Dissection of a nuclear localization signal.
The regulated process of protein import into the nucleus of a eukaryotic cell is mediated by specific nuclear localization signals (NLSs) that are recognized by protein import receptors. This study seeks to decipher the energetic details of NLS recognition by the receptor importin alpha through quantitative analysis of variant NLSs. The relative importance of each residue in two monopartite NLS sequences was determined using an alanine scanning approach. These measurements yield an energetic definition of a monopartite NLS sequence where a required lysine residue is followed by two other basic residues in the sequence K(K/R)X(K/R). In addition, the energetic contributions of the second basic cluster in a bipartite NLS ( approximately 3 kcal/mol) as well as the energy of inhibition of the importin alpha importin beta-binding domain ( approximately 3 kcal/mol) were also measured. These data allow the generation of an energetic scale of nuclear localization sequences based on a peptide's affinity for the importin alpha-importin beta complex. On this scale, a functional NLS has a binding constant of approximately 10 nm, whereas a nonfunctional NLS has a 100-fold weaker affinity of 1 microm. Further correlation between the current in vitro data and in vivo function will provide the foundation for a comprehensive quantitative model of protein import.
Hodel
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MR
Department of Biochemistry, Emory University School of Medicine, Atlanta, Georgia 30322, USA.
Corbett
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AH
Hodel
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AE
D000595:Amino Acid Sequence; D000952:Antigens, Polyomavirus Transforming / Q000737:chemistry; D001665:Binding Sites; D002151:Calorimetry; D014644:Genetic Variation; D049452:Green Fluorescent Proteins; D028884:Karyopherins; D008164:Luminescent Proteins / Q000032:analysis; D008958:Models, Molecular; D008969:Molecular Sequence Data; D019913:Nuclear Localization Signals / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D009687:Nuclear Proteins / Q000737:chemistry* / Q000378:metabolism; D011487:Protein Conformation; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013539:Simian virus 40; D013816:Thermodynamics
D000952:Antigens, Polyomavirus Transforming; D028884:Karyopherins; D008164:Luminescent Proteins; D019913:Nuclear Localization Signals; D009687:Nuclear Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins