RESUMO
Endoplasmic reticulum (ER) membrane junctions are formed by the dynamin-like GTPase atlastin (ATL). Deletion of ATL results in long unbranched ER tubules in cells, and mutation of human ATL1 is linked to hereditary spastic paraplegia. Here, we demonstrate that COPII formation is drastically decreased in the periphery of ATL-deleted cells. ER export of cargo proteins becomes defective; ER exit site initiation is not affected, but many of the sites fail to recruit COPII subunits. The efficiency of cargo packaging into COPII vesicles is significantly reduced in cells lacking ATLs, or when the ER is transiently fragmented. Cargo is less mobile in the ER in the absence of ATL, but the cargo mobility and COPII formation can be restored by ATL R77A, which is capable of tethering, but not fusing, ER tubules. These findings suggest that the generation of ER junctions by ATL plays a critical role in maintaining the necessary mobility of ER contents to allow efficient packaging of cargo proteins into COPII vesicles.
Assuntos
Vesículas Revestidas pelo Complexo de Proteína do Envoltório/genética , Retículo Endoplasmático/genética , Proteínas de Ligação ao GTP/genética , Proteínas de Membrana/genética , Transporte Proteico/genética , Animais , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/metabolismo , Células COS , Chlorocebus aethiops , Retículo Endoplasmático/metabolismo , Complexo de Golgi/genética , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas Mutantes/genética , Deleção de Sequência/genética , Paraplegia Espástica Hereditária/genética , Paraplegia Espástica Hereditária/patologiaRESUMO
Carbon tetrachloride (CCl4 ) causes hepatotoxicity in mammals, with its hepatocytic metabolism producing radicals that attack the intracellular membrane system and destabilize intracellular vesicle transport. Inhibition of intracellular transport causes lipid droplet retention and abnormal protein distribution. The intracellular transport of synthesized lipids and proteins from the endoplasmic reticulum (ER) to the Golgi apparatus is performed by coat complex II (COPII) vesicle transport, but how CCl4 inhibits COPII vesicle transport has not been elucidated. COPII vesicle formation on the ER membrane is initiated by the recruitment of Sar1 protein from the cytoplasm to the ER membrane, followed by that of the COPII coat constituent proteins (Sec23, Sec24, Sec13, and Sec31). In this study, we evaluated the effect of CCl4 on COPII vesicle formation using the RLC-16 rat hepatocyte cell line. Our results showed that CCl4 suppressed ER-Golgi transport in RLC-16 cells. Using a reconstituted system of rat liver tissue-derived cytoplasm and RLC-16 cell-derived ER membranes, CCl4 treatment inhibited the recruitment of Sar1 and Sec13 from the cytosolic fraction to ER membranes. CCl4 -induced changes in the ER membrane accordingly inhibited the accumulation of COPII vesicle-coated constituent proteins on the ER membrane, as well as the formation of COPII vesicles, which suppressed lipid and protein transport between the ER and Golgi apparatus. Our data suggest that CCl4 inhibits ER-Golgi intracellular transport by inhibiting COPII vesicle formation on the ER membrane in hepatocytes.