Corsi D, Galluzzi L, Crinelli R, Magnani M
Institute of Biological Chemistry G. Fornaini, University of Urbino, Italy.
J Biol Chem. 1995 Apr 14;270(15):8928-35. doi: 10.1074/jbc.270.15.8928.
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.
通过低渗透析、等渗重封和再退火的方法将125I-泛素包裹到人红细胞中，对红细胞（RBC）蛋白的泛素化进行了研究。将负载125I-泛素的细胞在37℃孵育长达2小时，结果在胞质蛋白（9.22±0.4微克/毫升红细胞）中回收了125I-泛素，并与膜蛋白结合（2.18±0.05微克/毫升红细胞）。这种结合是时间依赖性的，在SDS-聚丙烯酰胺凝胶电泳（PAGE）上显示被标记的主要膜蛋白条带的表观分子量为240 kDa。用三种不同的抗泛素抗体进行的蛋白质印迹实验表明，该蛋白在体内也被泛素化。无细胞实验表明，从成熟红细胞和网织红细胞中制备的组分II（用0.5 M KCl洗脱的DEAE结合蛋白组分）能够将泛素与该蛋白结合。泛素结合是ATP依赖性的（Km为0.09 mM）、时间依赖性的且依赖于组分II（8±0.5皮摩尔125I-泛素/小时/毫克组分II）。通过使用生物素化泛素分离出被泛素化的主要红细胞膜蛋白。一旦用这种衍生物进行泛素化，膜蛋白就通过固定化抗生物素蛋白的亲和色谱法进行提取和纯化。柱上保留的主要成分是分子量为220和240 kDa的两种肽。两种蛋白都能被抗血影蛋白单克隆抗体识别，但只有240 kDa的组分能被抗生物素蛋白过氧化物酶偶联物检测到。使用125I-泛素和抗血影蛋白单克隆抗体通过免疫亲和色谱法证实，240 kDa的泛素化膜蛋白确实是α-血影蛋白。抗原-抗体复合物通过蛋白A色谱法纯化，并通过SDS-PAGE和放射自显影进行分析。再次洗脱了240和220 kDa的两条带（α-和β-血影蛋白），但通过放射自显影仅检测到一条对应于α-血影蛋白电泳迁移率的带。因此，α-血影蛋白是ATP依赖性泛素化系统的底物，这表明在网织红细胞和成熟红细胞中，细胞骨架都通过泛素化进行共价修饰。
