title: Identification of the main ubiquitination site in human erythroid alpha-spectrin.
abstract: Erythroid spectrin is the main component of the red cell membrane skeleton, which is very important in determining the shape, resistance to mechanical stresses and deformability of red cells. Previously we demonstrated that human erythroid alpha-spectrin is ubiquitinated in vitro and in vivo, and using recombinant peptides we identified on repeat 17 the main ubiquitination site of alpha-spectrin. In order to identify the lysine(s) involved in the ubiquitination process, in the present study we mutated the lysines by site-directed mutagenesis. We found that ubiquitination was dramatically inhibited in peptides carrying the mutation of lysine 27 on repeat 17 (mutants K25,27R and K27R). We also demonstrated that the correct folding of this protein is fundamental for its recognition by the ubiquitin conjugating system. Furthermore, the region flanking lysine 27 showed a 75% similarity with the leucine zipper pattern present in many regulatory proteins. Thus, a new potential ubiquitin recognition motif was identified in alpha-spectrin and may be present in several other proteins.
authors.affiliation: Institute of Biological Chemistry G. Fornaini, University of Urbino, Italy.
authors.forename: L
authors.initials: L
authors.lastname: Galluzzi
authors.forename: M
authors.initials: M
authors.lastname: Paiardini
authors.forename: M C
authors.initials: MC
authors.lastname: Lecomte
authors.forename: M
authors.initials: M
authors.lastname: Magnani
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites / Q000235:genetics; D004912:Erythrocytes / Q000737:chemistry; D015870:Gene Expression; D006801:Humans; D008239:Lysine / Q000235:genetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020449:Repetitive Sequences, Amino Acid; D016415:Sequence Alignment; D020539:Sequence Analysis, Protein; D017386:Sequence Homology, Amino Acid; D013049:Spectrin / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D014452:Ubiquitins / Q000378:metabolism*
chemical_list: D011993:Recombinant Fusion Proteins; D014452:Ubiquitins; D013049:Spectrin; D008239:Lysine