Dynamin assembles into spirals under physiological salt conditions upon the addition of GDP and gamma-phosphate analogues.
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 receptor-mediated 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 gamma-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 approximately 90-kDa species, lacking the C terminus, or to two smaller fragments, a approximately 55-kDa species originating from the N-terminal half of the protein and a approximately 30-kDa species lacking both the N and C termini. This work indicates that the addition of GDP and gamma-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.
Carr
J F
JF
Laboratory of Cell Biochemistry and Biology, NIDDK, National Institutes of Health, Bethesda, Maryland 20892, USA.
Hinshaw
J E
JE
D000818:Animals; D002460:Cell Line; D034281:Dynamins; D020558:GTP Phosphohydrolases / Q000378:metabolism*; D006153:Guanosine Diphosphate / Q000378:metabolism*; D007313:Insecta; D008854:Microscopy, Electron; D010710:Phosphates / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D012965:Sodium Chloride / Q000378:metabolism*
D010710:Phosphates; D011994:Recombinant Proteins; D006153:Guanosine Diphosphate; D012965:Sodium Chloride; D020558:GTP Phosphohydrolases; D034281:Dynamins