Ran in Nucleocytoplasmic Transport - Ras Superfamily Small G Proteins: Biology and Mechanisms 2 - page 112

et al. 1998 ; Smith et al. 1998 ), that binds RanGDP with ~100 nM affinity but does
not bind RanGTP. The ~100 nM affinity of NTF2 for RanDGP is sufficient for it to
remain bound during the ~100 ms required to traverse the pore after which it can
dissociate in the nucleus to enable the Ran nucleotide to be exchanged (Bayliss
et al. 1999 ; Chaillan-Huntington et al. 2000 ). Like the karyopherins, NTF2 is able
to surmount the NPC barrier function by binding to FG nucleoporins, albeit in this
case only those that have a FxFG core motif (Bayliss et al. 1999 , 2002b ).
The selectivity exhibited by NTF2 for binding RanGDP but not Ran-GTP
derives from NTF2 binding primarily to the Ran switch II loop (Stewart
et al. 1998a ) that changes its conformation dramatically between the different
nucleotide states (Fig. 6.2 ). In RanGDP, the conformation taken up by the switch
II loop enables the aromatic side chain of Phe72 of Ran to insert into the central
hydrophobic cavity of NTF2 while also facilitating formation of a salt bridge
between Lys71 of Ran and Asp92, Glu93, and Asp94 of NTF2 (Stewart
et al. 1998a ; Kent et al. 1999 ). Mutations in NTF2 that impair its binding to Ran
result in Ran mislocalization and growth defects in yeast (Paschal and Gerace 1995 ;
Clarkson et al. 1997 ; Quimby et al. 2001 ) underlining the functional importance of
this interaction in vivo. Interestingly, NTF2 null mutants can be suppressed by
overexpression of Ran, indicating that, although RanGDP is sufficiently small to be
able to diffuse unaided through NPCs, its import needs to be accelerated in living
cells because of the high rate at which Ran is employed in nucleocytoplasmic
transport (Paschal et al. 1997 ). The switch II loop in the GDP-bound form of the
Ran Q69L mutant, in which the GTPase activity is impaired and which is used
extensively in functional studies, takes on a different conformation to wild type
which may complicate studies in which it is used in vivo (Stewart et al. 1998b ).
In addition to binding RanGDP, NTF2 is also able to bind to FG nucleoporins
and so facilitate the transport of RanGDP from the cytoplasm to the nucleus through
NPCs. Mutation of the FG-nucleoporin binding site impairs Ran nuclear import and
leads to growth defects in yeast (Bayliss et al. 1999 ; Quimby et al. 2001 ).
Mog1 is another Ran-binding protein that has a vague structural homology to
NTF2 and which binds to Ran in either nucleotide state (Stewart and Baker 2000 ).
Although NTF2 and Mog1 compete for binding to RanGDP, the binding sites do not
appear to be identical. The precise function of Mog1 remains somewhat specula-
tive, but the ability of Mog1 overexpression to suppress a number of temperature-
sensitive variants of the yeast Ran homologue Gsp1 would be consistent with
formation of the complex contributing to the thermal stability of Ran (Oki and
Nishimoto 1998 ).

6.5 Nuclear Import Pathways

Proteins are imported into the nucleus by karyopherin-family (Chook and Blobel
2001 ) transport factors such as importin- and transportin (Kap95 and Kap104,

respectively, in yeast). These transport factors bind their cargoes in the cytoplasm,

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