Mutations within the Ran/TC4 GTPase

doi:10.1074/jbc.271.51.32834

Mutations within the Ran/TC4 GTPase

EFFECTS ON REGULATORY FACTOR INTERACTIONS AND SUBCELLULAR LOCALIZATION*

From the Department of Pathology and Comprehensive Cancer Center, University of Vermont, Burlington, Vermont 05405

^‡Supported by National Research Service Award F32CA63801 from the NCI, National Institutes of Health. To whom correspondence should be addressed. E-mail: klounsbu{at}200.uvm.edu

Abstract

Ran, a member of the Ras superfamily of GTPases, is predominantly localized in the nucleus and is a necessary component in the active transport of proteins through nuclear pores. Disruption of Ran function affects the regulation of mitosis, DNA synthesis, and RNA processing and export. To explore the mechanisms of Ran function, mutants of the Ran GTPase were characterized, several of which are capable of dominantly interfering 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 addition the G19V Ran and effector domain mutants (L43E and E46G Ran) were not sensitive to the GTPase-activating 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 (Δ-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 predominantly localized at the nuclear envelope, and Δ-DE Ran was primarily cytosolic. Similar results were observed when permeabilized BHK21 cells were incubated with extracts of COS cells expressing the mutants. Thus mutations that affect the interaction of Ran with regulatory proteins and effectors can disrupt the normal subcellular localization of Ran, lending support for the current model of Ran-mediated nuclear import.

Footnotes

↵^§ Supported by National Institutes of Health Environmental Pathology Training Grant EST3207122.
↵* This work was supported in part by Public Service Award GM50526 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

RCC1

regulator of chromosome condensation 1

RanGAP

Ran GTPase-activating protein

RanBP

Ran-binding protein

RanBD

Ran-binding domain

Δ-DE Ran

C-terminally deleted Ran

BSA

bovine serum albumin

HSA

human serum albumin

GST

glutathione S-transferase

PAGE

polyacrylamide gel electrophoresis

MOPS

3-(N-morpholino)propanesulfonic acid

NLS

nuclear localization signal

TBS-T

Tris-buffered saline plus 0.1% Tween 20

GAP

GTPase-activating protein.
- Received April 30, 1996.
- Revision received September 24, 1996.