A 23-Amino Acid Motif Spanning the Basic Domain Targets Zebrafish Myogenic Regulatory Factor Myf5 into Nucleolus

Abstract

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.

Nucleolar localization and identification of nuclear/nucleolar localization signals of the calmodulin-binding protein nucleomorphin during growth and mitosis in Dictyostelium

“…nor CaM was required for proper localization of the NLS/ NoLS peptides. This appears to be the first precise delineation of both a NoLS and a NLS/NoLS in this eukaryote model and is in keeping with results from studies on mammalian cells where certain NLSs also function as NoLSs (Wang et al 2005). The nucleoplasmic localization of these NLS/NoLS peptides is thought to be due to nuclear retention via a nuclear binding protein rather than active transport across the nuclear pore complex since isolated nuclei displayed the same localization despite the lack of cytoplasm which would contain nuclear pore complex transporters such as importin.…”

“…Some NoLSs can also act as NLSs and are thus referred to as NLS/NoLSs. Examples of proteins containing NLS/ NoLSs include human NF-jB-inducing kinase, the novel human nucleolar protein, phosphatidylinositol 4-kinase, and myogenic regulatory factor (Birbach et al 2004;Kakuk et al 2008;Ueki et al 1998;Wang et al 2005). As the nucleolus gains more attention due to its newly discovered role in cancer, understanding the targeting of nucleolar proteins is of key importance (Maggi Jr and Weber 2006;Mijatovic et al 2008;Montanaro et al 2008).…”

Arabidopsis Ribosomal Proteins RPL23aA and RPL23aB Are Differentially Targeted to the Nucleolus and Are Disparately Required for Normal Development

“…One divergent region between the RPL23a isoforms occurs within a stretch of basic amino acids containing a putative NoLS/NLS (Kalderon et al, 1984;Dingwall and Laskey, 1991;Weber et al, 2000;Horke et al, 2004). In RPL23aA, this putative NoLS conforms to the core consensus sequence for nucleolin binding in mammals [(K/R) 2 XK; Xue et al, 1993;Lee et al, 1998;Intine et al, 2004;Wang et al, 2005]. Nucleolin is a major nucleolar protein involved in RNA polymerase I transcription of rDNA, rRNA processing, and nucleocytoplasmic trafficking of RNA and ribonucleoprotein particles (RNPs; Bouvet et al, 1998;Ginisty et al, 1998;Roger et al, 2003;Mongelard and Bouvet, 2007).…”

Multiple upstream modules regulate zebrafish myf5expression

“…Antibody labeling was performed as previously described, with minor modifications [42]. Embryos were fixed in 4% paraformaldehyde in phosphate buffered saline (PBS, pH 7.0) for 4 h at room temperature, or overnight at 4°C.…”