Alternate pleckstrin homology domain orientations regulate dynamin-catalyzed membrane fission.
The self-assembling GTPase dynamin catalyzes endocytic vesicle scission via membrane insertion of its pleckstrin homology (PH) domain. However, the molecular mechanisms underlying PH domain-dependent membrane fission remain obscure. Membrane-curvature-sensing and membrane-curvature-generating properties have been attributed, but it remains to be seen whether the PH domain is involved in either process independent of dynamin self-assembly. Here, using multiple fluorescence spectroscopic and microscopic techniques, we demonstrate that the isolated PH domain does not act to bend membranes but instead senses high membrane curvature through hydrophobic insertion into the membrane bilayer. Furthermore, we use a complementary set of short- and long-distance Förster resonance energy transfer approaches to distinguish PH-domain orientation from proximity at the membrane surface in full-length dynamin. We reveal, in addition to the GTP-sensitive "hydrophobic mode," the presence of an alternate, GTP-insensitive "electrostatic mode" of PH domain-membrane interactions that retains dynamin on the membrane surface during the GTP hydrolysis cycle. Stabilization of this alternate orientation produces dramatic variations in the morphology of membrane-bound dynamin spirals, indicating that the PH domain regulates membrane fission through the control of dynamin polymer dynamics.
Department of Physiology and Biophysics, Case Western Reserve University School of Medicine, Cleveland, OH 44106.
Niharika
N
Mehrotra
Justin
J
Nichols
Rajesh
R
Ramachandran
D000595:Amino Acid Sequence; D002462:Cell Membrane / Q000378:metabolism* / Q000648:ultrastructure; D034284:Dynamin I / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004705:Endocytosis; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D015870:Gene Expression; D006160:Guanosine Triphosphate / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D008081:Liposomes / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D055672:Static Electricity
D008081:Liposomes; D011994:Recombinant Proteins; D006160:Guanosine Triphosphate; D034284:Dynamin I