IDENTIFICATION OF ALPHA-SPECTRIN DOMAINS SUSCEPTIBLE TO UBIQUITINATION

Previously, we demonstrated that alpha-spectrin is a substrate for the ubiquitin system and that this conjugation is a dynamic process (Cors i, D., Galluzzi, L., Crinelli, R., and Magnani, M. (1995) J. Biol. Che m. 270, 8928-8935). In this study, we mapped the sites of ubiquitinati on on erythrocyte alpha-spectrin. A peptide map of digested alpha-spec trin, previously submitted to in vitro I-125-ubiquitin conjugation, re vealed the presence of four distinct labeled bands with M(r) 40,000, 3 6,000, 29,000, and 25,500. Western blotting experiments using antibodi es against each alpha-spectrin domain revealed that only IgG anti-alph a III domain recognized the I-125-labeled ubiquitin peptide of 29 kDa, whereas the IgG anti-alpha V domain recognized the M(r) 40,000 I-125- ubiquitin-labeled peptide. The other two labeled bands of M(r) 36,000 and M(r) 25,500 were identified as tetra and tri multiubiquitin chains . Ubiquitination of the alpha III and alpha V domains was further conf irmed by anti-alpha-spectrin domain immunoaffinity chromatography. End oprotease Lys C-digested spectrin conjugated previously to I-125-ubiqu itin was incubated with antibodies against each trypsin-resistant doma in of alpha-spectrin. Gamma counting of the radiolabeled antigen-antib ody complexes purified by protein A chromatography showed labeling in the IgG anti-alpha III and anti-alpha V complexes alone. Domain alpha III is not associated with any known function, whereas domain alpha V contains the nucleation site for the association of the alpha and beta chains. Ubiquitination of the latter domain suggests a role for ubiqu itin in the modulation of the stability, deformability, and viscoelast ic properties of the erythrocyte membrane.