The protein RCC1 possesses at least two distinct biological functions. The first and best-established function is to generate a Ran-GTP gradient across the nuclear pores (Bischoff and Ponstingl, 1991), which is used to drive the transport of a wide variety of cargo molecules against their concentration gradients (Mattaj and Englmeier 1998
; Melchior and Gerace 1998
; Pemberton et al. 1998
; Görlich and Kutay 1999
; Nakielny and Dreyfuss 1999
; Macara, 2000). A second, more recently characterized function is to create a Ran-GTP gradient near the chromatin surface, which is required for mitotic spindle formation (Carazo-Salas, 1999; Ohba et al. 1999
; Wilde and Zheng 1999
). In both cases, RCC1 behaves as a sort of identification tag for chromatin and hence, in interphase cells, as a marker for the nuclear compartment. It operates by efficiently catalyzing guanine-nucleotide exchange on the Ran GTPase (Bischoff and Ponstingl, 1991). The amount of RCC1 associated with the chromatin must double with each cell cycle. One can imagine two extreme mechanisms to achieve this result. In the first mechanism, RCC1 is only synthesized in S-phase, and must await breakdown of the nuclear envelope in prophase to associate with the chromatin. This mechanism would be problematic for cells such as those of the budding yeast, Saccharomyces cerevisiae
, that use a closed mitotic cycle. In the second mechanism, RCC1 is synthesized throughout the cell cycle and is imported continuously into the nucleus, either by passive diffusion or by a facilitated process.
Although the size of RCC1 (~45 kD) is such that it could, in principle, diffuse passively through the nuclear pores, the rate of diffusion, based on comparison with ovalbumin, which is of similar size (Featherstone et al. 1988
), is likely to be too low to be practicable. Also, accumulation of newly synthesized RCC1 within the cytoplasm would likely reduce the efficiency of nucleocytoplasmic transport and disrupt microtubule dynamics formation. Therefore, one might predict that RCC1 is imported by a facilitated mechanism throughout interphase. In support of this prediction, a conserved polybasic sequence that resembles a classic NLS is found near the NH2
terminus of RCC1 from many species ( A). Attachment of this NTD to β-galactosidase permits accumulation of the fusion protein within nuclei of transiently transfected COS 7 cells (Seino et al. 1992
). Also, a study of multiple importin-α isoforms showed that RCC1 can accumulate within the nuclei of digitonin-permeabilized cells in the presence of energy and soluble transport factors, and that accumulation was enhanced specifically by the importin-α3 isoform (Köhler et al. 1999
). These studies suggest that the import of RCC1 can be mediated by the classical importin-α/β pathway through a conventional NLS. However, these studies did not define the NH2
-terminal sequence as the signal recognized by importin-α3. The NTD has also been reported to bind DNA (Seino et al. 1992
), thus complicating analysis of the transfection data for the NTD-β-galactosidase construct described above. Because the transfected cells had undergone at least one cell cycle (Seino et al. 1992
), one could imagine that the NTD caused binding of the fusion protein to exposed chromatin during mitosis, and hence mediated trapping within nuclei rather than import through the nuclear pores. Moreover, an NH2
-terminal deletion mutant of RCC1 was not excluded from the nuclei of transfected tsBN2 cells and was capable of rescuing these cells at the nonpermissive temperature (Seino et al. 1992
). Either the deletion mutant could diffuse into the nuclei, where it would be retained by binding to chromatin, or there exists a second transport system that is independent of the putative NH2
Given the physiological importance of RCC1 import, we examined the mechanism by which it is translocated into the nucleus using a combination of intact and permeabilized cell assays. We find that the NTD of RCC1 is necessary for binding importin-α and that RCC1 preferentially recognizes the α3 isoform. Attachment of the first 25 amino acids from RCC1 are also sufficient to target a fluorescent reporter protein to the nucleus upon microinjection into the cytoplasm. The NTD of RCC1 is not sufficient, however, to target proteins to chromatin in vitro (data not shown). Furthermore, we show that NTD-mediated transport is dependent upon both a preexisting Ran gradient and energy in vivo. These data confirm that the NTD of RCC1 is a NLS and not a DNA-binding domain.
By time-lapse photography, we found that almost all of the cells concentrated the injected RCC1 protein within their nucleus before we could initiate the time lapse program (<1 min). Assuming that (a) each cell was injected with ~5 × 10−13
L of substrate, (b) there are ~3,000 nuclear pores per nucleus, and (c) ~80% of the injected protein became nuclear within one minute, we can estimate that ~72,000 RCC1 molecules were imported per second (~24 events/pore/s). To our knowledge, this is one of the most rapid nuclear import process recorded to date. In fact, kinetic analysis of RCC1 demonstrated that its import was at least 30-fold greater than the import of a classic NLS-containing import substrate (GGNLS) under similar conditions. Since RCC1 and GGNLS can bind to importin-α3 with similar affinity (), each can access the importin-α/β import pathway; however, GGNLS can both bind and be imported by every importin-α molecule tested to date while RCC1 can not (Köhler et al. 1999
; Welch et al. 1999
). The fact that RCC1 is imported much more rapidly than GGNLS thus suggests that RCC1 possesses another import pathway that is independent of that mediated by importin-α3.
