An Intramolecular Signaling Element that Modulates Dynamin Function In Vitro and In Vivo

• DOI: 10.1091/mbc.e09-04-0318
• Publication Date: 2009-06-11T01:29:00Z
• Authors: Joshua S. Chappie; Sharmistha Acharya; Ya-Wen Liu; Marilyn Leonard; Thomas J. Pucadyil; Sandra L. Schmid
• Keywords: Mice, Knockout; Models, Molecular; 0301 basic medicine; Binding Sites; Hydrolysis; Molecular Sequence Data; Clathrin-Coated Vesicles; Fibroblasts; Endocytosis; Cell Line; Dynamin II; Mice; Microscopy, Electron; 03 medical and health sciences; Animals; Humans; Amino Acid Sequence; Guanosine Triphosphate; Fluorescent Antibody Technique, Indirect; Cells, Cultured; Dynamin I; Protein Binding

Dynamin exhibits a high basal rate of GTP hydrolysis that is enhanced by self-assembly on lipid template. Dynamin's GTPase effector domain (GED) required for this stimulation, though its mechanism action poorly understood. Recent structural work has suggested GED may physically dock with the to exert stimulatory effects. To examine how these interactions activate dynamin, we engineered minimal GTPase-GED fusion protein (GG) reconstitutes dynamin's activity and utilized it define framework mediates GED's association domain. Chemical cross-linking GG mutagenesis full-length dynamin establishes interface comprised N- C-terminal helices C-terminus GED. We further show essential stability in dynamin. Finally, identify mutations disrupt assembly-stimulated dynamin-catalyzed membrane fission vitro impair late stages clathrin-mediated endocytosis vivo. These data suggest components act as an intramolecular signaling module, which term bundle element, can modulate function