Dynamin GTPase domain mutants that differentially affect GTP binding, GTP hydrolysis, and clathrin-mediated endocytosis.
The GTPase dynamin is essential for clathrin-mediated endocytosis. Unlike most GTPases, dynamin has a low affinity for nucleotide, a high rate of GTP hydrolysis, and can self-assemble, forming higher order structures such as rings and spirals that exhibit up to 100-fold stimulated GTPase activity. The role(s) of GTP binding and/or hydrolysis in endocytosis remain unclear because mutations in the GTPase domain so far studied impair both. We generated a new series of GTPase domain mutants to probe the mechanism of GTP hydrolysis and to further test the role of GTP binding and/or hydrolysis in endocytosis. Each of the mutations had parallel effects on assembly-stimulated and basal GTPase activities. In contrast to previous reports, we find that mutation of Thr-65 to Ala (or Asp or His) dramatically lowered both the rate of assembly-stimulated GTP hydrolysis and the affinity for GTP. The assemblystimulated rate of hydrolysis was lowered by the mutation of Ser-61 to Asp and increased by the mutation of Thr-141 to Ala without significantly altering the Km for GTP. For some mutants and to a lesser extent for WT dynamin, self-assembly dramatically altered the Km for GTP, suggesting that conformational changes in the active site accompany self-assembly. Analysis of transferrin endocytosis rates in cells overexpressing mutant dynamins revealed a stronger correlation with both the basal and assembly-stimulated rates of GTP hydrolysis than with the calculated ratio of dynamin-GTP/free dynamin, suggesting that GTP binding is not sufficient, and GTP hydrolysis is required for clathrin-mediated endocytosis in vivo.
Song
Byeong Doo
BD
Department of Cell Biology, The Scripps Research Institute, La Jolla, California 92037, USA.
Leonard
Marilyn
M
Schmid
Sandra L
SL
D000256:Adenoviridae / Q000235:genetics; D000409:Alanine / Q000737:chemistry; D016367:Baculoviridae / Q000378:metabolism; D001665:Binding Sites; D002850:Chromatography, Gel; D002966:Clathrin / Q000378:metabolism*; D034281:Dynamins / Q000737:chemistry* / Q000378:metabolism; D004705:Endocytosis; D020558:GTP Phosphohydrolases / Q000235:genetics / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D008055:Lipids / Q000737:chemistry; D008956:Models, Chemical; D009154:Mutation; D009841:Oligonucleotides / Q000737:chemistry; D017354:Point Mutation; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D012694:Serine / Q000737:chemistry; D013696:Temperature; D013912:Threonine / Q000737:chemistry; D013997:Time Factors; D014168:Transferrin / Q000737:chemistry
D002966:Clathrin; D008055:Lipids; D009841:Oligonucleotides; D014168:Transferrin; D013912:Threonine; D012694:Serine; D006160:Guanosine Triphosphate; D020558:GTP Phosphohydrolases; D034281:Dynamins; D000409:Alanine