19 MSX1 is one of the homeoproteins with the homeodomain (HD) sequence, which regulates 20 proliferation and differentiation of mesenchymal cells. In this study, we investigated the 21 nuclear localization signal (NLS) in the MSX1 HD by deletion and amino acid substitution 22 analyses. The web-based tool NLStradamus predicted two putative basic motifs in the N-and 23 C-termini of the MSX1 HD. Green fluorescent protein (GFP) chimera studies revealed that 24 NLS1 ( 161 RKHKTNRKPR 170 ) and NLS2 (

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... 216 NRRAKAKR 223 ) were independently 25 insufficient for robust nuclear localization. However, they can work cooperatively to promote 26 nuclear localization of MSX1, as was shown by the two tandem NLS motifs partially 27 restoring functional NLS, leading to a significant nuclear accumulation of the GFP chimera. 28 These results demonstrate a unique NLS motif in MSX1, which consists of an essential single 29 core motif in helix-I, with weak potency, and an auxiliary subdomain in helix-III, which alone 30 does not have nuclear localization potency. Additionally, other peptide sequences, other than 31 predicted two motifs in the spacer, may be necessary for complete nuclear localization in 32 MSX1 HD. 33