UBIQUITIN IS CONJUGATED TO THE CYTOSKELETAL PROTEIN ALPHA-SPECTRIN INMATURE ERYTHROCYTES

Ubiquitination of red blood cell (RBC) proteins was investigated by en capsulation of I-125-ubiquitin into human erythrocytes using a procedu re of hypotonic dialysis, isotonic resealing, and reannealing. Incubat ion (37 degrees C, up to 2 h) of I-125-ubiquitin-loaded cells resulted in the recovery of I-125-ubiquitin with the cytosolic proteins (9.22 +/- 0.4 mu g/ml RBC) and conjugated to membrane proteins (2.18 +/- 0.0 5 mu g/ml RBC). This conjugation was time-dependent, and the predomina nt membrane protein band that became labeled showed an apparent molecu lar mass of 240 kDa on SDS-polyacrylamide gel electrophoresis (PAGE). Western blotting experiments with three different anti-ubiquitin antib odies revealed that this protein is also ubiquitinated in vivo. Cell-f ree experiments have shown that fraction II (a DEAE-bound protein frac tion eluted by 0.5 M KCl) prepared from both mature erythrocytes and r eticulocytes is able to conjugate ubiquitin to this protein. Ubiquitin conjugation was ATP-dependent (K-m 0.09 mM), time-dependent, and frac tion II-dependent (8 +/- 0.5 pmol of I-125-ubiquitin/h/mg of fraction II). Isolation of the major RBC membrane protein that is ubiquitinated was obtained by using biotinylated ubiquitin. Membrane proteins, once ubiquitinated with this derivative, were extracted and purified by af finity chromatography on immobilized avidin. The major components reta ined by the column were two peptides of molecular masses 220 and 240 k Da. Both proteins are recognized by a monoclonal anti-spectrin antibod y, but only the 240-kDa component is detected by streptavidin peroxida se conjugate. That indeed the ubiquitinated membrane protein of 240-kD a is alpha-spectrin was confirmed by immunoaffinity chromatography usi ng I-125-ubiquitin and a monoclonal anti-spectrin antibody. Antigen-an tibody complexes were purified by protein A chromatography and analyze d by SDS-PAGE and autoradiography. Again two bands of 240 and 220 kDa were eluted (alpha- and beta-spectrin), but only one band correspondin g to the electrophoretic mobility of alpha-spectrin was detected by au toradiography. Thus, alpha-spectrin is a substrate for the ATP-depende nt ubiquitination system, suggesting that the cytoskeleton is convalen tly modified by ubiquitination both in reticulocytes and mature RBC.