INTERACTION OF COATOMER WITH AMINOGLYCOSIDE ANTIBIOTICS - EVIDENCE THAT COATOMER HAS AT LEAST 2 DILYSINE BINDING-SITES

Coatomer is the soluble precursor of the COPI coat (coat protein I) in volved in traffic among membranes of the endoplasmic reticulum and the Golgi apparatus. We report herein that neomycin precipitates coatomer from cell extracts and from purified coatomer preparations. Precipita tion first increased and then decreased as the neomycin concentration increased, analogous to the precipitation of a polyvalent antigen by d ivalent antibodies. This suggested that neomycin cross-linked coatomer into large aggregates and implies that coatomer has two or more bindi ng sites for neomycin. A variety of other aminoglycoside antibiotics p recipitated coatomer, or if they did not precipitate, they interfered with the ability of neomycin to precipitate. Coatomer is known to inte ract with a motif (KKXX) containing adjacent lysine residues at the ca rboxyl terminus of the cytoplasmic domains of some membrane proteins r esident in the endoplasmic reticulum. All of the antibiotics that inte racted with coatomer contain at least two close amino groups, suggesti ng that the antibiotics might be interacting with the di-lysine bindin g site of coatomer. Consistent with this idea, di-lysine itself blocke d the interaction of antibiotics with coatomer. Moreover, di-lysine an d antibiotics each blocked the coating of Golgi membranes by coatomer. These data suggest that certain aminoglycoside antibiotics interact w ith di-lysine binding sites on coatomer and that coatomer contains at least two of these di-lysine binding sites.