An internal GAP domain negatively regulates presynaptic dynamin in vivo: a two-step model for dynamin function

Area of Science:

• Cell Biology
• Molecular Biology
• Biochemistry

Background:

• Dynamin is a GTPase crucial for vesicle formation during endocytosis.
• Mutations in Drosophila dynamin (shibire) cause temperature-sensitive defects in endocytosis.
• The precise mechanism of dynamin's function and regulation is not fully understood.

Purpose of the Study:

• To elucidate the role of specific dynamin domains and mutations in GTP binding and GTPase activity.
• To investigate the function of the GTPase Effector Domain (GED) as a GTPase Activating Protein (GAP).
• To propose a model for dynamin's mechanism in vesicle formation.

Main Methods:

• Analysis of temperature-sensitive mutations in Drosophila dynamin (shibire).
• In vitro assays to measure GTP binding affinity and GTPase activity.
• In vivo studies to assess the rescue of endocytic defects.

Main Results:

• A mutation in the switch 2 region of dynamin's GTPase domain impairs GTP binding.
• Suppressor mutations in the GTPase domain and GED fully rescue endocytic defects.
• GED acts as an internal GAP for dynamin, negatively regulating its activity.

Conclusions:

• Dynamin's function is negatively regulated by its GAP activity, similar to other GTPases.
• A two-step model for dynamin function is proposed, involving early regulatory and late hydrolysis-dependent steps.