The Cellular Distribution of RanGAP1 Is Regulated by CRM1-Mediated Nuclear Export in Mammalian Cells.
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.
Department of Biological Sciences, Wayne State University, Detroit, Michigan, United States of America.
Keith
K
Cha
Department of Biological Sciences, Wayne State University, Detroit, Michigan, United States of America.
Progga
P
Sen
Department of Biological Sciences, Wayne State University, Detroit, Michigan, United States of America.
Sarita
S
Raghunayakula
Department of Biological Sciences, Wayne State University, Detroit, Michigan, United States of America.
Xiang-Dong
XD
Zhang
D021581:Active Transport, Cell Nucleus / Q000187:drug effects*; D000818:Animals; D003593:Cytoplasm / Q000235:genetics / Q000378:metabolism; D005231:Fatty Acids, Unsaturated / Q000494:pharmacology; D020690:GTPase-Activating Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D028884:Karyopherins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D022022:Nuclear Pore / Q000378:metabolism*; D034622:RNA Interference; D018160:Receptors, Cytoplasmic and Nuclear / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D058207:Sumoylation / Q000235:genetics; D000097606:Exportin 1 Protein
D005231:Fatty Acids, Unsaturated; D020690:GTPase-Activating Proteins; D028884:Karyopherins; D018160:Receptors, Cytoplasmic and Nuclear; D000097606:Exportin 1 Protein; C092317:RANGAP1 protein, human; C038753:leptomycin B