Multiple distinct coiled-coils are involved in dynamin self-assembly.
Dynamin, a 100-kDa GTPase, has been implicated to be involved in synaptic vesicle recycling, receptor-mediated endocytosis, and other membrane sorting processes. Dynamin self-assembles into helical collars around the necks of coated pits and other membrane invaginations and mediates membrane scission. In vitro, dynamin has been reported to exist as dimers, tetramers, ring-shaped oligomers, and helical polymers. In this study we sought to define self-assembly regions in dynamin. Deletion of two closely spaced sequences near the dynamin-1 C terminus abolished self-association as assayed by co-immunoprecipitation and the yeast interaction trap, and reduced the sedimentation coefficient from 7.5 to 4.5 S. Circular dichroism spectroscopy and equilibrium ultracentrifugation of synthetic peptides revealed coiled-coil formation within the C-terminal assembly domain and at a third, centrally located site. Two of the peptides formed tetramers, supporting a role for each in the monomer-tetramer transition and providing novel insight into the organization of the tetramer. Partial deletions of the C-terminal assembly domain reversed the dominant inhibition of endocytosis by dynamin-1 GTPase mutants. Self-association was also observed between different dynamin isoforms. Taken altogether, our results reveal two distinct coiled-coil-containing assembly domains that can recognize other dynamin isoforms and mediate endocytic inhibition. In addition, our data strongly suggests a parallel model for dynamin subunit self-association.
Okamoto
P M
PM
Department of Cell Biology, University of Massachusetts Medical School, Worcester, Massachusetts 01605, USA.
Tripet
B
B
Litowski
J
J
Hodges
R S
RS
Vallee
R B
RB
D000818:Animals; D019556:COS Cells; D002499:Centrifugation, Density Gradient; D002942:Circular Dichroism; D034284:Dynamin I; D034281:Dynamins; D004705:Endocytosis; D020558:GTP Phosphohydrolases / Q000737:chemistry* / Q000235:genetics; D008870:Microtubules / Q000737:chemistry* / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D010641:Phenotype; D011485:Protein Binding; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D013329:Structure-Activity Relationship; D014461:Ultracentrifugation; D015003:Yeasts
D034284:Dynamin I; D020558:GTP Phosphohydrolases; D034281:Dynamins