Real-time detection reveals that effectors couple dynamin's GTP-dependent conformational changes to the membrane.
The GTPase dynamin is a mechanochemical enzyme involved in membrane fission, but the molecular nature of its membrane interactions and their regulation by guanine nucleotides and protein effectors remain poorly characterized. Using site-directed fluorescence labeling and several independent fluorescence spectroscopic techniques, we have developed robust assays for the detection and real-time monitoring of dynamin-membrane and dynamin-dynamin interactions. We show that dynamin interacts preferentially with highly curved, PIP2-dense membranes and inserts partially into the lipid bilayer. Our kinetic measurements further reveal that cycles of GTP binding and hydrolysis elicit major conformational rearrangements in self-assembled dynamin that favor dynamin-membrane association and dissociation, respectively. Sorting nexin 9, an abundant dynamin partner, transiently stabilizes dynamin on the membrane at the onset of stimulated GTP hydrolysis and may function to couple dynamin's mechanochemical conformational changes to membrane destabilization. Amphiphysin I has the opposite effect. Thus, dynamin's mechanochemical properties on a membrane surface are dynamically regulated by its GTPase cycle and major binding partners.
Ramachandran
Rajesh
R
Department of Cell Biology, The Scripps Research Institute, La Jolla, CA 92037, USA.
Schmid
Sandra L
SL
D000818:Animals; D002462:Cell Membrane / Q000201:enzymology* / Q000235:genetics; D002478:Cells, Cultured; D034284:Dynamin I / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000502:physiology*; D006801:Humans; D007313:Insecta / Q000235:genetics; D008051:Lipid Bilayers / Q000737:chemistry / Q000378:metabolism; D008081:Liposomes; D016297:Mutagenesis, Site-Directed; D010713:Phosphatidylcholines / Q000737:chemistry / Q000378:metabolism; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000737:chemistry / Q000378:metabolism; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D013552:Swine
D008051:Lipid Bilayers; D008081:Liposomes; D010713:Phosphatidylcholines; D019269:Phosphatidylinositol 4,5-Diphosphate; D006160:Guanosine Triphosphate; D034284:Dynamin I; C017251:1,2-oleoylphosphatidylcholine