The Dynamic Association of RCC1 with Chromatin Is Modulated by Ran-dependent Nuclear Transport

Abstract

Regulator of chromosome condensation (RCC1) binding to chromatin is highly dynamic, as determined by fluorescence recovery after photobleaching analysis of GFP-RCC1 in stably transfected tsBN2 cells. Microinjection of wild-type or Q69L Ran markedly slowed the mobility of GFP-RCC1, whereas T24N Ran (defective in nucleotide loading) decreased it further still. We found significant alterations in the mobility of intranuclear GFP-RCC1 after treatment with agents that disrupt different Ran-dependent nuclear export pathways. Leptomycin B, which inhibits Crm1/RanGTP-dependent nuclear export, significantly increased the mobility of RCC1 as did high levels of actinomycin D (to inhibit RNA polymerases I, II, and III) or ␣-amanitin (to inhibit RNA polymerases II and III) as well as energy depletion. Inhibition of just mRNA transcription, however, had no affect on GFP-RCC1 mobility consistent with mRNA export being a Ran-independent process. In permeabilized cells, cytosol and GTP were required for the efficient release of GFP-RCC1 from chromatin. Recombinant Ran would not substitute for cytosol, and high levels of supplemental Ran inhibited the cytosol-stimulated release. Thus, RCC1 release from chromatin in vitro requires a factor(s) distinct from, or in addition to, Ran and seems linked in vivo to the availability of Ran-dependent transport cargo. INTRODUCTIONRegulator of chromosome condensation (RCC1) stimulates guanine nucleotide exchange by the small GTPase Ran (Bischoff and Ponstingl, 1991a). Inside the cell, RCC1 is essential for the efficient conversion of RanGDP to RanGTP. RanGTP in turn is essential for several key cellular processes, including nucleocytoplasmic transport, regulation of spindle formation and nuclear envelope reassembly at mitosis, and prevention of rereplication of DNA during S phase (Dasso, 2002;Yamaguchi and Newport, 2003).The local concentration of RanGTP is a positional cue used by nuclear import and export complexes to distinguish between the cytoplasm and nuclear interior, and this regulates the assembly and disassembly of these transport complexes in the correct compartment. RanGTP is kept high inside the nucleus by localization of RCC1 to chromatin in the nuclear interior, and low inside the cytoplasm by the RanGAP (GTPase activating protein) that is restricted to that compartment. The differential placement of these two Ran accessory factors forms a gradient of RanGTP across the nuclear pore complex essential for most nuclear transport during interphase . All nuclear carriers of the karyopherin-␤ (Kap-␤) (importin ␤) family bind RanGTP Gorlich and Kutay, 1999). Binding of RanGTP is required for Kap-␤ export carriers to simultaneously bind their cargo inside the nucleus. These export complexes disassemble after encounter with the RanGAP (and a cofactor RanBP1) in the cytoplasm and the subsequent conversion of complexed RanGTP to RanGDP. Conversely, import complexes consisting of the carrier with bound cargo assemble only in the absence of RanGTP (the cytoplasm) and disassemble upon encoun...

Nuclear Localization and Dynamic Properties of the Marek's Disease Virus Oncogene Products Meq and Meq/vIL8

“…2P to R; Table 2). FRAP analysis indicated that Meq had a recovery time of approximately 15 s in the nucleolus and nucleoplasm, which is similar to that seen for many other nuclear proteins (11,15,18,19,25,41,42,50). This recovery time was much slower than that of GFP alone, which had a t 1/2 of Ͻ0.3 s (not shown) within nuclei, suggesting that the mobility of Meq relative to that of GFP was reduced, perhaps due to specific and nonspecific interactions with nuclear components such as chromatin.…”

Regulation of the EBNA1 Epstein-Barr Virus Protein by Serine Phosphorylation and Arginine Methylation

“…An effect of methylation inhibitors on the localization of two other methylated proteins, Sam68 and HnRNP A2, has previously been reported, in both cases causing these normally nuclear proteins to accumulate in the cytoplasm (10,44). The nucleolar rings formed by EBNA1 upon MTA treatment or PRMT1 silencing are reminiscent of those formed by B23 upon inhibition of RNA polymerase I (11,57); however, MTA treatment did not affect B23 localization (Fig. 8A).…”

BMAL1 Shuttling Controls Transactivation and Degradation of the CLOCK/BMAL1 Heterodimer

“…However, the use of an NLS-tagged CLOCK with its own nuclear import ability fully compensated for the inhibitory effect of the BMAL1 NES mutation on transcriptional activity but had only a moderate effect on LMB-mediated inhibition of transcription. This could be due to unexpected LMB effects such as defects in the transcriptional machinery, since LMB treatment blocks shuttling not only of BMAL1 but also of a number of nuclear proteins (4,8,19). Thus, these findings indicate that nucleocytoplasmic shuttling of BMALl is essential for nuclear accumulation of CLOCK and that this in turn leads to transactivation of the CLOCK/BMAL1 heterodimer.…”