physiological significance. Results The IBB domain-lacking importin α mutant is localized in the nucleus when transiently expressed in mammalian cells Importin α is known to be a classical NLS receptor and to function as an adapter molecule between an NLS-bearing karyophile and importin β. Our previous studies showed, by indirect immunofluorescence with anti-importin α antibodies, that importin α is localized throughout the cytoplasm and nucleus, and that a portion of cytoplasmically injected anti-importin α antibodies migrated into the nucleus in a piggy-back fashion (Imamoto et al., 1995b), indicating that endogenous importin α traverses the nuclear envelope in living mammalian cells. In this study, to analyze the dynamic behavior of importin α more precisely in living cells, we first transiently expressed a variety of GFP–importin α constructs in HeLa cells. As shown in Figure 1, full-length importin α was predominantly localized in the nucleus, which is consistent with the current model in which importin α enters the nucleus via complex formation with a karyophile and importin β. However, we found that an importin α mutant (101–534) lacking the IBB domain, which is unable to bind to importin β, also accumulated in the nucleus, indicating that importin α is able to migrate into the nucleus in an IBB domain-independent manner. In addition, it was found that this migration was dependent on the C-terminal arm repeat domain containing arm repeat 9 and a portion of arm repeat 10 (Figure 1). Furthermore, this IBB domain-independent nuclear import of importin α is distinguished from the import through complex formation with importin β, because a mutant (1–413) lacking arm 9 and a portion of arm 10 but containing the IBB domain could accumulate in the nucleus. These findings suggest two possibilities: (i) importin α has the ability to migrate into the nucleus by itself; or (ii) importin α is carried into the nucleus in a piggy-back fashion by binding to multimeric karyophiles complexed with importin α/β. Figure 1.Nuclear accumulation of importin α lacking an IBB domain. (A) Summary and the cellular localization of the importin α (mNPI2) deletion mutants used in this transfection analysis. These mutants were constructed into pEGFP-C2 transfection vectors (see Materials and methods). (B) Cellular localization of these mutants in transiently transfected HeLa cells. After transfection (12 h), the cells were fixed with 3.7% formaldehyde in PBS and the subcellular localization of EGFP-fused proteins was observed. Download figure Download PowerPoint Importin α migrates into the nucleus in the absence of cytosol in vitro In order to characterize the IBB domain-independent nuclear import of importin α, we next used digitonin-permeabilized cells. Consistent with the previous results, the nuclear import of the SV40 T-antigen NLS fused with GST and GFP (GST–NLS–GFP) was observed only in the presence of cytosolic extract and an ATP regeneration system (Figure 2A). Surprisingly, we found that recombinant GFP–importin α accumulated in the nucleus, even in the absence of exogenous cytosolic extracts. The nuclear migration of importin α was sensitive to temperature and was inhibited by wheatgerm agglutinin (WGA), which is known to bind to glycosylated nucleoporins. These results indicate that the nuclear migration of importin α does not result from passive diffusion, but that this protein migrates into the nucleus through the gated channels of the NPC. Figure 2.Importin α is able to migrate into the nucleus in an importin β-independent manner in an in vitro assay. (A–D) Cells were treated with 40 μg/ml digitonin in TB (see Materials and methods) for 5 min on ice, and after washing with PBS twice, the cells were incubated with 10 μl of testing solution. (A) Digitonin-permeabilized MDBK cells were incubated with 2.5 μM GFP–importin α (mRch1) with TB alone or 2.5 μM GST–NLS–GFP with cytosolic extracts prepared from mouse Ehrlich ascites tumor cells and an ATP regeneration system for 20 min at 30°C or on ice. The other import reactions of GFP–importin α or GST–NLS–GFP were performed for 20 min at 30°C after pretreatment with 0.4 mg/ml WGA for 10 min at 30°C, or in the presence of 25 μM MBP–IBB for 20 min at 30°C. (B) Digitonin-permeabilized MDBK cells were incubated with 1 μM wild-type GST–importin α (NPI1) or 1 μM GST–ΔIBB importin α (NPI1; 78–534 amino acids) for 20 min at 30°C. As a control, 1 μM GST alone or 1 μM GST–importin β was used. To detect the GST portion, anti-GST–antibody (B-14; a mouse monoclonal IgG; Santa Cruz Biotechnology, Inc.) (2 μg/ml) was used and detected with RITC-conjugated goat anti-mouse IgG. (C) Digitonin-permeabilized MDBK cells were incubated with 2.5 μM GFP–mRch1, 2.5 μM GFP–mNPI2 and 2.5 μM GFP–mQip1 alone or 2.5 μM GST–NLS–GFP in the presence or absence of Ehrlich cytosolic extracts and an ATP regeneration system for 20 min at 30°C. (D) Digitonin-permeabilized MDBK cells were incubated with 2.5 μM GFP–importin α (mRch1) alone or 2.5 μM GST–NLS–GFP with Ehrlich ascites tumor cells cytosolic extracts and an ATP regeneration system in the presence of 25 μM T-BSA or 25 μM revT-BSA for 20 min at 30°C. Download figure Download PowerPoint To rule out the possibility that this cytosol-independent nuclear import of importin α is dependent upon endogenous importin β remaining in the digitonin-permeabilized cells, maltose binding protein (MBP)-fused IBB domain-containing protein (MBP–IBB) was added to the permeabilized cells. As shown in Figure 2A, we found that the addition of the IBB domain had no effect on the nuclear accumulation of importin α, indicating that the remaining importin β in permeabilized cells is not involved in the nuclear migration of importin α and that importin α is not transported into the nucleus in a piggy-back fashion by binding to another karyophile/importin α/β complex. In order to confirm that the cytosol-independent import of importin α is independent of the IBB domain, we employed a GST–importin α mutant lacking the IBB domain (GST–ΔIBB importin α) in the in vitro assay. As shown in Figure 2B, the IBB domain-lacking mutant of importin α efficiently migrated into the nucleus. In addition, three isoforms of importin α from the mouse, importin α2/mRch1, importin αS2/mNPI2 and importin α3/mQip1, entered the nucleus in a similar manner (Figure 2C), indicating that the three distinct importin α subfamilies have the same activity with respect to their ability to enter the nucleus. We next examined the effect of basic type NLS-containing substrates on the import of importin α. When an excess amount of unlabeled T-BSA, which is the SV40 T-antigen NLS conjugated to BSA, was added to the assay, the cytosol-independent nuclear import of importin α was strongly inhibited, while it was not affected at all by the addition of unlabeled reverse T-BSA (revT-BSA) (Figure 2D). In addition, the presence of an excess amount of T-antigen NLS peptide or of GST–NLS–GFP recombinant protein also dramatically inhibited the nuclear import of importin α (data not shown). These results indicate that importin α has the ability to migrate into the nucleus by itself in an importin β-independent manner when it is not bound to basic type NLS-containing proteins, and that importin α itself possesses the necessary and sufficient information to be translocated via the NPC. Nuclear import of importin α occurs without the GTP hydrolysis of Ran To further verify that the migration of importin α into the nucleus is not dependent on Ran, we examined the effect of a dominant-negative Ran mutant that is defective in GTP hydrolysis, Q69LRanGTP, which has been demonstrated to strongly inhibit the Ran-dependent import of known substrates (Palacios et al., 1996), on the cytosol-independent nuclear migration of importin α. As shown in Figure 3, Q69LRanGTP had no effect on importin α import, while the cytosol-dependent nuclear import of GST–NLS–GFP was significantly inhibited. Furthermore, it was found that a non-hydrolyzable GTP analog, GTPγS, did not inhibit the nuclear migration of importin α. These data strongly suggest that Ran does not play a role in the nuclear import of importin α. Figure 3.Imporin β-independent nuclear import of importin α occurs without the support of GTP hydrolysis of Ran. Digitonin-permeabilized MDBK cells were incubated with 2.5 μM GFP–importin α (mRch1) alone, or 2.5 μM GST–NLS–GFP with Ehrlich cytosolic extracts and an ATP regeneration system, in the presence of 25 μM Q69LRanGTP or 1 mM GTPγS for 20 min at 30°C. Download figure Download PowerPoint Importin α import does not require ATP hydrolysis As discussed above, importin α migrated into the nucleus without the exogenous addition of ATP. In order to determine the energy requirement for importin α import, digitonin-permeabilized cells were treated with apyrase. As shown in Figure 4, the pre-incubation of the cells with apyrase had no effect on importin α import, whereas the import of GST–NLS–GFP was completely abolished in the apyrase-pretreated permeabilized cells. In addition, a non-hydrolyzable ATP analog, ATPγS, had no effect on the nuclear import of importin α. These data suggest that the requirement