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Mutations in human dynamin block an intermediate stage in coated vesicle formation
A M van der Bliek1, T E Redelmeier, H Damke
1Division of Biology, California Institute of Technology, Pasadena 91125.
Abstract:
The role of human dynamin in receptor-mediated endocytosis was investigated by transient expression of GTP-binding domain mutants in mammalian cells. Using assays which detect intermediates in coated vesicle formation, the dynamin mutants were found to block endocytosis at a stage after the initiation of coat assembly and preceding the sequestration of ligands into deeply invaginated coated pits. Membrane transport from the ER to the Golgi complex was unaffected indicating that dynamin mutants specifically block early events in endocytosis. These results demonstrate that mutations in the GTP-binding domain of dynamin block Tfn-endocytosis in mammalian cells and suggest that a functional dynamin GTPase is required for receptor-mediated endocytosis via clathrin-coated pits.
Insights
Human dynamin is crucial for receptor-mediated endocytosis. Mutations in its GTP-binding domain block this process, specifically at early stages of coated vesicle formation in mammalian cells.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Receptor-mediated endocytosis is a vital cellular process for nutrient uptake and signal transduction.
- Dynamin, a GTPase, is implicated in various membrane trafficking events, including endocytosis.
- The precise role of dynamin's GTP-binding domain in endocytosis remains to be fully elucidated.
Purpose of the Study:
- To investigate the function of human dynamin in receptor-mediated endocytosis.
- To determine the specific stage of endocytosis affected by dynamin GTP-binding domain mutants.
- To assess the necessity of a functional dynamin GTPase for clathrin-coated pit-mediated endocytosis.
Main Methods:
- Transient expression of GTP-binding domain mutants of human dynamin in mammalian cells.
- Utilizing assays to detect intermediates in coated vesicle formation.
- Monitoring transferrin (Tfn) endocytosis and membrane transport from the ER to the Golgi.
Main Results:
- Dynamin mutants blocked endocytosis after coat assembly initiation but before ligand sequestration.
- The observed blockage occurred specifically in early endocytic events.
- Membrane transport from the ER to the Golgi remained unaffected, indicating specificity.
- Mutations in the dynamin GTP-binding domain inhibited transferrin endocytosis.
Conclusions:
- A functional dynamin GTPase is essential for receptor-mediated endocytosis via clathrin-coated pits.
- Mutations in the dynamin GTP-binding domain disrupt early stages of endocytosis.
- Dynamin plays a critical role in the scission of coated vesicles during endocytosis.