Functional analysis of the hydrophobic patch on nuclear transport factor 2 involved in interactions with the nuclear pore in vivo.
Nuclear transport factor 2 (NTF2) is a small homodimeric protein that interacts simultaneously with both RanGDP and FxFG nucleoporins. The interaction between NTF2 and Ran is essential for the import of Ran into the nucleus. Here we use mutational analysis to dissect the in vivo role of the interaction between NTF2 and nucleoporins. We identify a series of surface residues that form a hydrophobic patch on NTF2, which when mutated disrupt the NTF2-nucleoporin interaction. Analysis of these mutants in vivo demonstrates that the strength of this interaction can be significantly reduced without affecting cell viability. However, cells cease to be viable if the interaction between NTF2 and nucleoporins is abolished completely, indicating that this interaction is essential for the function of NTF2 in vivo. In addition, we have isolated a dominant negative mutant of NTF2, N77Y, which has increased affinity for nucleoporins. Overexpression of the N77Y protein blocks nuclear protein import and concentrates Ran at the nuclear rim. These data support a mechanism in which NTF2 interacts transiently with FxFG nucleoporins to translocate through the pore and import RanGDP into the nucleus.
Quimby
B B
BB
Department of Biochemistry, Emory University School of Medicine, Atlanta, Georgia 30322, USA.
Leung
S W
SW
Bayliss
R
R
Harreman
M T
MT
Thirumala
G
G
Stewart
M
M
Corbett
A H
AH
D021581:Active Transport, Cell Nucleus; D000483:Alleles; D000595:Amino Acid Sequence; D000818:Animals; D002467:Cell Nucleus / Q000378:metabolism*; D002470:Cell Survival; D004252:DNA Mutational Analysis; D019281:Dimerization; D005799:Genes, Dominant; D049452:Green Fluorescent Proteins; D015151:Immunoblotting; D008164:Luminescent Proteins / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D019913:Nuclear Localization Signals; D022022:Nuclear Pore / Q000378:metabolism*; D028861:Nuclear Pore Complex Proteins / Q000378:metabolism; D029543:Nucleocytoplasmic Transport Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D010641:Phenotype; D011485:Protein Binding; D051381:Rats; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013997:Time Factors; D020931:ran GTP-Binding Protein / Q000378:metabolism
D008164:Luminescent Proteins; D019913:Nuclear Localization Signals; D028861:Nuclear Pore Complex Proteins; D029543:Nucleocytoplasmic Transport Proteins; C503612:Nutf2 protein, rat; D049452:Green Fluorescent Proteins; D020931:ran GTP-Binding Protein