Figure 7

Nuclear variants of Bmp4 and Gdf5 are also translated from downstream alternative start codons and contain NLSs that overlap the sites of proprotein processing. (a) Map of the Bmp4 preproprotein showing the signal peptide, propeptide, and mature chain. The amino acid sequence and location of the bipartite NLS are shown, and the alternative start codon and site of proteolytic cleavage are marked. (b) Endogenous Bmp4 is detectable in the nuclei of three cultured cell lines. Non-transfected 10T1/2, BALB/3T3, and RCS cells were cultured on slides and immunostained using an anti-Bmp4 antibody (green). Nuclei were stained with TO-PRO-3 (red), and cells were examined by laser confocal microscopy. (c) Bmp4/GFP fusion constructs containing targeted mutations in the NLS, the conventional start codon 1, or the alternative start codon 83, were transfected into RCS cells to examine the effects of these mutations on nuclear localization of Bmp4. (d) Map of the Gdf5 preproprotein showing the signal peptide, propeptide, and mature chain. The amino acid sequence and location of the bipartite NLS are shown, and the alternative start codon and site of proteolytic cleavage are marked. (e) Endogenous Gdf5 is detectable in the nuclei of two of the three cultured cell lines shown. Non-transfected 10T1/2, BALB/3T3, and RCS cells were cultured on slides and immunostained using an anti-Gdf5 antibody (green). Nuclei were stained with TO-PRO-3 (red), and cells were examined by laser confocal microscopy. (f) GDF5/GFP fusion constructs containing targeted mutations in the NLS, the conventional start codon 1, or the alternative start codon 173, were transfected into RCS cells to examine the effects of these mutations on nuclear localization of Gdf5.