GTP-binding proteins in rat liver nuclear envelopes.
GTP-binding proteins in rat liver nuclear envelopes.
复制标题
大鼠肝核膜中的 GTP 结合蛋白。
DOI：
10.1073/pnas.87.18.7080
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发表时间：
1990
影响因子：
11.1
通讯作者：
Riedel,N
中科院分区：
文献类型：
--
作者：
Rubins,JB;Benditt,JO;Dickey,BF;Riedel,N
核运输以及有丝分裂完成后核膜（NE）的重新组装是已被证明需要GTP和ATP的过程。为了研究的存在和定位的GTP结合蛋白在NE中，我们结合了互补技术的[α-32 P]GTP结合蛋白质印迹和UV交联的[α-32 P]GTP与完善的程序NE亚分级。GTP结合到印迹NE蛋白揭示了五个低分子量的GTP结合蛋白26，25，24.5，24，和23 kDa，和[α-32 P]GTP光亲和标记揭示了主要蛋白质的表观分子量为140，53，47，33，和31 kDa。所有GTP结合蛋白似乎优先定位于内核膜，可能内核膜和纤层之间的界面。尽管NE和粗面内质网之间的进化保守，GTP结合蛋白确定这两个车厢之间的不同。最值得注意的是，68-和30-kDa的GTP-结合亚基的信号识别颗粒受体，其中光标记与[α-32 P]GTP在粗面内质网部分，被完全排除在NE馏分。相反，一个主要的53-kDa的光标记蛋白质在NE是不存在的粗面内质网。而蛋白质印迹NE蛋白结合GTP特异性，所有的[α-32 P]GTP光标记的蛋白质可以通过与ATP的竞争而被阻断，尽管与GTP获得的竞争概况不同。在比较交联研究与[α-32 P]ATP，我们已经确定了三个特定的ATP结合蛋白的分子量为160，78和74 kDa。GTP-和ATP-结合蛋白的NE内的本地化出现适当的参与核运输和在有丝分裂后的细胞核的重新组装所需的核膜囊泡的GTP依赖性融合。
Nuclear transport as well as reassembly of the nuclear envelope (NE) after completion of mitosis are processes that have been shown to require GTP and ATP. To study the presence and localization of GTP-binding proteins in the NE, we have combined complementary techniques of [alpha-32P]GTP binding to Western-blotted proteins and UV crosslinking of [alpha-32P]GTP with well-established procedures for NE subfractionation. GTP binding to blotted NE proteins revealed five low molecular mass GTP-binding proteins of 26, 25, 24.5, 24, and 23 kDa, and [alpha-32P]GTP photoaffinity labeling revealed major proteins with apparent molecular masses of 140, 53, 47, 33, and 31 kDa. All GTP-binding proteins appear to localize preferentially to the inner nuclear membrane, possibly to the interface between inner nuclear membrane and lamina. Despite the evolutionary conservation between the NE and the rough endoplasmic reticulum, the GTP-binding proteins identified differed between these two compartments. Most notably, the 68- and 30-kDa GTP-binding subunits of the signal recognition particle receptor, which photolabeled with [alpha-32P]GTP in the rough endoplasmic reticulum fraction, were totally excluded from the NE fraction. Conversely, a major 53-kDa photolabeled protein in the NE was absent from rough endoplasmic reticulum. Whereas Western-blotted NE proteins bound GTP specifically, all [alpha-32P]GTP photolabeled proteins could be blocked by competition with ATP, although with a competition profile that differed from that obtained with GTP. In comparative crosslinking studies with [alpha-32P]ATP, we have identified three specific ATP-binding proteins with molecular masses of 160, 78, and 74 kDa. The localization of GTP- and ATP-binding proteins within the NE appears appropriate for their involvement in nuclear transport and in the GTP-dependent fusion of nuclear membrane vesicles required for reassembly of the nucleus after mitosis.