Identification of ubiquitinated repeats in human erythroid α-spectrin

The spectrin role(s) is (are) very important for the shape and the physical properties of red cells, such as deformability and resistance to mechanical stresses. Moreover a variety of spectrin diseases are known. We have previously demonstrated [Corsi, D., Galluzzi, L., Crinelli, R. & Magnani, M. (1995) J. Biol. Chem. 270, 8928-8935] that human erythroid alpha-spectrin is ubiquitinated in vitro and in vivo. In order to define the ubiquitinated repeats of this long protein and find out a possible function, we have produced recombinant peptides encompassing the alpha III-, alpha IV-, alpha V- and EF hand domains of alpha-spectrin chain. These peptides were tested in in vitro ubiquitin conjugation assays and two regions susceptibles to ubiquitination were found. The first one, in the alpha IV-domain, includes the repeat 17 and the second one, in the alpha V-domain, includes the repeat 20 and a part of repeat 21. We also demonstrated that the susceptibility to ubiquitination of the alpha V-domain is reduced by interaction with the corresponding portion of beta-spectrin chain (beta IV-domain). Thus, at least ubiquitination of alpha V-domain is susceptible to cytoskeleton assembly and spectrin dimerization.