Bmp2 contains a functional bipartite NLS that overlaps the site of proteolytic processing. (a) Map of the Bmp2 preproprotein showing the signal peptide, propeptide, and mature chain. The amino acid sequence and location of each predicted NLS is shown, and the site of proteolytic cleavage is marked by an arrow. (b) Four GFP/NLS fusion genes were constructed as shown to test the ability of each predicted NLS to direct GFP to the nucleus. These expression vectors were transfected into RCS cells, and GFP localization (green) was visualized using laser confocal microscopy. Nuclei were stained with TO-PRO-3 (red). Only the bipartite NLSc directed strong nuclear localization. (c) Alignment of Bmp2 bipartite NLSc sequences from five different species. The six basic amino acids that characterize this sequence as a bipartite NLS are boxed. (d) To determine whether the bipartite NLSc is functional within Bmp2, GFP was fused to the C-terminus of the full-length Bmp2 preproprotein (wtBmp2/GFP). In a parallel fusion construct, the bipartite NLSc was mutated (NLSmtBmp2/GFP). These plasmids were transfected into RCS cells, and GFP localization was visualized using laser confocal microscopy. Nuclei were stained with TO-PRO-3 (red).