The methylated N-terminal tail of RCC1 is required for stabilisation of its interaction with chromatin by Ran in live cells

Abstract

Background: Regulator of chromosome condensation 1 (RCC1) is the guanine nucleotide exchange factor for Ran GTPase. Localised generation of Ran-GTP by RCC1 on chromatin is critical for nucleocytoplasmic transport, mitotic spindle assembly and nuclear envelope formation. Both the N-terminal tail of RCC1 and its association with Ran are important for its interaction with chromatin in cells. In vitro, the association of Ran with RCC1 induces a conformational change in the N-terminal tail that promotes its interaction with DNA.

Results: We have investigated the mechanism of the dynamic interaction of the alpha isoform of human RCC1 (RCC1alpha) with chromatin in live cells using fluorescence recovery after photobleaching (FRAP) of green fluorescent protein (GFP) fusions. We show that the N-terminal tail stabilises the interaction of RCC1alpha with chromatin and this function can be partially replaced by another lysine-rich nuclear localisation signal. Removal of the tail prevents the interaction of RCC1alpha with chromatin from being stabilised by RanT24N, a mutant that binds stably to RCC1alpha. The interaction of RCC1alpha with chromatin is destabilised by mutation of lysine 4 (K4Q), which abolishes alpha-N-terminal methylation, and this interaction is no longer stabilised by RanT24N. However, alpha-N-terminal methylation of RCC1alpha is not regulated by the binding of RanT24N. Conversely, the association of Ran with precipitated RCC1alpha does not require the N-terminal tail of RCC1alpha or its methylation. The mobility of RCC1alpha on chromatin is increased by mutation of aspartate 182 (D182A), which inhibits guanine-nucleotide exchange activity, but RCC1alphaD182A can still bind nucleotide-free Ran and its interaction with chromatin is stabilised by RanT24N.

Conclusions: These results show that the stabilisation of the dynamic interaction of RCC1alpha with chromatin by Ran in live cells requires the N-terminal tail of RCC1alpha. alpha-N-methylation is not regulated by formation of the binary complex with Ran, but it promotes chromatin binding through the tail. This work supports a model in which the association of RCC1alpha with chromatin is promoted by a conformational change in the alpha-N-terminal methylated tail that is induced allosterically in the binary complex with Ran.

Figure 1

The N-terminal region (NTR) or tail of RCC1α is required for stable interaction with chromatin in live cells. (A) Schematic diagrams of N-terminal and C-terminal GFP fusion constructs. (B) Representative images of live HeLa cells expressing the N-terminal and C-terminal GFP fusion constructs. (C) Fluorescence recovery after photobleaching (FRAP) of RCC1α-GFP. Images of a cell during the timecourse are shown (left). Circles indicate the region of photobleaching. Output of data (right) with FRAP half-time (t_1/2). (D) Mean of the FRAP t_1/2 for each GFP fusion construct. Data from 3 independent experiments were combined and tested for significant difference in FRAP t_1/2 using the one-way ANOVA test. P-value less than 0.05 was considered to be statistically different (p-value < 0.001 shows as ***).

Figure 2

Effect of Ran on the interaction of RCC1α with chromatin in live cells. Representative images of live U2OS cells co-expressing wild-type RCC1α-GFP (A), Δ27RCC1-GFP (B), RCC1α^K4Q-GFP (C) or RCC1α^D182A-GFP (D) with mCherry empty vector (top), mCherry-Ran^WT (middle) or mCherry-Ran^T24N (bottom) during fluorescence recovery after photobleaching (FRAP).

Figure 3

Stabilisation of the interaction of RCC1α with chromatin by Ran requires the methylated N-terminal tail of RCC1α. Bar chart showing the mean FRAP t_1/2 +/- SD of live U2OS cells co-expressing wild-type RCC1α-GFP, Δ27RCC1-GFP, RCC1α^K4Q-GFP or RCC1α^D182A-GFP with mCherry empty vector (green), mCherry-Ran^WT (pink) or mCherry-Ran^T24N (red). Data were tested for significant difference in FRAP t_1/2 using the Student's t-test. P-values considered to be statistically different (< 0.001) are indicated as ***.

Figure 4

α-N-terminal methylation of RCC1α is not affected by the stable association of Ran. U2OS cells were co-transfected with GFP, RCC1α-GFP or RCC1α^K4Q-GFP and mCherry, mCherry-Ran^WT or mCherry-Ran^T24N. Cell lysates were analysed by Western blotting for α-dimethylated RCC1, RCC1, Ran and actin as a loading control. Methylated RCC1α-GFP was revealed on a light exposure of the α-dimethylated RCC1 blot and endogenous RCC1 isoforms on a dark exposure. A non-specific reactive band noted by [18] is indicated by *. The migration position of molecular mass markers (kDa) are shown left.

Figure 5

Mutations of RCC1 that remove the N-terminal tail, block N-terminal methylation or inhibit GEF activity do not prevent its interaction with apoRan. RCC1α wild-type (WT) and mutants (Δ27, K4Q and D182A) fused at the C-terminal with GFP-FLAG were expressed in U2OS cells (A) and immunoprecipitated with α-FLAG agarose beads (B). RCC1α-GFP-FLAG proteins were analysed by Western blotting using antibodies from Santa Cruz Biotechnology (sc-1162; does not recognize Δ27RCC1) (middle panel) and Transduction Laboratories (R35420) (bottom panel). The Transduction antibody detects both full-length RCC1α-GFP-FLAG and a lower band that is not detected by the Santa Cruz antibody. The latter therefore probably represents a truncated form lacking the N-terminal tail of RCC1α. A prominent non-specific band reacting with the Transduction antibody is indicated by *. Endogenous Ran present in the cell lysates (A) or co-immunoprecipitated with RCC1α-GFP-FLAG proteins (B) was detected by a specific antibody (top panel).

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 methylated N-terminal tail undergoes a conformational change that allows it to interact with DNA and stabilise the association of RCC1. Binding of GTP to Ran causes dissociation of the binary complex and the release of RCC1 from the nucleosome.