Investigation of the subcellular distribution of the bcl-2 oncoprotein: residence in the nuclear envelope, endoplasmic reticulum, and outer mitochondrial membranes.
Investigation of the subcellular distribution of the bcl-2 oncoprotein: residence in the nuclear envelope, endoplasmic reticulum, and outer mitochondrial membranes.
复制标题
DOI：
--
复制
发表时间：
1993-10
期刊：
影响因子：
11.2
通讯作者：
S. Krajewski;S. Tanaka;S. Takayama;M. J. Schibler;W. Fenton;John Calvin Reed
中科院分区：
文献类型：
--
作者：
S. Krajewski;S. Tanaka;S. Takayama;M. J. Schibler;W. Fenton;John Calvin Reed
一个多学科的方法被用来调查由bcl-2基因编码的26 kDa的整合膜蛋白的细胞内位置。含t（14;18）的淋巴瘤细胞系的亚细胞分级分析显示Bcl-2蛋白存在于细胞核、重膜和轻膜组分中，但不存在于胞浆中。在Nycodenz和Percoll连续梯度的重膜组分的沉淀证明了p26-Bcl-2与线粒体但不是其他细胞器相关蛋白的共迁移。分馏轻膜馏分使用不连续的蔗糖梯度显示协会的Bcl-2蛋白主要与轻密度微粒体（滑面内质网），而不是重密度微粒体（粗面内质网）。免疫显微镜研究，采用激光扫描显微镜，前和postebedding电子显微镜的方法，和6种不同的抗Bcl-2抗体表现出Bcl-2免疫反应性的核被膜和外线粒体膜中的斑片状分布。此外，抗Bcl-2抗体的免疫反应性一般出现直接覆盖在高放大率的电子显微镜研究的核包膜，让人想起核孔复合物。此外，在体外翻译的p26-Bcl-2分离的易位能力的线粒体显示跨膜结构域依赖的Bcl-2蛋白与线粒体的关联，但没有提供证据进口到蛋白酶抗性隔室，符合免疫显微镜定位到线粒体外膜。两者合计，研究结果表明，p26-Bcl-2主要驻留在核膜，内质网，和外线粒体膜的不均匀分布，暗示参与蛋白质复合物可能参与运输的某些方面。
A multidisciplinary approach was taken to investigate the intracellular locations of the 26-kDa integral membrane protein encoded by the bcl-2 gene. Subcellular fractionation analysis of a t(14;18)-containing lymphoma cell line revealed the presence of Bcl-2 protein in nuclear, heavy-membrane, and light-membrane fractions but not in cytosol. Sedimentation of heavy-membrane fractions in Nycodenz and Percoll continuous gradients demonstrated comigration of p26-Bcl-2 with mitochondrial but not other organelle-associated proteins. Fractionation of light-membrane fractions using discontinuous sucrose-gradients revealed association of Bcl-2 protein primarily with lighter-density microsomes (smooth endoplasmic reticulum) as opposed to heavy-density microsomes (rough endoplasmic reticulum). Immune microscopy studies using laser-scanning microscopy, pre- and postembedding electron microscopic methods, and six different anti-Bcl-2 antibodies demonstrated Bcl-2 immunoreactivity in the nuclear envelope and outer mitochondrial membrane in a patchy distribution. Furthermore, anti-Bcl-2 antibody immunoreactivity generally appeared to directly overlie the nuclear envelope in high magnification electron microscopic studies, reminiscent of nuclear pore complexes. Addition of in vitro translated p26-Bcl-2 to isolated translocation-competent mitochondria revealed transmembrane domain-dependent association of Bcl-2 protein with mitochondria but provided no evidence for import into a protease-resistant compartment, consistent with immunomicroscopic localization to the outer mitochondrial membrane. Taken together, the findings demonstrate that p26-Bcl-2 resides primarily in the nuclear envelope, endoplasmic reticulum, and outer mitochondrial membrane in a nonuniform distribution suggestive of participation in protein complexes perhaps involved in some aspect of transport.