Moon R T, Lazarides E
J Cell Biol. 1984 May;98(5):1899-904. doi: 10.1083/jcb.98.5.1899.
Ankyrin is an extrinsic membrane protein in human erythrocytes that links the alpha beta-spectrin-based extrinsic membrane skeleton to the membrane by binding simultaneously to the beta-spectrin subunit and to the transmembrane anion transporter. To analyse the temporal and spatial regulation of assembly of this membrane skeleton, we investigated the kinetics of synthesis and assembly of ankyrin ( goblin ) with respect to those of spectrin in chicken embryo erythroid cells. Electrophoretic analysis of Triton X-100 soluble and cytoskeletal fractions show that at steady state both ankyrin and spectrin are detected exclusively in the cytoskeleton. In contrast, continuous labeling of erythroid cells with [35S]methionine, and immunoprecipitation of ankyrin and alpha- and beta-spectrin, reveals that newly synthesized ankyrin and spectrin are partitioned into both the cytoskeletal and Triton X-100 soluble fractions. The soluble pools of ankyrin and beta-spectrin reach a plateau of labeling within 1 h, whereas the soluble pool of alpha-spectrin is substantially larger and reaches a plateau more slowly, reflecting an approximately 3:1 ratio of synthesis of alpha- to beta-spectrin. Ankyrin and beta-spectrin enter the cytoskeletal fraction within 10 min of labeling, and the amount assembled into the cytoskeletal fraction exceeds the amount present in their respective soluble pools within 1 h of labeling. Although alpha-spectrin enters the cytoskeletal fraction with similar kinetics to beta-spectrin and ankyrin, and in amounts equimolar to beta-spectrin, the amount of cytoskeletal alpha-spectrin does not exceed the amount of soluble alpha-spectrin even after 3 h of labeling. Pulse-chase labeling experiments reveal that ankyrin and alpha- and beta-spectrin assembled into the cytoskeleton exhibit no detectable turnover, whereas the Triton X-100 soluble polypeptides are rapidly catabolized, suggesting that stable assembly of the three polypeptides is dependent upon their association with their respective membrane receptor(s). The existence in the detergent-soluble compartment of newly synthesized ankyrin and alpha- and beta-spectrin that are catabolized, rather than assembled, suggests that ankyrin and spectrin are synthesized in excess of available respective membrane binding sites, and that the assembly of these polypeptides, while rapid, is not tightly coupled to their synthesis. We hypothesize that the availability of the high affinity receptor(s) localized on the membrane mediates posttranslationally the extent of assembly of the three cytoskeletal proteins in the correct stoichiometry, their stability, and their spatial localization.
锚蛋白是人类红细胞中的一种外在膜蛋白，它通过同时与β-血影蛋白亚基和跨膜阴离子转运体结合，将基于αβ-血影蛋白的外在膜骨架与膜连接起来。为了分析这种膜骨架组装的时间和空间调控，我们研究了鸡胚红细胞中锚蛋白（小鬼蛋白）与血影蛋白合成和组装的动力学关系。对Triton X-100可溶性组分和细胞骨架组分进行电泳分析表明，在稳态时，锚蛋白和血影蛋白仅在细胞骨架中被检测到。相反，用[35S]甲硫氨酸对红细胞进行连续标记，并对锚蛋白以及α-和β-血影蛋白进行免疫沉淀，结果显示新合成的锚蛋白和血影蛋白被分配到细胞骨架和Triton X-100可溶性组分中。锚蛋白和β-血影蛋白的可溶性池在1小时内达到标记平台期，而α-血影蛋白的可溶性池则大得多，达到平台期的速度更慢，这反映出α-血影蛋白与β-血影蛋白的合成比例约为3:1。标记后10分钟内，锚蛋白和β-血影蛋白进入细胞骨架组分，标记1小时内组装到细胞骨架组分中的量超过其各自可溶性池中的量。尽管α-血影蛋白以与β-血影蛋白和锚蛋白相似的动力学进入细胞骨架组分，且量与β-血影蛋白等摩尔，但即使标记3小时后，细胞骨架中的α-血影蛋白量也不超过可溶性α-血影蛋白量。脉冲追踪标记实验表明，组装到细胞骨架中的锚蛋白以及α-和β-血影蛋白没有可检测到的周转，而Triton X-100可溶性多肽迅速被分解代谢，这表明这三种多肽的稳定组装依赖于它们与各自膜受体的结合。新合成的、被分解代谢而非组装的锚蛋白以及α-和β-血影蛋白存在于去污剂可溶区室中，这表明锚蛋白和血影蛋白的合成量超过了可用的各自膜结合位点，并且这些多肽的组装虽然迅速，但与其合成并没有紧密耦合。我们推测，定位在膜上的高亲和力受体的可用性在翻译后介导了这三种细胞骨架蛋白以正确的化学计量比进行组装的程度、它们的稳定性以及它们的空间定位。
