A novel nuclear localization signal in the human single-minded proteins SIM1 and SIM2.
Human Single-minded 1 (SIM1) and SIM2 genes were found as homologs of Drosophila sim gene which plays a key role in the midline cell lineage of the central nervous system. SIM proteins belong to a family of transcription factors, called bHLH/PAS. Here we examined the intracellular localization of SIM proteins using the expression constructs of whole SIM2 or SIM1 protein fused with enhanced green fluorescent protein (EGFP). The transient expression analysis revealed the nuclear localization of SIM proteins in the cultured cells. To identify the nuclear localization signal, we made expression constructs of EGFP-fusion protein consisting of various portions of SIM proteins. Transfection assay showed the presence of NLS activity in the small region of 23 and 21 amino acid residues at the central part of SIM2 and SIM1 proteins, respectively. Further analysis with amino acid substitution of this small region of SIM2 protein revealed the critical role of five amino acid residues (Arg367, Lys373, Pro385, Tyr386, and Gln389) in NLS activity. The consensus sequence of RKxxKx[K/R]xxxxKxKxRxxPY was estimated as a presumptive NLS in SIM proteins from various species. Thus, the NLS consisting of a cluster of basic amino acids with Pro and Tyr at the C-terminal end is novel and well conserved in the SIM proteins during evolution.
Yamaki
Akiko
A
Department of Medical Genetics, Kyorin University School of Health Sciences, 146 Miyashita-cho, Hachioji, Tokyo 192-8508, Japan.
Kudoh
Jun
J
Shimizu
Nobuyoshi
N
Shimizu
Yoshiko
Y
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D051792:Basic Helix-Loop-Helix Proteins; D002460:Cell Line; D045744:Cell Line, Tumor; D003593:Cytoplasm / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D049452:Green Fluorescent Proteins; D006367:HeLa Cells; D006801:Humans; D008164:Luminescent Proteins / Q000378:metabolism; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D009154:Mutation; D019913:Nuclear Localization Signals; D010957:Plasmids / Q000378:metabolism; D011392:Proline / Q000737:chemistry; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012097:Repressor Proteins / Q000737:chemistry*; D017386:Sequence Homology, Amino Acid; D014157:Transcription Factors / Q000737:chemistry*; D014162:Transfection; D014443:Tyrosine / Q000737:chemistry
D051792:Basic Helix-Loop-Helix Proteins; D018076:DNA, Complementary; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D019913:Nuclear Localization Signals; D011392:Proline; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D014157:Transcription Factors; D014443:Tyrosine; C491094:SIM1 protein, human; C417032:SIM2 protein, human; C486578:Sim2 protein, mouse