GTP HYDROLYSIS IS REQUIRED FOR VESICLE FUSION DURING NUCLEAR-ENVELOPE ASSEMBLY INVITRO
GTP HYDROLYSIS IS REQUIRED FOR VESICLE FUSION DURING NUCLEAR-ENVELOPE ASSEMBLY INVITRO
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DOI：
10.1083/jcb.116.2.281
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发表时间：
1992-01-01
影响因子：
7.8
通讯作者：
WILSON, KL
中科院分区：
文献类型：
--
作者：
BOMAN, AL;DELANNOY, MR;WILSON, KL
用非洲爪蛙卵提取液体外研究了核膜组装。核特异性小泡与去膜精子染色质结合，但在没有胞浆的情况下不融合。胞浆的加入促进了囊泡融合、毛孔复合体组装和最终核膜的生长。光镜和电子显微镜观察囊泡结合和融合情况。在结合的小泡中加入ATP和GTP可导致有限的小泡融合，但没有观察到染色质的封闭。这一结果表明，核泡融合需要不可透析的可溶性成分。GTP-γ-S和鸟苷酰亚胺二磷酸显着抑制小泡融合，但不影响小泡与染色质的结合。用新鲜的胞浆检测，1 mM GTP-γ-S或GTP预先孵育的膜不影响囊泡的结合或融合。然而，用GTP-γ-S和胞浆预先孵育的膜对融合有不可逆转的抑制作用。介导GTP-γ-S抑制作用的可溶性因子，我们称之为GTP依赖的可溶性因子(GSF)，是可滴定的，并通过与多余的膜与GTP-γ-S孵育而从细胞质中消失，表明GSF与膜组分之间存在化学计量比相互作用。在初步实验中，耗尽了GSF的胞浆对染色质结合的小泡的融合仍然活跃，这表明融合反应本身可能不需要GSF。我们认为GTP在核泡融合之前的一个步骤中是必需的。
Nuclear envelope assembly was studied in vitro using extracts from Xenopus eggs. Nuclear-specific vesicles bound to demembranated sperm chromatin but did not fuse in the absence of cytosol. Addition of cytosol stimulated vesicle fusion, pore complex assembly, and eventual nuclear envelope growth. Vesicle binding and fusion were assayed by light and electron microscopy. Addition of ATP and GTP to bound vesicles caused limited vesicle fusion, but enclosure of the chromatin was not observed. This result suggested that nondialyzable soluble components were required for nuclear vesicle fusion. GTP-gamma-S and guanylyl imidodiphosphate significantly inhibited vesicle fusion but had no effect on vesicle binding to chromatin. Preincubation of membranes with 1 mM GTP-gamma-S or GTP did not impair vesicle binding or fusion when assayed with fresh cytosol. However, preincubation of membranes with GTP-gamma-S plus cytosol caused irreversible inhibition of fusion. The soluble factor mediating the inhibition by GTP-gamma-S, which we named GTP-dependent soluble factor (GSF), was titratable and was depleted from cytosol by incubation with excess membranes plus GTP-gamma-S, suggesting a stoichiometric interaction between GSF and a membrane component in the presence of GTP-gamma-S. In preliminary experiments, cytosol depleted of GSF remained active for fusion of chromatin-bound vesicles, suggesting that GSF may not be required for the fusion reaction itself. We propose that GTP hydrolysis is required at a step before the fusion of nuclear vesicles.