Abstract
We have used a range of complementary biochemical and biophysical methods to investigate the interactions between nuclear transport factor 2 (NTF2), the Ras family GTPase Ran, and XFXFG nucleoporin repeats that are crucial for nuclear trafficking. Microcalorimetry, microtiter plate binding, and fluorescence quenching in solution are all consistent with the binding constant for the NTF2-RanGDP interaction being in the 100 nM range, whereas the interaction between NTF2 and XFXFG repeat-containing nucleoporins such as Nsp1p is in the 1 microM range. Although the accumulation of NTF2 at the nuclear envelope is enhanced by RanGDP, we show that Ran binding does not alter the affinity of NTF2 for nucleoporins nor does the binding of nucleoporins alter the affinity of NTF2 for RanGDP. These results indicate that, instead, Ran increases the binding of NTF2 to nucleoporins by another mechanism, most probably by Ran itself binding to nucleoporins and NTF2 binding to this nuclear pore-associated Ran.
Highlights
The Ras family GTPase Ran is a key component of the nuclear trafficking machinery and is important in cell cycle progression [1, 2]
The accumulation of nuclear transport factor 2 (NTF2) at the nuclear envelope is enhanced by RanGDP, we show that Ran binding does not alter the affinity of NTF2 for nucleoporins nor does the binding of nucleoporins alter the affinity of NTF2 for RanGDP
Using microtiter plates coated with 0.7 M NTF2, S-tagged RanGDP showed a Kd of 240 Ϯ 90 nM (Fig. 1A), whereas plates coated with the NTF2 E42K mutant (which does not bind RanGDP (Ref. 8)) did not bind RanGDP
Summary
Protein Expression and Purification—Rat NTF2, canine RanGDP, and the 18 XFXFG repeat-containing fragment of nucleoporin Nsp1p (18R-Nsp1) were expressed and purified as described [7, 17, 14]. After blocking with PBS-I-block (0.2%), S-tagged Ran diluted in PBS containing 0.2% I-Block was added to the wells at a range of concentrations (0 – 8 M) and incubated for either 2 h at room temper-. NBD Labeling of NTF2—NTF2 dialyzed against PBS buffer containing 2 mM MgCl2 was incubated with a 10-fold excess of NBD-Cl overnight at 4 °C. The interaction of NTF2 with Ran was assayed using NBD-labeled NTF2 (1 M) and monitoring the changes in NBD fluorescence emission followed at 506 nm after excitation at 435 nm. After five column washes with 20 mM Tris, pH 8.0, 2 mM MgCl2, 1 mM dithiothreitol, 0.1 mM phenylmethylsulfonyl fluoride, radioactive NTF2 was eluted with 40 ml of a 0 –50% ethylene glycol linear gradient in the same buffer. All the concentrations given for NTF2 are given according to the amount of monomeric chains present
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