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 site 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 immunofluorescence and cell fractionation, ARNO was found to be mostly cytosolic in HeLa cells. Its overexpression led to a strong inhibition of the secretion of SEAP (secreted form of alkaline phosphatase). Newly synthesized SEAP failed to acquire endoglycosidase H resistance, indicating a block in the early secretory pathway. This effect on secretion was accompanied by a disassembly of the Golgi complex and a redistribution of Golgi resident proteins into the endoplasmic reticulum (ER). On the other hand, ARNO overexpression did not affect the early endocytic pathway. These results show that ARNO functions in vivo in Golgi to ER transport. Its behavior is then consistent with ARNO being an exchange factor for ARF1.
Monier
S
S
Institut Curie, CNRS UMR 144, 75248 Paris Cedex 5, France. smonier@curie.fr
Chardin
P
P
Robineau
S
S
Goud
B
B
D020823:ADP-Ribosylation Factor 1; D020727:ADP-Ribosylation Factors; D001692:Biological Transport / Q000187:drug effects / Q000502:physiology; D020126:Brefeldin A / Q000494:pharmacology; D003594:Cytoplasmic Granules / Q000737:chemistry / Q000201:enzymology*; D003600:Cytosol / Q000737:chemistry / Q000201:enzymology; D004705:Endocytosis / Q000502:physiology; D004721:Endoplasmic Reticulum / Q000737:chemistry / Q000201:enzymology; D019204:GTP-Binding Proteins / Q000032:analysis / Q000235:genetics*; D020690:GTPase-Activating Proteins; D015971:Gene Expression Regulation, Enzymologic; D006056:Golgi Apparatus / Q000737:chemistry / Q000201:enzymology*; D006367:HeLa Cells; D006801:Humans; D007369:Interferon Inducers / Q000494:pharmacology; D037102:Lectins / Q000494:pharmacology; D037121:Plant Lectins; D010957:Plasmids; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D013347:Subcellular Fractions / Q000737:chemistry / Q000201:enzymology; D014168:Transferrin / Q000493:pharmacokinetics
D020690:GTPase-Activating Proteins; D007369:Interferon Inducers; D037102:Lectins; D037121:Plant Lectins; D011500:Protein Synthesis Inhibitors; D014168:Transferrin; C102474:cytohesin-2; C043696:jacalin; D020126:Brefeldin A; D019204:GTP-Binding Proteins; D020823:ADP-Ribosylation Factor 1; D020727:ADP-Ribosylation Factors