The Mitotic Function Study Of Mog1/Ran Complex And The Structural Study Of Mog1/Ran/RanBP1 Complex

Ran belongs the Ras superfamily of small GTPase that is highly conserved in eukaryotic cells and acts in diverse cellular processes including nucleocytoplasmic transport,mitotic spindle assembly,and nuclear envelope formation.The cycling of Ran between its GTP-and GDP-bound forms is controlled by a pair of Ran-specific regulators RanGEF(guanine nucleotide exchange factor)and RanGAP(GTPase activating protein).Gradients of RanGTP are established through the localization of its regulators.During the cell cycle of interphase,RanGEF mostly distributes in the nucleus,while RanGAP is in the cytoplasm.Therefore,Ran is GTP-bound in the nucleus and GDP-bound in the cytoplasm at interphase.Mogl was initially identified as a suppressor that was able to rescue the temperature-sensitive defect of Saccharomyces cerevisiae.Later on,it was discovered that Mogl is a regulatory protein for Ran.Biochemical activity of Mogl is similar to,but distinct from that of RanGEF.Both Mogl and RanGEF stimulate GTP release from Ran in vitro.RanGEF permits nucleotide re-binding(nucleotide exchange)of Ran,whereas Mogl inhibits nucleotide re-binding of Ran and it only bound to nucleotide-free Ran in a complex.Hence,Mogl functions as a nucleotide release factor,rather than a nucleotide exchange factor.Recently,It is reported that hMogl is a new factor of the cardiac sodium channel,Nav1.5.Overexpression of mogl in Nav1.5-expressing cells increases sodium current markedly.Moreover,hMogl can form stable complex with Ran and acts as a nucleotide release factor.The critical residues of hMogl for interaction with Ran were identified by NMR chemical shift perturbation,GST-pull down and mutagenesis method.Mogl interacts with Ran in nucleus of living cells observed by using BiFC.Furthermore,using a Rango2 biosensor together with real time fluorescence imaging for the RanGTP gradient,we found that hMogl plays a vital role in regulating the RanGTP gradient to modulate the function of Ran during mitosis.Together,our structural,biochemical and functional studies provide new insights into the functions of hMogl.We have studied the structure of the ternary complex form by Mogl,Ran and RanBPl using EM.We use stereoselective lebel method to label the methyl group of the ternary complex to simplify the spectrum.A BiFC-FRET assay is applicated to explore the function of the presence of the ternary complex.