FIG 4.
PDCD2L is a nucleocytoplasmic shuttling protein that exports the nucleus by using a leucine-rich NES. (A) U-2 OS cells induced to express GFP-tagged versions of PDCD2L (a to f) and PRMT3 (g to l) were treated (d to f and j to l) or not treated (a to c and g to i) with LMB for 3 h. DNA staining with 4′,6-diamidino-2-phenylindole (DAPI) shows the nucleus of each cell (b, e, h, and k). Bar, 20 μm. (B) Alignment of putative NESs of PDCD2L proteins across multicellular organisms. The NES consensus sequence ϕx2-3ϕx2-3ϕxϕ is indicated at the top, where ϕ is a large, hydrophobic amino acid and x represents any amino acid. H. sapiens, Homo sapiens; M. musculus, Mus musculus; G. gallus, Gallus gallus; D. rerio, Danio rerio; X. tropicalis; Xenopus tropicalis. (C) Visual analysis of U-2 OS cells that were transiently transfected with DNA constructs expressing GFP-PDCD2L (a to c) and a version of GFP-PDCD2L with substitutions of leucines 162, 165, and 167 for alanines (NESmut) (d to f). (D) Visual analysis of U-2 OS cells transiently transfected with DNA constructs expressing GFP (a to f) as well as GFP fused to wild-type (g to l) and mutant (m to r) versions of the PDCD2L NES (amino acids 153 to 181) (see panel E). Cells were treated (+) or not treated (−) with LMB. (E) Schematic of wild-type and mutant versions of GFP-NES-PDCD2L fusions used for panel D. (F) Direct interaction between PDCD2L and CRM1 in vitro. Totals of 50 pmol GST, GST-PDCD2L, and GST-Rev fusion proteins were immobilized on beads and incubated with 9 pmol Cy3-labeled CRM1 alone or in the presence of 180 pmol RanGTPQ69L and 70 pmol Nup2141916–2033. Bound Cy3-CRM1 was analyzed by flow cytometry, and the fluorescence intensity relative to GST was plotted. Data and error bars represent the means and standard deviations, respectively, from six independent experiments. ***, P < 0.001; **, P < 0.01 (as determined by Student's t test).