title: Different binding domains for Ran-GTP and Ran-GDP/RanBP1 on nuclear import factor p97.
abstract: Several proteins are required for the transport of nuclear proteins from the cytoplasm to the nucleus, including the nuclear location sequence receptor (NLS receptor), p97, the small nuclear GTPase Ran/TC4, and several nucleoporins. The interaction of Ran with p97 is thought to regulate the interaction of these transport components. Ran-GTP alone binds p97, but Ran-GDP binds p97 only in conjunction with RanBP1. Using site-directed mutagenesis and deletion analysis, we have identified two distinct but overlapping binding domains for Ran-GTP and Ran-GDP/RanBP1 on p97. A short acidic sequence in p97 is part of the Ran-GDP/RanBP1 binding domain, possibly functioning in a similar manner as the C-terminal acidic sequence in Ran. A conserved cysteine residue in p97, Cys-158, is required for binding Ran-GDP/RanBP1, but not for binding of Ran-GTP to p97. In a permeabilized cell protein import assay, a mutant p97 with alanine substituted for Cys-158 is unable to support import in the presence of NLS receptor and Ran. These results support a direct active role for Ran-GDP in the receptor complex and provide evidence that the activity of downstream effectors of small GTPases may be regulated by both GTP- and GDP-bound forms of the protein.
authors.affiliation: Department of Cell and Molecular Biology, Northwestern University Medical School, Chicago, Illinois 60611, USA.
authors.forename: N C
authors.initials: NC
authors.lastname: Chi
authors.forename: E J
authors.initials: EJ
authors.lastname: Adam
authors.forename: S A
authors.initials: SA
authors.lastname: Adam
mesh_terms: D000409:Alanine; D001665:Binding Sites; D001692:Biological Transport; D002451:Cell Compartmentation; D002467:Cell Nucleus / Q000378:metabolism*; D003545:Cysteine; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006801:Humans; D009687:Nuclear Proteins / Q000378:metabolism*; D017354:Point Mutation; D011994:Recombinant Proteins; D013329:Structure-Activity Relationship; D028961:beta Karyopherins; D020931:ran GTP-Binding Protein
chemical_list: D009687:Nuclear Proteins; D011994:Recombinant Proteins; D028961:beta Karyopherins; C091775:ran-binding protein 1; D019204:GTP-Binding Proteins; D020931:ran GTP-Binding Protein; D003545:Cysteine; D000409:Alanine