for ATP and its hydrolysis is much less for the import of importin α, compared with conventional NLS-mediated nuclear import. Figure 4.Nuclear import of importin α is distinct from that of a conventional NLS-containing karyophile in terms of its requirement for ATP hydrolysis. Digitonin-permeabilized MDBK cells were incubated with TB containing 0.1 U/ml apyrase (Sigma) and 2% BSA for 5 min at 30°C. After rinsing the cells with TB, they were incubated with import mixtures containing 2.5 μM GFP–importin α (mRch1) alone or 2.5 μM GST–NLS–GFP with Ehrlich ascites tumor cell cytosolic extracts and ATP regeneration system for 20 min at 30°C. The permeabilized cells were also incubated with the import mixtures in the presence of 1 mM ATPγS for 20 min at 30°C. Download figure Download PowerPoint Nuclear import of importin α is saturable but does not compete with importin β To further characterize importin α import, we examined the saturability of the import process. For this experiment, an excess amount of untagged importin α was added, in the presence of GFP–importin α, to the in vitro assay system. Under these conditions, the nuclear accumulation of GFP–importin α was greatly diminished, while no effect was observed for the nuclear import of GST–NLS–GFP (Figure 5). In contrast, an importin β mutant (1–449 amino acids), which is known to inhibit the nuclear import of both the importin α/β/NLS-substrate complex and importin β alone through a specific interaction with nucleoporins (Kose et al., 1997), had no effect on importin α import. Collectively, these results clearly demonstrate that importin α import is saturable and involves specific interactions, probably with the NPC, that are distinct from that of both importin α/β/NLS-substrate complex and importin β alone. Figure 5.Nuclear import of importin α is saturable but does not compete with importin β. Digitonin-permeabilized MDBK cells were incubated with 2.5 μM GFP–importin α (mRch1) alone, or 2.5 μM GST–NLS–GFP with Ehrlich ascites tumor cells cytosolic extracts and ATP regeneration system, in the presence of an excess (∼10×) amount of untagged importin α (mRch1), or 25 μM importin β mutant (1–449 amino acids) for 20 min at 30°C. Download figure Download PowerPoint Importin β-independent nuclear import activity of importin α is conserved in yeast To further examine the process of importin β-independent nuclear import of importin α, we examined whether it also occurred in yeast. Results shown in Figure 6 indicate that yeast importin α that lacks the IBB domain (ΔIBB importin α) is efficiently localized to the nucleus and this localization is indistinguishable from that of wild-type importin α. Similar results are obtained whether importin α proteins are visualized as C-terminal GFP fusion proteins (Figure 6A) or epitope tagged and detected with an anti-myc antibody (Figure 6B). To confirm that import of ΔIBB importin α is not mediated by binding to NLS cargo that contains multiple NLSs, we examined whether a mutant of importin α that cannot bind NLS cargo (Gruss et al., 2001), ΔIBB ED importin α, could still enter the nucleus. Results shown in Figure 6A indicate that ΔIBB ED importin α is localized to the nucleus as efficiently as either wild-type or ΔIBB importin α. This suggests that yeast ΔIBB importin α does not enter the nucleus by binding to cargo that contains more than one nuclear targeting signal and piggy-backing into the nucleus via another import receptor that recognizes that cargo. Taken together, these results support the finding that there is an importin β-independent mechanism for nuclear import of importin α and suggest that this mechanism has been conserved through evolution. Figure 6.Saccharomyces cerevisiae importin α enters the nucleus in an importin β-independent manner. (A) Wild-type yeast cells were transformed with importin α–GFP, ΔIBB importin α–GFP or ΔIBB ED importin α–GFP. GFP fusion proteins were visualized by direct fluorescence microscopy. Corresponding differential interference contrast (DIC) images are shown. (B) Myc-tagged importin α proteins were detected by indirect immunofluorescence using an anti-myc antibody. Cells were also stained with DAPI to show the position of the nucleus. Corresponding DIC images are shown. Download figure Download PowerPoint The rate of import of importin α differs among individual nuclei of permeabilized cells To estimate the rate of entry of importin α into the nucleus in permeabilized cells, we monitored its import into the nucleus by time-lapse photography using confocal microscopy. For control experiments, the