Engineered mutants in the switch II loop of Ran define the contribution made by key residues to the interaction with nuclear transport factor 2 (NTF2) and the role of this interaction in nuclear protein import.
Nuclear protein import requires a precisely choreographed series of interactions between nuclear pore components and soluble factors such as importin-beta, Ran, and nuclear transport factor 2 (NTF2). We used the crystal structure of the GDPRan-NTF2 complex to design mutants in the switch II loop of Ran to probe the contribution of Lys71, Phe72 and Arg76 to this interaction. X-ray crystallography showed that the F72Y, F72W and R76E mutations did not introduce major structural changes into the mutant Ran. The GDP-bound form of the switch II mutants showed no detectable binding to NTF2, providing direct evidence that salt bridges involving Lys71 and Arg76 and burying Phe72 are all crucial for the interaction between Ran and NTF2. Nuclear protein accumulation in digitonin-permeabilzed cells was impaired with Ran mutants deficient in NTF2 binding, confirming that the NTF2-Ran interaction is required for efficient transport. We used mutants of the yeast Ran homologue Gsp1p to investigate the effect of the F72Y and R76E mutations in vivo. Although neither mutant was viable when integrated into the genome as a single copy, yeast mildly overexpressing the Gsp1p mutant corresponding Ran F72Y on a centromeric plasmid were viable, confirming that this mutant retained the essential properties of wild-type Ran. However, yeast expressing the Gsp1p mutant corresponding to R76E to comparable levels were not viable, although strains overexpressing the mutant to higher levels using an episomal 2micrometers plasmid were viable, indicating that the R76E mutation may also have interfered with other interactions made by Gsp1p.
Kent
H M
HM
MRC Laboratory of Molecular Biology, Hills Road, Cambridge, CB2 2QH, England.
Moore
M S
MS
Quimby
B B
BB
Baker
A M
AM
McCoy
A J
AJ
Murphy
G A
GA
Corbett
A H
AH
Stewart
M
M
D000818:Animals; D001692:Biological Transport; D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism*; D002460:Cell Line / Q000378:metabolism; D002463:Cell Membrane Permeability; D002467:Cell Nucleus / Q000378:metabolism*; D018360:Crystallography, X-Ray; D020558:GTP Phosphohydrolases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism; D015966:Gene Expression Regulation, Fungal; D006160:Guanosine Triphosphate / Q000378:metabolism; D008958:Models, Molecular; D020559:Monomeric GTP-Binding Proteins; D009154:Mutation; D009687:Nuclear Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D029543:Nucleocytoplasmic Transport Proteins; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D015003:Yeasts / Q000235:genetics / Q000378:metabolism; D020931:ran GTP-Binding Protein
D002352:Carrier Proteins; C080324:GSP1 protein, S cerevisiae; D009687:Nuclear Proteins; D029543:Nucleocytoplasmic Transport Proteins; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D006160:Guanosine Triphosphate; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins; D020559:Monomeric GTP-Binding Proteins; D020931:ran GTP-Binding Protein