2 NUCLEAR-LOCALIZATION SIGNALS PRESENT IN THE BASIC-HELIX-1 DOMAINS OF MYOD PROMOTE ITS ACTIVE NUCLEAR TRANSLOCATION AND CAN FUNCTION INDEPENDENTLY

MyoD, a member of the family of helix-loop-helix myogenic factors that plays a crucial role in skeletal muscle differentiation, is a nuclear phosphoprotein, Using microinjection of purified MyoD protein into rat fibroblasts, we show that the nuclear import of MyoD is a rapid and active process, being ATP and temperature dependent, Two nuclear localization signals (NLSs), one present in the basic region and the other in the helix 1 domain of MyoD protein, are demonstrated to be functional: in promoting the active nuclear transport of MyoD, Synthetic peptides spanning these two NLSs and biochemically coupled to IgGs can promote the nuclear import of microinjected IgG conjugates in muscle and nonmuscle cells, Deletion analysis reveals that each sequence can function independently within the MyoD protein since concomittant deletion of both sequences is required to alter the nuclear import of this myogenic factor, In addition, the complete cytoplasmic retention of a beta-galactosidase-MyoD fusion mutant protein, double deleted at these two NLSs, argues against the existence of another functional NLS motif in MyoD.