Overexpression of the ARF1 exchange factor ARNO inhibits the early secretory pathway and causes the disassembly of the Golgi complex

The small GTPase ARF1 is a key regulator of intracellular membrane traffic. In its active, GTP-bound form, ARF1 is associated with Golgi membranes and promotes the recruitment of the cytosolic coat protein complex, which will result in membrane budding and vesicle formation. ARNO (ARF nucleotide sit e opener) has been shown to act in vitro as a GTP exchange factor for ARF1, Here, we have investigated the function of ARNO in vivo. By immunofluoresc ence and cell fractionation, ARNO was found to be mostly cytosolic in HeLa cells. Its overexpression led to a strong inhibition of the secretion of SE AP (secreted form of alkaline phosphatase). Newly synthesized SEAP failed t o acquire endoglycosidase H resistance, indicating a block in the early sec retory pathway. This effect on secretion was accompanied by a disassembly o f the Golgi complex and a redistribution of Golgi resident proteins into th e endoplasmic reticulum (ER), On the other hand, ARNO overexpression did no t affect the early endocytic pathway. These results show that ARNO function s in vivo in Golgi to ER transport. Its behavior is then consistent with AR NO being an exchange factor for ARF1.