Mutations within the Ran/TC4 GTPase

Abstract

Ran, a member of the Ras superfamily of GTPases, is predominantly localized in the nucleus and is a neces- sary component in the active transport of proteins through nuclear pores. Disruption of Ran function af- fects the regulation of mitosis, DNA synthesis, and RNA processing and export. To explore the mechanisms of Ran function, mutants of the Ran GTPase were charac- terized, several of which are capable of dominantly in- terfering with nuclear protein import. Unlike wild-type Ran, the putative gain-of-function mutant (G19V Ran) was not sensitive to the exchange factor, RCC1. In addi- tion the G19V Ran and effector domain mutants (L43E and E46G Ran) were not sensitive to the GTPase-activat- ing protein, Fug1. Epitope-tagged G19V Ran and L43E Ran isolated from transfected BHK21 cells were each about 50% GTP-bound, whereas the wild-type and a C- terminal deletion mutant (-DE Ran) were primarily bound to GDP. While G19V Ran interacted with known Ran-binding proteins and with an isolated Ran-binding domain, the T24N Ran did not, and binding by L43E Ran was substantially reduced. Wild-type HA1-tagged Ran expressed in BHK21 cells was nuclear, whereas the G19V, T24N, L43E, and E46G forms of Ran were predom- inantly localized at the nuclear envelope, and -DE Ran was primarily cytosolic. Similar results were observed when permeabilized BHK21 cells were incubated with extracts of COS cells expressing the mutants. Thus mu- tations that affect the interaction of Ran with regula- tory proteins and effectors can disrupt the normal sub- cellular localization of Ran, lending support for the current model of Ran-mediated nuclear import.