rate of entry of GST–NLS–GFP and GFP–importin β was examined. The nuclear import of GST–NLS–GFP was monitored at intervals of 3 s in the presence of importin α/β, Ran, NTF2 and an ATP regenerating system, and GFP–importin β, GFP–importin α and GFP–ΔIBB importin α were monitored at intervals of 1 s in the absence of soluble factors and ATP. As shown in Figure 7, we observed a steady increase in nuclear fluorescence of GST–NLS–GFP that continued for ∼25 min and found that the fluorescence was concentrated in almost all of the nuclei at nearly the same rate. Nuclear import of GFP–importin β occurred more rapidly than that of GST–NLS–GFP and reached a plateau within ∼4 min and, like GST–NLS–GFP, the nuclear accumulation of GFP–importin β occurred at almost the same rate in almost all of the nuclei. The nuclear import of importin α, however, was very unique and significantly different from that of GST–NLS–GFP and importin β. That is, GFP–importin α accumulated in individual nuclei at distinctly different rates, although the nuclear accumulation of importin α was observed to be as rapid as that of importin β in a few cells. For the most unique example, we observed a nucleus (shown by an arrowhead in Figure 7) where GFP–importin α was rapidly concentrated in the nucleus after an incubation of ∼6 min. Importantly, GFP–ΔIBB importin α also accumulated in individual nuclei in a similar manner to the wild-type importin α, which clearly eliminates the possibility that endogenous residual importin β remaining in the individual permeabilized cells may be recycled and thus affect the influx kinetics of GFP–importin α. These results suggest that each nucleus may be functionally different with respect to the import of importin α. Furthermore, more careful observation indicated that GFP–importin α transiently concentrated at the nuclear periphery, suggesting that importin α may interact with nucleoporins at the nuclear pore before translocation into the nucleoplasm and that the functional heterogeneity of each nucleus for importin α import may be the result of structural and/or functional heterogeneity of the NPCs of individual nuclei. Figure 7.Time-lapse analysis of importin α nuclear import. Four micromolar GST–NLS–GFP, 4 μM GFP–importin β, 4 μM GFP–importin α (mRch1) and 4 μM GFP–ΔIBB importin α (hRch1) were added to the digitonin-permeabilized MDBK cells and their accumulation into nuclei was recorded in real time by confocal microscopy. The nuclear import of GST–NLS–GFP was monitored in the presence of recombinant importin α (mRch1), importin β, RanGDP, p10/NTF2 and an ATP regeneration system, and that of GFP–importin β, GFP–importin α and GFP–ΔIBB importin α was monitored in the absence of any soluble factors and exogenous ATP. The image of GST–NLS–GFP was captured 500 times at intervals of 3 s. The images of GFP–importin β, GFP–importin α and GFP–ΔIBB importin α were captured 500 times at intervals of 1 s. A nucleus indicated by an arrowhead showed that GFP–importin α was rapidly concentrated in the nucleus just after an incubation of ∼6 min. The change in the mean fluorescence intensity of the nucleus with time was plotted. Download figure Download PowerPoint The heterogeneity of the nuclear import rate of importin α in individual permeabilized cells can be recovered by the addition of ATP To determine whether the heterogeneity of the import rate of importin α for individual nuclei is affected by residual amounts of endogenous soluble transport factors such as importin β, CAS, Ran and NTF2 remaining in the permeabilized cells, we performed indirect immunofluorescence on permeabilized cells using specific antibodies. These factors remained in each permeabilized cell to almost the same extent, not only just after the permeabilization (data not shown) but also after the import reaction (Figure 8A), suggesting that the heterogeneity was not due to these residual, soluble factors. Figure 8.Incubation of permeabilized cells with ATP affects the nuclear import efficiency of importin α. (A) After digitonin-permeabilized HeLa cells were fixed with 3.7% formaldehyde in PBS, endogenous importin β, CAS, Ran and NTF2 were stained with respective specific antibodies (mouse monoclonal antibodies; Transduction Laboratories). These antibodies were detected by Alexa 546-conjugated goat anti-mouse IgG (Molecular Probes). (B) Digitonin-permeabilized MDBK cells were incubated with 2.5 μM GFP–importin α (mRch1) in the absence or presence of an ATP regeneration system for 20 min at 30°C. Download figure Download P
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