Translation of Bmp2 from an alternative start codon downstream of the signal peptide produces the uncleaved nuclear variant of Bmp2. (a) The N-terminus of the rat Bmp2 protein with corresponding DNA sequence is shown. The conventional (codon 1) and predicted alternative (codon 58) start codons are marked (arrowheads), and the signal peptide is shown in italics. (b) In vitro synthesis of Bmp2 produced the expected 42 kDa protein and some lower molecular weight proteins (lane 1). Mutation of codon 58 eliminated one of the smaller proteins, indicating that it was initiated at codon 58 (arrow). (c) Bmp2/GFP fusion constructs containing substitutions in either the conventional start codon 1 (ATG1 mtBmp2/GFP) or the alternative start codon 58 (ATG58 mtBmp2/GFP), were transfected into cells, and the percentage of transfected cells showing nuclear localization was quantified. (d) HA tags were inserted into the propeptide or fused to the C-terminus of Bmp2 as shown. (e) The HA tagged expression vectors were transfected into RCS cells, and nuclear extracts were analyzed by western blotting. Both vectors produced an HA-tagged nuclear protein of ~50 kDa (top left panel). When nuclear extracts from non-transfected RCS cells were analyzed by western blotting using an anti-Bmp2 antibody, a ~50 kDa endogenous nuclear protein was labeled (top right panel--note that this panel contains only one wide lane). Nuclear (N) and cytoplasmic (C) extracts were probed using a Golgi-specific antibody to verify that the nuclear extracts were not contaminated with cytoplasmic proteins (bottom panels).