Fluorescence Resonance Energy Transfer Biosensors That Detect Ran Conformational Changes and a Ran·GDP-Importin-β-RanBP1 Complexin Vitro and in Intact Cells

Kendra Plafker,Ian G Macara

doi:10.1074/jbc.m203006200

Abstract

The Ran GTPase plays a central role in nucleocytoplasmic transport. Association of Ran x GTP with transport carriers (karyopherins) triggers the loading/unloading of export or import cargo, respectively. The C-terminal tail of Ran x GTP is deployed in an extended conformation when associated with a Ran binding domain or importins. To monitor tail orientation, a Ran-GFP fusion was labeled with the fluorophore Alexa546. Fluorescence resonance energy transfer (FRET) occurs efficiently between the green fluorescent protein (GFP) and Alexa546 for Ran x GDP and Ran x GTP, suggesting that the tail is tethered in both states. However, Ran x GTP complexes with importin-beta, RanBP1, and Crm1 all show reduced FRET consistent with tail extension. Displacement of the C-terminal tail of Ran by karyopherins may be a general mechanism to facilitate RanBP1 binding. A Ran x GDP-RanBP1-importin-beta complex also displayed a low FRET signal. To detect this complex in vivo, a bipartite biosensor consisting of Ran-Alexa546 plus GST-GFP-RanBP1, was co-injected into the cytoplasm of cells. The Ran redistributed predominantly to the nucleus, and RanBP1 remained cytoplasmic. Nonetheless, a robust cytoplasmic FRET signal was detectable, which suggests that a significant fraction of cytoplasmic Ran.GDP may exist in a ternary complex with RanBP1 and importins.

Highlights

The flux of macromolecules between the nucleus and cytoplasm of a fibroblast has been estimated to be in excess of 10 million/min [1]
A robust cytoplasmic Fluorescence resonance energy transfer (FRET) signal was detectable, which suggests that a significant fraction of cytoplasmic Ran1⁄7GDP may exist in a ternary complex with Ran-binding protein 1 (RanBP1) and importins
This huge traffic flows through about 3,000 nuclear pore complexes (NPCs) that are plugged into the double membrane that encompasses the nucleus [2,3,4,5]

Summary

Introduction

The flux of macromolecules between the nucleus and cytoplasm of a fibroblast has been estimated to be in excess of 10 million/min [1]. For both importins and exportins, a carrier-Ran1⁄7GTP complex is delivered to the cytoplasm, where RanGAP, in co-operation with a factor called Ran-binding protein 1 (RanBP1),1 triggers hydrolysis of the GTP and release of Ran1⁄7GDP from the carrier.

Results

Conclusion