Removal of the NTD from RCC1 abolished binding to importin-α but did not prevent the RCC1 deletion mutant from entering the nucleoplasm ( C). Kinetic analysis of RCC1(23-421)-GFP in vivo is complicated by the fact that a subset of cells did not import the protein within the time frame of the time-lapse experiment; analysis of those cells that imported, however, showed that the rate was indistinguishable from that of RCC1-GFP. The fact that not all of the cells import RCC1(23-421)-GFP within 25 min suggests that the import might be a regulated process. Efforts to either increase or decrease the import of RCC1 (23-421)-GFP have only been successful by treatment at low temperature, the addition of importin-β(45-462), and by competition with excess, unlabeled RCC1 in vitro (, data not shown). The heterogeneity of import may also reflect differences in individual cells to import under time-lapse conditions; however, since cells that did not import RCC1(23-421)-GFP were excluded from kinetic analysis, the overall mean import rate for RCC1-GFP in vivo must be more rapid than for RCC1(23-421)-GFP. This observation, plus the fact that the NTD of RCC1 confers high-affinity binding to importin-α3, suggests that the NTD can facilitate the import of RCC1 in vivo.
Interestingly, all of the RCC1 constructs that were tested accumulated at the nuclear periphery before import (). In our hands, the import of molecules by classic transport pathways, such as GGNLS () and RCC1 (1-25)-NPC (data not shown), do not show this phenotype. A similar accumulation at the nuclear envelope was observed during the import of the RCC1 constructs into the nuclei of temperature-shifted tsBN2 cells (data not shown); therefore, this phenotype is not due to import by a Ran-dependent process. This phenomenon could represent either RCC1 that is bound either directly or indirectly to the nuclear pore, or RCC1 that is binding to chromatin just as it enters the nucleus. The latter hypothesis, however, predicts that accumulation of RCC1 in the nucleus would proceed from the nuclear periphery to the nuclear center, i.e., that the ring of RCC1 would thicken over time, and this process is not observed. If RCC1 is docking at the nuclear pore, then this may be the rate-limiting step in the import of RCC1. The fact that the import of RCC1 was inhibited by importin-β(45-462) suggests that either RCC1 binds to some of the same nucleoporins as importin-β or that access to RCC1-binding sites on nuclear pores is obscured by importin-β. The identity of the putative nucleoporin docking sites for RCC1 remains to be investigated. It is noteworthy that staining of RCC1 at the nuclear periphery was not prominent in digitonin-permeabilized cells incubated with RCC1 at 4°C. Therefore, either binding is of very low affinity or access to the docking site is reduced at low temperatures. Low-affinity binding to nucleoporins is also a characteristic of NTF2, which has an ~25 μM affinity for nucleoporin-FxFG repeats (Chaillan-Huntington et al. 2000
A surprising feature of the factor-independent RCC1 import pathway is that, compared with GGNLS, it is much less sensitive to inhibition by WGA. WGA binds with high affinity to O-linked glycoproteins of the nuclear pore (Stoffler et al. 1999
), including the p62 complex which is localized near the central gated channel of the pore (Guan et al. 1995
). WGA is presumed not to occlude the channel because it does not abrogate the passive diffusion of small dextrans (Finlay et al. 1987
). For this reason, WGA has been regarded as a marker to distinguish active from passive transport. However, different transport pathways use different nucleoporins (Shah and Forbes 1998
; Shah et al. 1998
; Moy and Silver 1999
; Seedorf et al. 1999
), and WGA may inhibit selectively those pathways that require the p62 complex. RCC1 may represent the first member of a class of proteins that are translocated across the nuclear pore complex independently of interactions with O-linked glycoproteins.
In digitonin-permeabilized cells, the import of RCC1 could only be inhibited by importin-β(45-462), low temperature, or competition with excess, unlabeled RCC1. The fact that the import reaction was inhibited by low temperature suggests strongly that import does not proceed by passive diffusion, and that the nuclear envelope was not disrupted during permeabilization of the plasma membrane ( B). The inhibition due to excess RCC1 could be explained by competition for a saturable transport process and/or competition for chromatin-binding sites. Evidence from time-lapse photography, however, suggests that the rate-limiting step for the import of RCC1 in vivo is transit across the nuclear pore.
Interestingly, the process by which RCC1 traverses the nuclear envelope does not require energy in vivo (). These data cannot be explained by the assertion that energy depletion alters the permeability of the nuclear envelope, because neither the injection site marker, GGNLS, nor RCC1(1-25)-NPC entered the nucleoplasm. Furthermore, since RCC1-GFP is beyond the diffusion limit of the nuclear pore, import in energy-depleted cells can not proceed by passive diffusion. Energy-independent translocation of this sort is not without precedent. Evidence from in vitro import assays suggest that single rounds of transport mediated by transportin, Crm1, and importin-β are energy independent (Schwoebel et al. 1998
; Englmeier et al. 1999
; Ribbeck et al. 1999
). In addition, proteins such as β-catenin (Yokoya et al. 1999
) and hnRNP K (Michael et al. 1997
) are able to translocate across the nuclear envelope in the absence of soluble factors. It is noteworthy that transport of these proteins is inhibited by the dominant-negative importin-β(45-462), as is the case for RCC1. It seems likely that the translocation of all these proteins occurs by a process of facilitated diffusion that requires specific, low-affinity interactions with nucleoporins that line the central channel of the pores. Facilitated diffusion cannot occur against a concentration gradient. Thus, nuclear pores must import and export RCC1 at the same rate and equilibrate the levels of free RCC1 in the cytoplasm to a concentration identical to that free in the nucleoplasm. The fact that RCC1 is >90% nuclear at steady state and does not shuttle detectably in vivo implies that the binding of RCC1 to chromatin is much more rapid than translocation across the nuclear envelope. In addition, it suggests that binding to chromatin can be sufficient to establish and maintain the nuclear localization of RCC1 and, hence, the Ran gradient. In effect, RCC1 acts as a chromatin marker, an activity key for the evolution of the nuclear compartment and the open mitotic cycle.
Key questions that remain to be explored include the identity of the RCC1-binding proteins on chromatin and at the nuclear pores. It will also be of interest to determine whether other import cargoes use the same pathway for transit across the nuclear envelope as RCC1.