In vitro, the stable association of nucleotide-free Ran with RCC1α produces a conformational change in the N-terminal tail that promotes binding to DNA and reduces affinity for core histones [13
]. In cells, however, the interaction of RCC1 isoforms with chromatin is highly dynamic and is affected by post-translational modification of the tail. Our results confirm that this dynamic interaction is regulated by the association of Ran with RCC1α. A Ran mutant (RanT24N
) that associates stably with RCC1α in a binary complex strongly reduces the mobility of RCC1α on chromatin, and this effect requires the methylated N-terminal tail of RCC1α.
Our results are consistent with a model in which apoRan (or RanT24N
) associates with the core domain of RCC1α and induces allosterically a conformational change in the N-terminal tail that stabilises interaction with chromatin. RCC1α associates weakly with chromatin through its core domain, possibly through interactions with core histones [13
]. The interaction of nucleotide-bound Ran with RCC1α releases the nucleotide from Ran, forming a transient binary complex in which a conformational change in RCC1α exposes its N-terminal domain and stabilises its interaction with chromatin (Figure ), possibly through direct interaction with DNA, although this remains to be confirmed in vivo. Other isoforms of RCC1 differ in the length of their N-terminal tails and in the turnover rate of their dynamic interactions with chromatin in cells [21
], but it is likely that all isoforms interact with chromatin through a similar mechanism, albeit with differing affinities depending on the composition of the tail.
Figure 6 Model of the dynamic interaction of RCC1 with chromatin. RCC1 interacts weakly with a nucleosome through its core domain. Interaction of Ran-GDP releases GDP and leads to the transient formation of a binary complex between apoRan and RCC1 in which the (more ...)
Our results with the D182A mutant of RCC1α, which inhibits guanine nucleotide exchange activity, are consistent with a model in which the dynamic interaction of RCC1α with chromatin is linked to its interaction with Ran. Previous experiments by Azuma and colleagues [12
] using purified proteins have shown that the D182A mutant of RCC1α forms a binary complex with apoRan at a reduced rate compared to wild-type RCC1α, and the apoRan-RCC1αD182A
complex dissociates slowly even in the absence of free guanine nucleotide whereas the wild-type binary complex is stable. However, addition of guanine nucleotide causes rapid dissociation of both the RCC1α wild-type and D182A binary complexes [12
]. Therefore the T24N mutation of Ran (which prevents nucleotide binding) can be envisaged to stabilise even the interaction with RCC1αD182A
to an extent and thereby promote the chromatin-binding conformation of this binary complex in cells. Nevertheless, the combination of reduced association rate of apoRan with RCC1αD182A
and likely increased dissociation rate of apoRan from RCC1αD182A
results in the interaction of RCC1αD182A
with chromatin being more dynamic than wild-type RCC1α even when RanT24N
In agreement with the partial stabilising effect of RanT24N
on the interaction of RCC1αD182A
with chromatin, we have also observed a complex formed between RCC1αD182A
and Ran under Mg2+
-chelating conditions, which promote the nucleotide-free form of Ran (apoRan). This suggests that the apoRan-RCC1αD182A
binary complex might be more stable in cells than was predicted. Indeed, the stability of the wild-type apoRan-RCC1α binary complex when it interacts with chromatin is not yet known. A recent study of the interaction of RCC1α with nucleosomes in solution has suggested the interesting possibility that Ran could interact with RCC1α on chromatin in a different conformation from the crystallised binary complex [28
], although the formation of such a distinct complex under cellular conditions remains to be confirmed. Whatever its conformation, the assembly of a complex between Ran and RCC1αD182A
that does not result in the efficient loading of Ran with GTP provides an explanation for the inhibitory effect of this mutant on mitosis [5
The precise relationship between the dynamics of the interaction of RCC1α with chromatin and its guanine nucleotide exchange activity is, however, not yet certain. In one model, they are tightly coupled: binding of nucleotide to apoRan causes Ran-GTP (or Ran-GDP) to dissociate from RCC1α, then RCC1α is released from chromatin and the N-terminal tail folds back against the core domain (Figure ). Although RCC1α can catalyse the reaction equally well from GDP to GTP and vice versa, the presence of accessory factors and the higher concentration of GTP than GDP in cells results in net loading of Ran with GTP. Alternatively, RCC1α could remain associated with chromatin for more than one guanine nucleotide exchange reaction if its dissociation from chromatin is slower than the release of Ran.
The conformational change in the N-terminal tail and/or its interaction with chromatin may be regulated by post-translational mechanisms as well as through the binding of Ran to RCC1 isoforms. Throughout the cell cycle, mono-, di- or tri-methylation of the α-amino group promotes the interaction with chromatin [18
], whereas in mitosis, phosphorylation of serines 2 and 11 makes the interaction more dynamic [19
]. We have found that α-N-terminal methylation of RCC1α is not affected by its stable association with Ran. We therefore favour the idea that post-translational modification of the N-terminal tail is relatively stable and is not tightly linked to the cycle of association and dissociation of RCC1 with chromatin, but rather alters the equilibrium of this interaction towards association with chromatin.