Abstract
Ubiquitination of red blood cell (RBC) proteins was investigated by encapsulation of 125I-ubiquitin into human erythrocytes using a procedure of hypotonic dialysis, isotonic resealing, and reannealing. Incubation (37 degrees C, up to 2 h) of 125I-ubiquitin-loaded cells resulted in the recovery of 125I-ubiquitin with the cytosolic proteins (9.22 +/- 0.4 micrograms/ml RBC) and conjugated to membrane proteins (2.18 +/- 0.05 micrograms/ml RBC). This conjugation was time-dependent, and the predominant membrane protein band that became labeled showed an apparent molecular mass of 240 kDa on SDS-polyacrylamide gel electrophoresis (PAGE). Western blotting experiments with three different anti-ubiquitin antibodies revealed that this protein is also ubiquitinated in vivo. Cell-free experiments have shown that fraction II (a DEAE-bound protein fraction eluted by 0.5 M KCl) prepared from both mature erythrocytes and reticulocytes is able to conjugate ubiquitin to this protein. Ubiquitin conjugation was ATP-dependent (Km 0.09 mM), time-dependent, and fraction II-dependent (8 +/- 0.5 pmol of 125I-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 affinity chromatography on immobilized avidin. The major components retained by the column were two peptides of molecular masses 220 and 240 kDa. Both proteins are recognized by a monoclonal anti-spectrin antibody, but only the 240-kDa component is detected by streptavidin peroxidase conjugate. That indeed the ubiquitinated membrane protein of 240-kDa is alpha-spectrin was confirmed by immunoaffinity chromatography using 125I-ubiquitin and a monoclonal anti-spectrin antibody. Antigen-antibody complexes were purified by protein A chromatography and analyzed by SDS-PAGE and autoradiography. Again two bands of 240 and 220 kDa were eluted (alpha- and beta-spectrin), but only one band corresponding to the electrophoretic mobility of alpha-spectrin was detected by autoradiography. Thus, alpha-spectrin is a substrate for the ATP-dependent ubiquitination system, suggesting that the cytoskeleton is covalently modified by ubiquitination both in reticulocytes and mature RBC.
Highlights
Cell-free experiments have shown that fraction II prepared from both mature erythrocytes and reticulocytes is able to conjugate ubiquitin to this protein
The first dialysis step was performed using a dialysis tube with a 12-k.Da cutoff that allowed the removal of unbound ubiquitin
Most of ubiquitin is conjugated to endogenous proteins
Summary
Ubiquitin; PMSF, phenylmethylhighly conserved, and found both free and covalently conjugated to other proteins in all eukaryotic cells [1]. Mature erythrocytes have virtually no Ub- and ATP-dependent protein degradation, but substantial levels of Db-protein conjugates sulfonyl fluoride; RBC, red blood cell; PAGE, polyacrylamide gel electrophoresis. Mature erythrocytes represent an ideal system for investigating protein ubiquitination when this process is not followed by protein degradation This fact, and the availability of a procedure for the encapsulation of labeled ubiquitin without affecting the main cellular functions, allowed us to investigate ubiquitin conjugation in intact mature erythrocytes providing clear evidence for the conjugation of Ub to a previously unidentified substrate, namely the cytoskeletal protein a-spectrin. The functional role of this process is not yet clear; spectrin represents the major ubiquitinatable substrate in mature red cells
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