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Mutations within the Ran/TC4 GTPase. Effects on regulatory factor interactions and subcellular localization
K M Lounsbury1, S A Richards, K L Carey
1Department of Pathology and Comprehensive Cancer Center, University of Vermont, Burlington, Vermont 05405, USA. klounsbu@200.uvm.edu
The Journal of Biological Chemistry
|December 20, 1996
Summary
Mutant forms of Ran GTPase disrupt nuclear protein import by altering interactions with regulatory proteins. These mutations affect Ran
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- Ran GTPase is crucial for nuclear transport, regulating mitosis, DNA synthesis, and RNA processing.
- Ran's function is mediated by its interactions with regulatory proteins like RCC1 and Fug1.
- Disruptions in Ran function can lead to significant cellular dysregulation.
Purpose of the Study:
- To investigate the mechanisms of Ran GTPase function through the characterization of its mutants.
- To explore how specific mutations affect Ran's interaction with regulatory proteins and its subcellular localization.
- To determine the impact of these mutations on nuclear protein import.
Main Methods:
- Characterization of various Ran GTPase mutants, including G19V, L43E, E46G, T24N, and Delta-DE.
- Assessing mutant sensitivity to the exchange factor RCC1 and GTPase-activating protein Fug1.
- Determining GTP/GDP binding states and interactions with Ran-binding proteins.
- Analyzing subcellular localization of wild-type and mutant Ran proteins in transfected cells.
Main Results:
- Gain-of-function mutant G19V Ran and effector domain mutants (L43E, E46G) showed altered sensitivity to RCC1 and Fug1.
- G19V Ran and L43E Ran exhibited increased GTP-bound states, while wild-type and Delta-DE Ran were GDP-bound.
- Mutations affected Ran's interaction with binding proteins, with T24N Ran showing no interaction and L43E reduced binding.
- Mutant Ran proteins displayed altered subcellular localization, with G19V, T24N, L43E, and E46G accumulating at the nuclear envelope, and Delta-DE Ran in the cytosol.
Conclusions:
- Mutations impacting Ran's interaction with regulatory proteins and effectors disrupt its normal subcellular localization.
- Altered localization of Ran mutants supports the model of Ran-mediated nuclear import.
- These findings provide insights into the molecular mechanisms governing nuclear transport regulation by Ran GTPase.