Bar-Zvi D, Mosley S T, Branton D
Department of Cellular and Developmental Biology, Harvard University, Cambridge, Massachusetts 02138.
J Biol Chem. 1988 Mar 25;263(9):4408-15.
We have studied the in vivo phosphorylation of clathrin-coated vesicle proteins from rat reticulocytes. The major 32P-labeled polypeptides of clathrin-coated vesicles isolated from metabolically labeled cells were the the 165-, 100-110-, and 50-kDa polypeptides of the assembly protein, the clathrin beta-light chain, and to a lesser extent the clathrin alpha-light chain. The phosphorylation of the assembled (particulate) and unassembled (soluble) pools of clathrin and assembly protein was compared by immunoprecipitating the respective protein complexes from particulate and soluble cell fractions. Although all the phosphorylated polypeptides were present in both fractions, the extent of labeling was protein and fraction specific: the apparent specific activities of the assembly protein 50-kDa polypeptide and clathrin light chain were higher in the unassembled pool, whereas those of the 100-110-kDa polypeptides were higher in the assembled pool. The amino acids and polypeptide fragments labeled in vivo appeared similar to those labeled in vitro.
我们研究了来自大鼠网织红细胞的网格蛋白包被小泡蛋白的体内磷酸化情况。从经代谢标记的细胞中分离出的网格蛋白包被小泡的主要32P标记多肽是组装蛋白的165 kDa、100 - 110 kDa和50 kDa多肽、网格蛋白β轻链，以及程度较轻的网格蛋白α轻链。通过从颗粒状和可溶性细胞组分中免疫沉淀各自的蛋白质复合物，比较了网格蛋白和组装蛋白的组装（颗粒状）和未组装（可溶性）库的磷酸化情况。尽管所有磷酸化多肽都存在于两个组分中，但标记程度具有蛋白质和组分特异性：组装蛋白50 kDa多肽和网格蛋白轻链在未组装库中的表观比活性较高，而100 - 110 kDa多肽在组装库中的表观比活性较高。体内标记的氨基酸和多肽片段与体外标记的相似。
