Figure 3

CRM1-dependent cytoplasmic localization of 16.4.1. HeLa cells were transfected with plasmids directing expression of IgG1-16.4.1 or 16.4.1-GFP fusion proteins and subcellular distribution of tagged proteins analysed 24 hours later in fixed cells. (A) Subcellular distribution of IgG1 fusion proteins containing full length 16.4.1 or various segments of 16.4.1. IgG1-16.4.1 proteins were detected by immunocytochemistry with a Cy3-conjugated anti human IgG1 antibody. A schematic diagram of the IgG1-16.4.1 fusion proteins and a summary of their localization behavior are shown at the top. Representative images of cells expressing IgG1 fusion proteins containing the indicated amino acid regions of 16.4.1 are shown below. IgG1 fusion proteins containing amino acids 2–171 (full-length), 2–133, 39–171 and 74–171 of 16.4.1 were predominantly cytoplasmic, whereas fusion proteins with amino acids 2–38 or 134–171 and unfused IgG1 were both cytoplasmic and nuclear. Scale bars: 20 μm. (B) Disruption of predominantly cytoplasmic localization of 16.4.1-GFP by treatment of cells with the CRM1-inhibitor Leptomycin B (LMB). HeLa cells were transiently transfected with plasmids for expression of 16.4.1-GFP, PKIα-GFP, Rev(52–116)-GFP and unfused GFP. Cells were treated with LMB (5 nM) for two hours. Representative images of the subcellular distribution of the GFP fusion proteins in untreated (-) and LMB treated cells (+) are shown at the top. Symbols in the graph indicate the nuclear proportion of fluorescence (%) in individual cells and horizontal lines and numbers the median of the cell population. LMB treatment increased the median nuclear proportion of 16.4.1-GFP from 25% to 44%. LMB had a similar effect on localization of GFP fusion proteins containing PKIα or the carboxyterminal region of Rev (aa 52–116), which are known transport substrates of CRM1. In contrast, LMB only marginally affects subcellular distribution of unfused GFP. Scale bars: 10 μm.