title: Live-cell visualization of intracellular interaction between a nuclear migration protein (hNUDC) and the thrombopoietin receptor (Mpl).
abstract: We previously demonstrated that endogenous hNUDC and Mpl co-localized in the perinuclear and cytoplasmic regions of megakaryocyte cells by indirect immunofluorescence. We further reported that hNUDC accumulated in the Golgi when NIH 3T3 cells were transfected with an hNUDC expression vector alone. However, co-transfection with hNUDC and Mpl expression vectors caused both proteins to co-localize predominantly in the cytosol. These observations led us to hypothesize that a complex containing hNUDC and Mpl may alter hNUDC subcellular location and induce its secretion. In the present study, we test this hypothesis by employing bimolecular fluorescence complementation (BiFC) to detect and visualize the complex formation of hNUDC/Mpl in living cells. We further examined in detail the subcellular locations of the hNUDC/Mpl complex by co-transfection of BiFC chimeras with known subcellular markers. The distribution of hNUDC/Mpl in the endoplasmic reticulum (ER), Golgi and cell surface was determined. Furthermore, the N-terminal 159 amino acids of hNUDC, but not C-terminal half, bound to Mpl in vivo and exhibited a similar localization pattern to that of full-length hNUDC in Cos-1 cells. Adenovirus-mediated overexpression of hNUDC or its N-terminal 159 residues in a human megakaryocyte cell line (Dami) resulted in increased levels of hNUDC or hNUDC(1-159) secretion. In contrast, depletion of Mpl by transfecting Dami cells with adenovirus bearing Mpl-targeting siRNA significantly blocked hNUDC secretion. Thus, we provide the first evidence that the N-terminal region of hNUDC contains all of the necessary information to complex with Mpl and traffic through the secretory pathway.
authors.affiliation: The Key Laboratory of Gene Engineering of Education Ministry, Zhongshan University, Guangzhou, The People's Republic of China.
authors.forename: Yuan-Bin
authors.initials: YB
authors.lastname: Zheng
authors.forename: Ying-Ying
authors.initials: YY
authors.lastname: Xiao
authors.forename: Peng
authors.initials: P
authors.lastname: Tan
authors.forename: Qing
authors.initials: Q
authors.lastname: Zhang
authors.forename: Peilin
authors.initials: P
authors.lastname: Xu
mesh_terms: D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D015153:Blotting, Western; D019556:COS Cells / Q000378:metabolism / Q000648:ultrastructure; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism*; D002522:Chlorocebus aethiops; D017077:Culture Media, Conditioned / Q000494:pharmacology; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D005434:Flow Cytometry; D031541:Fluorescence Resonance Energy Transfer; D006056:Golgi Apparatus / Q000378:metabolism*; D006801:Humans; D018613:Microscopy, Confocal; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000235:genetics; D053628:Receptors, Thrombopoietin / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D013347:Subcellular Fractions
chemical_list: D018797:Cell Cycle Proteins; D017077:Culture Media, Conditioned; C512648:NUDC protein, human; D009687:Nuclear Proteins; D034741:RNA, Small Interfering; D053628:Receptors, Thrombopoietin