Ran in Nucleocytoplasmic Transport - Ras Superfamily Small G Proteins: Biology and Mechanisms 2 - page 111

6.3 The Spatial Distribution of Ran Nucleotide State

The nucleotide state of Ran is determined by the action of a guanine nucleotide
exchange factor (RanGEF or RCC1, Prp20 in yeast) and a Ran GTPase activating
protein (RanGAP, Rna1 in yeast). RCC1 mediates nucleotide exchange on Ran,
whereas RanGAP stimulates the Ran GTPase reaction. In both cases these acces-
sory proteins stimulate their respective processes by roughly five orders of magni-
tude (Klebe et al. 1995a ). The asymmetric distribution of these proteins is crucial
for imposing directionality on karyopherin-based nuclecytoplasmic transport path-
ways (Izaurralde et al. 1997 ).
The RanGEF, RCC1 (yeast Prp20), has a 7-bladed

-propeller structure (Renault
et al. 1998 ) and catalyzes the exchange of nucleotides on Ran (Bischoff and
Ponstingl 1991 ) by stabilizing the nucleotide-free transition state (Klebe
et al. 1995b ; Renault et al. 2001 ). Although Ran has a slightly higher affinity for
GDP than GTP, because the nucleotide in cells is almost entirely GTP, RCC1
essentially results in RanGDP rapidly being converted into RanGTP (Klebe
et al. 1995b ). Moreover, because RCC1 is bound to chromatin (Ohtsubo
et al. 1989 ), nuclear Ran is primarily in the GTP-bound form.
Conversely, RanGAP, in conjunction with Ran-binding protein 1 (RanBP1),
stimulates the Ran GTPase activity resulting in the conversion of RanGTP to
RanGDP (Bischoff and G¨rlich 1997 ). In metazoans, RanGAP binds to RanBP2
that is present in cytoplasmic fibrils emanating from NPCs in an interaction that
requires RanGAP to be modified with the small ubiquitin-related protein, SUMO
(Mahajan et al. 1997 ; Matunis et al. 1996 , 1998 ). Because RanGAP is located in the
cytoplasm, cytoplasmic Ran is primarily in the GDP-bound form. In common with
many other Ras-family GTPases, the enzymatic activity of Ran is impaired by
mutation of a key glutamine (Q69L mutation), albeit this mutation also introduces
changes into the structure of Ran switch II loop in the GDP-bound state (Stewart
et al. 1998b ). Although most GAPs for Ras-family GTPases employ an arginine
finger to catalyze GTP hydrolysis (Ahmadian et al. 1997 ), RanGAP has a novel fold
(Hillig et al. 1999 ) and employs a novel mechanism in which the correct positioning
of the catalytic machinery on Ran itself and its shielding from the solvent are
sufficient (Hillig et al. 1999 ; Seewald et al. 2002 ).

β

6.4 NTF2 Facilitates Return of RanGDP to the Nucleus

Following RanGAP-mediated GTP hydrolysis in the cytoplasm, it is necessary for
RanGDP to return to the nucleus to be recharged with GTP by RCC1. Although
RanGDP is sufficiently small to be able to diffuse through the NPC transport
channel, it appears that this simple diffusion is not sufficiently rapid to maintain
adequate levels of nuclear RanGTP and so the nuclear import of RanGDP is
expedited by a specialized import factor, NTF2 (Wong et al. 1997 ; Ribbeck