Jacotot E, Ferri K F, El Hamel C, Brenner C, Druillennec S, Hoebeke J, Rustin P, Métivier D, Lenoir C, Geuskens M, Vieira H L, Loeffler M, Belzacq A S, Briand J P, Zamzami N, Edelman L, Xie Z H, Reed J C, Roques B P, Kroemer G
Centre National de la Recherche Scientifique, UMR 1599, Institut Gustave Roussy, F-94805 Villejuif, France.
J Exp Med. 2001 Feb 19;193(4):509-19. doi: 10.1084/jem.193.4.509.
Viral protein R (Vpr), an apoptogenic accessory protein encoded by HIV-1, induces mitochondrial membrane permeabilization (MMP) via a specific interaction with the permeability transition pore complex, which comprises the voltage-dependent anion channel (VDAC) in the outer membrane (OM) and the adenine nucleotide translocator (ANT) in the inner membrane. Here, we demonstrate that a synthetic Vpr-derived peptide (Vpr52-96) specifically binds to the intermembrane face of the ANT with an affinity in the nanomolar range. Taking advantage of this specific interaction, we determined the role of ANT in the control of MMP. In planar lipid bilayers, Vpr52-96 and purified ANT cooperatively form large conductance channels. This cooperative channel formation relies on a direct protein-protein interaction since it is abolished by the addition of a peptide corresponding to the Vpr binding site of ANT. When added to isolated mitochondria, Vpr52-96 uncouples the respiratory chain and induces a rapid inner MMP to protons and NADH. This inner MMP precedes outer MMP to cytochrome c. Vpr52-96-induced matrix swelling and inner MMP both are prevented by preincubation of purified mitochondria with recombinant Bcl-2 protein. In contrast to König's polyanion (PA10), a specific inhibitor of the VDAC, Bcl-2 fails to prevent Vpr52-96 from crossing the mitochondrial OM. Rather, Bcl-2 reduces the ANT-Vpr interaction, as determined by affinity purification and plasmon resonance studies. Concomitantly, Bcl-2 suppresses channel formation by the ANT-Vpr complex in synthetic membranes. In conclusion, both Vpr and Bcl-2 modulate MMP through a direct interaction with ANT.
病毒蛋白R（Vpr）是一种由HIV-1编码的具有凋亡作用的辅助蛋白，它通过与通透性转换孔复合物的特异性相互作用诱导线粒体膜通透性改变（MMP），该复合物由外膜（OM）中的电压依赖性阴离子通道（VDAC）和内膜中的腺嘌呤核苷酸转位酶（ANT）组成。在此，我们证明一种合成的Vpr衍生肽（Vpr52-96）以纳摩尔范围内的亲和力特异性结合到ANT的膜间隙面。利用这种特异性相互作用，我们确定了ANT在MMP控制中的作用。在平面脂质双分子层中，Vpr52-96和纯化的ANT协同形成大电导通道。这种协同通道形成依赖于直接的蛋白质-蛋白质相互作用，因为添加对应于ANT的Vpr结合位点的肽可消除该作用。当添加到分离的线粒体中时，Vpr52-96使呼吸链解偶联，并诱导质子和NADH快速发生内膜MMP。这种内膜MMP先于细胞色素c的外膜MMP出现。Vpr52-96诱导的基质肿胀和内膜MMP都可通过用重组Bcl-2蛋白预孵育纯化的线粒体来预防。与VDAC的特异性抑制剂König多阴离子（PA10）不同，Bcl-2不能阻止Vpr52-96穿过线粒体OM。相反，如亲和纯化和表面等离子体共振研究所确定的，Bcl-2降低了ANT-Vpr相互作用。同时，Bcl-2抑制合成膜中ANT-Vpr复合物形成通道。总之，Vpr和Bcl-2都通过与ANT的直接相互作用调节MMP。
