Abstract
Ran is a small GTPase that
cycles between a guanosine diphosphate (GDP)-bound form (RanGDP) and a
guanosine triphosphate (GTP)-bound form (RanGTP) and plays important roles in
nuclear transport and mitosis. For studies of Ran function and its interactions
with partner proteins, pure RanGDP and RanGTP complexes are critical. Ran
complexed with the nonhydrolyzable GTP analog, GMPPNP (RanGMPPNP), is used
instead of RanGTP when inhibition of hydrolysis is required. In this study, we
demonstrate that the binding of Ran to a UNO Q ion exchange column is
remarkably sensitive to small shifts in MgCl2 concentration, and we use this
property to purify recombinant RanGTP, RanGMPPNP, and RanGDP complexes. At 10mM
MgCl2, Ran was found predominantly in the flow-through and, thus, was separated
from the vast majority of bacterial proteins. After reducing the concentration
of MgCl2 to 5mM, further purification of RanGTP, RanGMPPNP, and RanGDP was
achieved by loading onto ion exchange columns and elution with an NaCl
gradient. Purity of the resulting preparations was confirmed by releasing the
bound nucleotide and checking it against a known nucleotide by high-performance
liquid chromatography (HPLC). To further confirm the purity and function of the
Ran preparations, appropriate protein-binding, enzymatic, and nuclear import
assays were carried out. These methods should facilitate studies of cellular
processes involving Ran by providing pure functional Ran–nucleotide complexes.
Comment
This is work from Nabeel Yaseen's lab at Northwestern
University. He is seeking to
understand the function of the Ran GTPase involved in the pathways of nuclear
import and export.