ADP-ribosylation factor-1 is sensitive to N-ethylmaleimide.
The treatment of normal rat kidney cells with N-ethylmaleimide caused the release of beta-COP, a component of coatomer, from the Golgi apparatus without causing disassembly of the organelle. The release of beta-COP, which was not due to depolymerization of microtubules, was markedly blocked by the activation of GTP-binding proteins by aluminum fluoride or a nonhydrolyzable analogue of GTP. To determine which component is N-ethylmaleimide-sensitive, we reconstituted the recruitment of coatomer from the bovine brain cytosol onto the Golgi apparatus in digitonin-permeabilized cells. In cells treated with N-ethylmaleimide before permeabilization, beta-COP was still recruited onto the Golgi apparatus. In contrast, beta-COP was not recruited when N-ethylmaleimide-treated bovine brain cytosol was used. These results suggest that the N-ethylmaleimide-sensitive factor(s) are present in the cytosol. It is known that coatomer and ADP-ribosylation factor-1 (ARF1) are the only cytoplasmic proteins needed for the assembly of Golgi-derived coated vesicles. N-Ethylmaleimide treatment of a coatomer-rich fraction did not affect the binding of beta-COP to the Golgi apparatus, whereas the same treatment 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 contains only one cysteine residue (Cys-159), which is located near the base moiety of the bound guanine nucleotide.
Yamaguchi
T
T
School of Life Science, Tokyo University of Pharmacy and Life Science, Horinouchi, Hachioji, Tokyo, 192-0392, Japan.
Nakayama
K
K
Hatsuzawa
K
K
Tani
K
K
Himeno
M
M
Tagaya
M
M
D020823:ADP-Ribosylation Factor 1; D020727:ADP-Ribosylation Factors; D033982:Adaptor Protein Complex gamma Subunits; D000818:Animals; D002417:Cattle; D002478:Cells, Cultured; D020756:Coatomer Protein; D003545:Cysteine; D003600:Cytosol / Q000737:chemistry; D005033:Ethylmaleimide / Q000737:chemistry / Q000494:pharmacology*; D019204:GTP-Binding Proteins / Q000737:chemistry / Q000187:drug effects* / Q000378:metabolism; D006056:Golgi Apparatus / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D007425:Intracellular Membranes / Q000378:metabolism; D007668:Kidney / Q000166:cytology; D008565:Membrane Proteins / Q000187:drug effects / Q000378:metabolism; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D017239:Paclitaxel / Q000494:pharmacology; D051381:Rats
D033982:Adaptor Protein Complex gamma Subunits; D020756:Coatomer Protein; D008565:Membrane Proteins; D008869:Microtubule-Associated Proteins; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D019204:GTP-Binding Proteins; D020823:ADP-Ribosylation Factor 1; D020727:ADP-Ribosylation Factors; D003545:Cysteine; D005033:Ethylmaleimide; D017239:Paclitaxel