ADP-ribosylation factor-1 is sensitive to N-Ethylmaleimide

The treatment of normal rat kidney cells with N-ethylmaleimide caused the r elease of beta-COP, a component of coatomer, from the Golgi apparatus witho ut causing disassembly of the organelle, The release of beta-COP, which was not due to depolymerization of microtubules, was markedly blocked by the a ctivation of GTP-binding proteins by aluminum fluoride or a nonhydrolyzable analogue of GTP, To determine which component is N-ethylmaleimide-sensitiv e, we reconstituted the recruitment of coatomer from the bovine brain cytos ol onto the Golgi apparatus in digitonin-permeabilized cells. In cells trea ted with N-ethylmaleimide before permeabilization, beta-COP was still recru ited onto the Golgi apparatus. In contrast, beta-COP was not recruited when N-ethylmaleimide-treated bovine brain cytosol was used, These results sugg est that the N-ethylmaleimide-sensitive factor(s) are present in the cytoso l, It is known that coatomer and ADP-ribosylation factor-1 (ARF1) are the o nly cytoplasmic proteins needed for the assembly of Golgi-derived coated ve sicles, N-Ethylmaleimide treatment of a coatomer-rich fraction did not affe ct the binding of beta-COP to the Golgi apparatus, whereas the same treatme nt of an ARF-rich fraction abolished beta-COP binding. Similar results were obtained using purified recombinant ARF1, Concomitant with inactivation, 0 .85 mol of N-ethylmaleimide was incorporated into 1 mol of ARF1, ARF1 conta ins only one cysteine residue (Cys-159), which is located near the base moi ety of the bound guanine nucleotide.