TY - JOUR
T1 - Rapid constriction of lipid bilayers by the mechanochemical enzyme dynamin
AU - Danino, Dganit
AU - Moon, Kwan Hoon
AU - Hinshaw, Jenny E.
N1 - Funding Information:
We thank D. Sackett for his help with the light scattering experiments; M. Marino for his technical assistance; and A.R. Robbins for critical reading of the manuscript. This work was supported in part by the Israel Science Foundation of the Israel Academy of Sciences and Humanities (No. 530/03).
PY - 2004/9
Y1 - 2004/9
N2 - Dynamin, a large GTPase, is located at the necks of clathrin-coated pits where it facilitates the release of coated vesicles from the plasma membrane upon GTP binding, and hydrolysis. Previously, we have shown by negative stain electron microscopy that wild-type dynamin and a dynamin mutant lacking the C-terminal proline-rich domain, ΔPRD, form protein-lipid tubes that constrict and vesiculate upon addition of GTP. Here, we show by time-resolved cryo-electron microscopy (cryo-EM) that ΔPRD dynamin in the presence of GTP rapidly constricts the underlying lipid bilayer, and then gradually disassembles from the lipid. In agreement with the negative stain results, the dynamin tubes constrict from 50 to 40nm, and their helical pitch decreases from ∼13 to 9.4nm. However, in contrast to the previous results, examination by cryo-EM shows that the lipid bilayer remains intact and small vesicles or fragments do not form upon GTP binding and hydrolysis. Therefore, the vesicle formation seen by negative stain may be due to the lack of mobility of the dynamin tubes on the grid during the GTP-induced conformational changes. Our results confirm that dynamin is a mechanochemical enzyme and suggest that during endocytosis dynamin is directly responsible for membrane constriction. In the cell, other proteins may enhance the activity of dynamin or the constraints induced by the surrounding coated pit and plasma membrane during constriction may cause the final membrane fission event.
AB - Dynamin, a large GTPase, is located at the necks of clathrin-coated pits where it facilitates the release of coated vesicles from the plasma membrane upon GTP binding, and hydrolysis. Previously, we have shown by negative stain electron microscopy that wild-type dynamin and a dynamin mutant lacking the C-terminal proline-rich domain, ΔPRD, form protein-lipid tubes that constrict and vesiculate upon addition of GTP. Here, we show by time-resolved cryo-electron microscopy (cryo-EM) that ΔPRD dynamin in the presence of GTP rapidly constricts the underlying lipid bilayer, and then gradually disassembles from the lipid. In agreement with the negative stain results, the dynamin tubes constrict from 50 to 40nm, and their helical pitch decreases from ∼13 to 9.4nm. However, in contrast to the previous results, examination by cryo-EM shows that the lipid bilayer remains intact and small vesicles or fragments do not form upon GTP binding and hydrolysis. Therefore, the vesicle formation seen by negative stain may be due to the lack of mobility of the dynamin tubes on the grid during the GTP-induced conformational changes. Our results confirm that dynamin is a mechanochemical enzyme and suggest that during endocytosis dynamin is directly responsible for membrane constriction. In the cell, other proteins may enhance the activity of dynamin or the constraints induced by the surrounding coated pit and plasma membrane during constriction may cause the final membrane fission event.
KW - Dynamin
KW - Endocytosis
KW - GTPase
KW - Membrane fission
KW - Self-assembly
KW - Time-resolved cryo-EM
UR - http://www.scopus.com/inward/record.url?scp=4344586301&partnerID=8YFLogxK
U2 - 10.1016/j.jsb.2004.04.005
DO - 10.1016/j.jsb.2004.04.005
M3 - 文章
C2 - 15450295
AN - SCOPUS:4344586301
SN - 1047-8477
VL - 147
SP - 259
EP - 267
JO - Journal of Structural Biology
JF - Journal of Structural Biology
IS - 3
ER -