Two 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.
Vandromme
M
M
Cell Biology Unit, Institut National de la Santé et de la Recherche Médicale, Montpellier, France.
Cavadore
J C
JC
Bonnieu
A
A
Froeschlé
A
A
Lamb
N
N
Fernandez
A
A
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism*; D017931:DNA Primers; D005347:Fibroblasts; D018257:Helix-Loop-Helix Motifs; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D016296:Mutagenesis; D017570:MyoD Protein / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D010446:Peptide Fragments / Q000737:chemistry; D016133:Polymerase Chain Reaction; D011499:Protein Processing, Post-Translational; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D017384:Sequence Deletion
D017931:DNA Primers; D046911:Macromolecular Substances; D017570:MyoD Protein; D010446:Peptide Fragments; D011994:Recombinant Proteins