Yu W H, Wolfgang W, Forte M
Vollum Institute for Advanced Biomedical Research, Portland, Oregon, USA.
J Biol Chem. 1995 Jun 9;270(23):13998-4006. doi: 10.1074/jbc.270.23.13998.
The voltage-dependent anion channel of the outer mitochondrial membrane, VDAC (also known as mitochondrial porin), is a small abundant protein which forms a voltage-gated pore when incorporated into planar lipid bilayers. This protein forms the primary pathway for movement of major metabolites through the outer membrane. Recently, it has been demonstrated that two human VDAC genes, HVDAC1 and HVDAC2, produce three proteins that differ most significantly at their amino termini. These results suggest that the distinct amino termini lead to the targeting of individual VDAC isoforms to different cellular compartments. Consistent with this hypothesis, recent reports suggest that HIV-DAC1 is found in the plasma membrane of mammalian cells. To define the subcellular location of HVDAC isoforms, HVDAC genes were modified so that the encoded proteins contain COOH-terminal epitopes recognized by either of two monoclonal antibodies. Introduction of these epitope tags had no effect on the function of modified VDAC proteins. Epitope-tagged proteins were then individually expressed in COS7 cells or rat astrocytes and the intracellular location of each isoform subsequently identified by subcellular fractionation, light level immunofluorescence, and immunoelectron microscopy. Our results demonstrate that each HVDAC protein is exclusively located in fractions or subcellular regions containing mitochondrial marker proteins. In addition, immunofluorescence and immunoelectron microscopy show that an individual mitochondrion can contain both HVDAC1 and HVDAC2. Our results call into question previous reports demonstrating VDAC molecules in the plasma membrane and suggest that functional differences between individual VDAC isoforms may result in distinct regulatory processes within a single mitochondrion.
线粒体外膜的电压依赖性阴离子通道，即VDAC（也称为线粒体孔蛋白），是一种含量丰富的小蛋白，当整合到平面脂质双分子层中时会形成电压门控孔。该蛋白构成了主要代谢物通过外膜移动的主要途径。最近，已证明两个人类VDAC基因，HVDAC1和HVDAC2，产生三种蛋白质，它们在氨基末端差异最为显著。这些结果表明，不同的氨基末端导致各个VDAC异构体靶向不同的细胞区室。与这一假设一致，最近的报道表明HIV-DAC1存在于哺乳动物细胞的质膜中。为了确定HVDAC异构体的亚细胞定位，对HVDAC基因进行了修饰，使得编码的蛋白质含有可被两种单克隆抗体之一识别的羧基末端表位。这些表位标签的引入对修饰后的VDAC蛋白的功能没有影响。然后将带有表位标签的蛋白质分别在COS7细胞或大鼠星形胶质细胞中表达，随后通过亚细胞分级分离、光镜免疫荧光和免疫电子显微镜确定每种异构体的细胞内定位。我们的结果表明，每种HVDAC蛋白仅位于含有线粒体标记蛋白的级分或亚细胞区域中。此外，免疫荧光和免疫电子显微镜显示，单个线粒体可以同时含有HVDAC1和HVDAC2。我们的结果对先前关于质膜中存在VDAC分子的报道提出了质疑，并表明各个VDAC异构体之间的功能差异可能导致单个线粒体内不同的调节过程。
