An intramolecular signaling element that modulates dynamin function in vitro and in vivo.
Dynamin exhibits a high basal rate of GTP hydrolysis that is enhanced by self-assembly on a lipid template. Dynamin's GTPase effector domain (GED) is required for this stimulation, though its mechanism of action is poorly understood. Recent structural work has suggested that GED may physically dock with the GTPase domain to exert its stimulatory effects. To examine how these interactions activate dynamin, we engineered a minimal GTPase-GED fusion protein (GG) that reconstitutes dynamin's basal GTPase activity and utilized it to define the structural framework that mediates GED's association with the GTPase domain. Chemical cross-linking of GG and mutagenesis of full-length dynamin establishes that the GTPase-GED interface is comprised of the N- and C-terminal helices of the GTPase domain and the C-terminus of GED. We further show that this interface is essential for structural stability in full-length dynamin. Finally, we identify mutations in this interface that disrupt assembly-stimulated GTP hydrolysis and dynamin-catalyzed membrane fission in vitro and impair the late stages of clathrin-mediated endocytosis in vivo. These data suggest that the components of the GTPase-GED interface act as an intramolecular signaling module, which we term the bundle signaling element, that can modulate dynamin function in vitro and in vivo.
Chappie
Joshua S
JS
Department of Cell Biology, The Scripps Research Institute, La Jolla, CA 92037, USA.
Acharya
Sharmistha
S
Liu
Ya-Wen
YW
Leonard
Marilyn
M
Pucadyil
Thomas J
TJ
Schmid
Sandra L
SL
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D002460:Cell Line; D002478:Cells, Cultured; D022163:Clathrin-Coated Vesicles / Q000378:metabolism; D034284:Dynamin I / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D034285:Dynamin II / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004705:Endocytosis; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism* / Q000648:ultrastructure; D019084:Fluorescent Antibody Technique, Indirect; D006160:Guanosine Triphosphate / Q000378:metabolism; D006801:Humans; D006868:Hydrolysis; D051379:Mice; D018345:Mice, Knockout; D008854:Microscopy, Electron; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction; D018411:Spodoptera
D006160:Guanosine Triphosphate; D034284:Dynamin I; D034285:Dynamin II