Abstract
The Ran GTPase activating protein RanGAP1 plays an essential role in nuclear transport by stimulating RanGTP hydrolysis in the cytoplasmic compartment. In mammalian cells, unmodified RanGAP1 is predominantly cytoplasmic, whereas modification by small ubiquitin-related modifier protein (SUMO) targets RanGAP1 to the cytoplasmic filaments of nuclear pore complex (NPC). Although RanGAP1 contains nine putative nuclear export signals and a nuclear localization signal, little is known if RanGAP1 shuttles between the nuclear and cytoplasmic compartments and how its primary localization in the cytoplasm and at the NPC is regulated. Here we show that inhibition of CRM1-mediated nuclear export using RNAi-knockdown of CRM1 and inactivation of CRM1 by leptomycin B (LMB) results in nuclear accumulation of RanGAP1. LMB treatment induced a more robust redistribution of RanGAP1 from the cytoplasm to the nucleoplasm compared to CRM1 RNAi and also uniquely triggered a decrease or loss of RanGAP1 localization at the NPC, suggesting that LMB treatment is more effective in inhibiting CRM1-mediated nuclear export of RanGAP1. Our time-course analysis of LMB treatment reveals that the NPC-associated RanGAP1 is much more slowly redistributed to the nucleoplasm than the cytoplasmic RanGAP1. Furthermore, LMB-induced nuclear accumulation of RanGAP1 is positively correlated with an increase in levels of SUMO-modified RanGAP1, suggesting that SUMOylation of RanGAP1 may mainly take place in the nucleoplasm. Lastly, we demonstrate that the nuclear localization signal at the C-terminus of RanGAP1 is required for its nuclear accumulation in cells treated with LMB. Taken together, our results elucidate that RanGAP1 is actively transported between the nuclear and cytoplasmic compartments, and that the cytoplasmic and NPC localization of RanGAP1 is dependent on CRM1-mediated nuclear export.
Highlights
The Ras-like GTPase Ran plays an essential role in various cellular processes including nuclear transport, mitotic spindle assembly, and nuclear envelope reformation [1,2,3,4,5]
To examine the effect of CRM1 RNA interference (RNAi) on the subcellular distribution of RanGAP1, Human cervical cancer cells (HeLa) cells were transfected with control or CRM1-specific siRNAs for 24 h and the plasmid encoding Myc-tagged RanGAP1 [12] for another 24 h followed by immunofluorescence microscopy using anti-Myc monoclonal antibody (mAb) (9E10)
The identification of the nuclear localization signal (NLS) sequence may allow us to generate the RanGAP1 mutant with a defect in nuclear import but not in vitro SUMOylation and Ubc9 interaction through site-directed mutagenesis. Such RanGAP1 mutant will enable us to further test if the nuclear import of RanGAP1 is critical for its SUMOylation. By using both CRM1 RNAi and leptomycin B (LMB) treatment to inhibit CRM1-mediated nuclear export, we demonstrate that CRM1 is responsible for the primary distribution of RanGAP1 in the cytoplasm and at the nuclear pore complex (NPC) in mammalian cells
Summary
The Ras-like GTPase Ran plays an essential role in various cellular processes including nuclear transport, mitotic spindle assembly, and nuclear envelope reformation [1,2,3,4,5]. Ran is unable to exchange between the two states at a physiologically significant rate by itself and requires interaction with two essential regulators, the Ran GTPaseactivating protein RanGAP and the Ran guanine nucleotide exchange factor RanGEF ( called RCC1) [6,7,8]. Because RanGAP is primarily cytoplasmic whereas RCC1 is exclusively nuclear, this asymmetry creates a steep concentration gradient from high RanGTP levels in the nucleoplasm to low RanGTP levels in the cytoplasm [10]. This gradient provides the driving force for nuclear transport of numerous proteins and RNAs across the nuclear pore complex (NPC) at the nuclear envelope [2]
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