Dynamin Assembles into Spirals under Physiological Salt Conditions upon the Addition of GDP and γ-Phosphate Analogues

Abstract

Dynamin is a 100-kDa GTPase that is believed to be involved in the constriction of clathrin-coated pits and the fission of clathrin-coated vesicles during receptormediated endocytosis and during membrane retrieval in nerve termini. It has been shown that purified dynamin incubated under low salt conditions forms rings and spirals that, in dimension and appearance, resemble the dense material occasionally observed at the necks of coated pits. In this report we show that purified dynamin forms spirals under physiological salt conditions when incubated with GDP and ␥-phosphate analogues (beryllium and aluminum fluoride) or when dialyzed into guanosine 5 -3-O-(thio)triphosphate. Moreover, spirals still form when dynamin is proteolyzed to either a predominant ϳ90-kDa species, lacking the C terminus, or to two smaller fragments, a ϳ55-kDa species originating from the N-terminal half of the protein and a ϳ30-kDa species lacking both the N and C termini. This work indicates that the addition of GDP and ␥-phosphate analogues arrests dynamin in a GTP or transition state that markedly stabilizes the spiral conformation under physiological ionic strength conditions and thereby suggests that dynamin in the absence of a receptor is capable of assembly into spirals at the necks of coated pits prior to vesicle fission.

The Pleckstrin Homology Domains of Dynamin Isoforms Require Oligomerization for High Affinity Phosphoinositide Binding

“…Analysis of proteolytic fragments indicated that the PH domain of dynaminϪ1 is required for its assembly on membrane tubes, while the PRD was necessary for neither assembly nor vesiculation. The helical tubes observed were similar in dimensions to the collars formed around the necks of clathrincoated pits in shibire Drosophila (8), and formed under conditions that do not favor assembly in the absence of lipids (19,20). Thus, Sweitzer and Hinshaw (55) argue that the lipid vesicles provide a surface on which dynamin self-assembly is favored, as previously suggested by chemical cross-linking studies (26), and further that the PH domain participates in this process.…”

“…In the absence of membranes, purified dynamin forms a tetramer (14), which further self-assembles into rings or spirals when subjected to low ionic strength conditions (19) or (at physiological ionic strength) to GDP plus metallofluorides (20). The assemblies are morphologically similar to the collars seen in constricted coated vesicles in vivo, and their formation requires the PRD, but not the PH domain (14,19).…”

Chapter 13 Visualization of Dynamins

“…In addition, incubation with GDP/BeF, under physiological salt conditions, results in dynamin rings and spirals (Carr and Hinshaw, 1997). To make spirals, dynamin (~0.2 mg/ml) in HCB100 (Hepes Column BuVer (20 mM Hepes, pH 7.2, 1 mM MgCl 2 , 2 mM EGTA, 1 mM DTT) with 100 mM NaCl) is incubated with 1 mM GDP, 5 mM NaF, and 500 µM BeCl 2 for 15 min at room temperature ( Fig.…”

“…A common method used to quantify the amount of oligomer formation in solution is a sedimentation assay (Carr and Hinshaw, 1997;Danino et al, 2004;Hinshaw and Schmid, 1995). Samples incubating at room temperature are transferred to an ice bath to prevent any additional reactions.…”

Dynamin Functions and Ligands: Classical Mechanisms Behind

“…Purified dynamin always polymerizes/self-assembles into rings and helices in solutions of suitable ionic strength (Carr and Hinshaw, 1997). Dynamin tubulates membrane bilayers …”