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GTP hydrolysis by Ran is required for nuclear envelope assembly
M Hetzer1, D Bilbao-Cortés, T C Walther
1European Molecular Biology Laboratory, Heidelberg, Germany.
Molecular Cell
|July 27, 2000
Summary
Nuclear envelope formation in Xenopus egg extracts needs GTPase activity. Ran GTPase and its exchange factor RCC1 are crucial for nuclear envelope vesicle fusion, requiring GTP generation and hydrolysis.
Area of Science:
- Cell Biology
- Molecular Biology
- Developmental Biology
Background:
- Nuclear envelope formation is essential for cell division and requires complex molecular machinery.
- GTPase activity, particularly involving the Ran protein, is implicated in nuclear processes.
Purpose of the Study:
- To investigate the specific roles of Ran GTPase and its regulators in nuclear envelope formation and vesicle fusion in Xenopus egg extracts.
- To elucidate the mechanism by which Ran GTPase activity drives nuclear envelope assembly.
Main Methods:
- Xenopus egg extracts were used as a cell-free system to study nuclear formation.
- GTPase activity was manipulated using GTPase inhibitors (GTP gamma S) and dominant-negative mutants (Q69L, T24N).
- Depletion and complementation experiments were performed for Ran and RCC1 (Ran exchange factor).
Main Results:
- Nuclear envelope (NE) vesicle fusion was inhibited by GTP gamma S and dominant-negative Ran mutants, confirming the requirement for GTPase activity.
- Depletion of Ran or RCC1 significantly inhibited NE formation, highlighting their essential roles.
- Complementation studies revealed that Ran requires GTP-bound form for function, and RCC1 is necessary for generating this active state.
Conclusions:
- Ran GTPase, regulated by RCC1, plays a critical role in nuclear envelope formation through the promotion of vesicle fusion.
- Both the generation of RanGTP by RCC1 and the GTP hydrolysis by Ran are indispensable for the membrane fusion events during NE assembly.