A 23-amino acid motif spanning the basic domain targets zebrafish myogenic regulatory factor myf5 into nucleolus.
Myf5 is a nuclear protein and one of the basic helix-loop-helix (bHLH) myogenic factors that play an important role in muscle specification and differentiation. The motif responsible for the nuclear translocation of Myf5 was unknown. Using on-line monitoring of EGFP (enhanced green fluorescent protein)-tagged zebrafish Myf5 translocation, we demonstrated that Myf5-EGFP protein resided in the nucleoplasm and nucleolus of zebrafish fibroblast cell lines (ZEM2S and ZF4), mammalian nonmuscle cell line (COS1), and muscle cell lines (RD and C2C12). In contrast, zebrafish MyoD-EGFP was localized in the nucleus but did not condense in the nucleolus. Using indirect immunofluorescent staining, we determined that zebrafish Myf5 was colocalized with nucleophosmin/B23, a nucleolus protein. Deletion analysis revealed that amino acid residues 60 to 82 (60KRKASTVDRRRAATMRERRRLKK82) of Myf5 were sufficient and necessary for nucleolus targeting. A GST pulldown assay followed by Western analysis showed that nucleolin/C23 could be pulled down specifically by GST-Myf5, but not by GST-MyoD. Based on these findings, we propose that the distinct functions of Myf5 and MyoD may result from their differential binding affinity to nucleolin/C23.
Wang
Yun-Hsin
YH
Institute of Molecular and Cellular Biology, National Taiwan University, Taipei, Taiwan, Republic of China. cmkuo@gate.sinica.edu.tw
Chen
Yau-Hung
YH
Lu
Jun-Hung
JH
Tsai
Huai-Jen
HJ
D000818:Animals; D015153:Blotting, Western; D019556:COS Cells; D002466:Cell Nucleolus / Q000378:metabolism*; D002522:Chlorocebus aethiops; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D019084:Fluorescent Antibody Technique, Indirect; D005982:Glutathione Transferase / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D009124:Muscle Proteins / Q000235:genetics / Q000378:metabolism*; D009132:Muscles / Q000166:cytology / Q000378:metabolism; D017570:MyoD Protein / Q000378:metabolism; D051787:Myogenic Regulatory Factor 5; D019913:Nuclear Localization Signals / Q000378:metabolism*; D009687:Nuclear Proteins / Q000378:metabolism; D000090243:Nucleophosmin; D015388:Organelles / Q000378:metabolism; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism*; D010750:Phosphoproteins / Q000378:metabolism; D010957:Plasmids; D021381:Protein Transport; D016601:RNA-Binding Proteins / Q000378:metabolism; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism*; D015027:Zebrafish / Q000254:growth & development / Q000378:metabolism*; D000097796:Nucleolin
D004268:DNA-Binding Proteins; D005982:Glutathione Transferase; D049452:Green Fluorescent Proteins; D009124:Muscle Proteins; D017570:MyoD Protein; D051787:Myogenic Regulatory Factor 5; D019913:Nuclear Localization Signals; D009687:Nuclear Proteins; D000090243:Nucleophosmin; D010446:Peptide Fragments; D010750:Phosphoproteins; D016601:RNA-Binding Proteins; D015534:Trans-Activators; D000097796:Nucleolin; C485184:enhanced green fluorescent protein