Deretic D, Puleo-Scheppke B, Trippe C
Department of Pathology, University of Texas Health Sciences Center at San Antonio 78284-7750, USA.
J Biol Chem. 1996 Jan 26;271(4):2279-86. doi: 10.1074/jbc.271.4.2279.
In retinal photoreceptors, highly polarized organization of the light-sensitive organelle, the rod outer segment, is maintained by the sorting of rhodopsin and its associated proteins into distinct post-Golgi vesicles that bud from the trans-Golgi network (TGN) and by their vectorial transport toward the rod outer segment. We have developed an assay that reconstitutes the formation of these vesicles in a retinal cell-free system. Vesicle formation in this cell-free assay is ATP-, GTP-, and cytosol-dependent. In frog retinas vesicle budding also proceeds at 0 degrees C, both in vivo and in vitro. Vesicles formed in vitro are indistinguishable from the vesicles formed in vivo by their buoyant density, protein composition, topology, and morphology. In addition to the previously identified G-proteins, these vesicles also contain rab11. Concurrently with vesicle budding, resident proteins are retained in the TGN. Collectively these data suggest that rhodopsin and its associated proteins are sorted upon exit from the TGN in this cell-free system. Removal of membrane-bound GTP-binding proteins of the rab family by rab GDP dissociation inhibitor completely abolishes formation of these vesicles and results in the retention of rhodopsin in the Golgi. A monoclonal antibody to the cytoplasmic (carboxy-terminal) domain of rhodopsin and its Fab fragments strongly inhibit vesicle formation and arrest newly synthesized rhodopsin in the TGN rather than the Golgi. Therefore rhodopsin sorting at the exit from the TGN is mediated by the interaction of its cytoplasmic domain with the intracellular sorting machinery.
在视网膜光感受器中，光敏感细胞器视杆外段的高度极化组织，是通过将视紫红质及其相关蛋白分选到从反式高尔基体网络（TGN）出芽的不同高尔基体后囊泡中，并通过它们向视杆外段的定向运输来维持的。我们开发了一种检测方法，可在无细胞视网膜系统中重建这些囊泡的形成。这种无细胞检测中的囊泡形成依赖于ATP、GTP和胞质溶胶。在青蛙视网膜中，无论是在体内还是体外，囊泡出芽在0摄氏度时也会进行。体外形成的囊泡与体内形成的囊泡在浮力密度、蛋白质组成、拓扑结构和形态上无法区分。除了先前鉴定出的G蛋白外，这些囊泡还含有rab11。与囊泡出芽同时，驻留蛋白保留在TGN中。这些数据共同表明，在这个无细胞系统中，视紫红质及其相关蛋白在从TGN出来时就被分选了。通过rab GDP解离抑制剂去除rab家族的膜结合GTP结合蛋白，会完全消除这些囊泡的形成，并导致视紫红质保留在高尔基体中。一种针对视紫红质细胞质（羧基末端）结构域的单克隆抗体及其Fab片段强烈抑制囊泡形成，并使新合成的视紫红质停滞在TGN而非高尔基体中。因此，视紫红质在TGN出口处的分选是由其细胞质结构域与细胞内分选机制的相互作用介导的。
