Gundersen G G, Khawaja S, Bulinski J C
J Cell Biol. 1987 Jul;105(1):251-64. doi: 10.1083/jcb.105.1.251.
Tyrosinated (Tyr) and detyrosinated (Glu) alpha-tubulin, species interconverted by posttranslational modification, are largely segregated in separate populations of microtubules in interphase cultured cells. We sought to understand how distinct Tyr and Glu microtubules are generated in vivo, by examining time-dependent alterations in Tyr and Glu tubulin levels (by immunoblots probed with antibodies specific for each species) and distributions (by immunofluorescence) after microtubule regrowth and stabilization. When microtubules were allowed to regrow after complete depolymerization by microtubule antagonists, Glu microtubules reappeared with a delay of approximately 25 min after the complete array of Tyr microtubules had regrown. In these experiments, Tyr tubulin immunofluorescence first appeared as an aster of distinct microtubules, while Glu tubulin staining first appeared as a grainy pattern that was not altered by detergent extraction, suggesting that Glu microtubules were created by detyrosination of Tyr microtubules. Treatments with taxol, azide, or vinblastine, to stabilize polymeric tubulin, all resulted in time-dependent increases in polymeric Glu tubulin levels, further supporting the hypothesis of postpolymerization detyrosination. Analysis of monomer and polymer fractions during microtubule regrowth and in microtubule stabilization experiments were also consistent with postpolymerization detyrosination; in each case, Glu polymer levels increased in the absence of detectable Glu monomer. The low level of Glu monomer in untreated or nocodazole-treated cells (we estimate that Glu tubulin comprises less than 2% of the monomer pool) also suggested that Glu tubulin entering the monomer pool is efficiently retyrosinated. Taken together these results demonstrate that microtubules are polymerized from Tyr tubulin and are then rapidly converted to Glu microtubules. When Glu microtubules depolymerize, the resulting Glu monomer is retyrosinated. This cycle generates structurally, and perhaps functionally, distinct microtubules.
酪氨酸化（Tyr）和去酪氨酸化（Glu）的α-微管蛋白可通过翻译后修饰相互转化，在间期培养细胞中，它们在很大程度上分隔于不同的微管群体中。我们试图通过检测微管重新生长和稳定后，Tyr和Glu微管蛋白水平（通过用针对每种类型的特异性抗体进行免疫印迹）和分布（通过免疫荧光）随时间的变化，来了解在体内如何产生不同的Tyr和Glu微管。当通过微管拮抗剂使微管完全解聚后再让其重新生长时，Glu微管在完整的Tyr微管阵列重新生长约25分钟后延迟出现。在这些实验中，Tyr微管蛋白免疫荧光最初表现为清晰微管组成的星状体，而Glu微管蛋白染色最初表现为颗粒状模式，去污剂提取对此模式无影响，这表明Glu微管是由Tyr微管的去酪氨酸化产生的。用紫杉醇、叠氮化物或长春花碱处理以稳定聚合态微管蛋白，均导致聚合态Glu微管蛋白水平随时间增加，进一步支持了聚合后去酪氨酸化的假说。对微管重新生长过程中以及微管稳定实验中的单体和聚合物组分分析也与聚合后去酪氨酸化一致；在每种情况下，在未检测到Glu单体的情况下，Glu聚合物水平增加。未处理或用诺考达唑处理的细胞中Glu单体水平较低（我们估计Glu微管蛋白占单体池的比例不到2%）也表明进入单体池的Glu微管蛋白能有效地重新酪氨酸化。综合这些结果表明，微管由Tyr微管蛋白聚合而成，然后迅速转化为Glu微管。当Glu微管解聚时，产生的Glu单体被重新酪氨酸化。这个循环产生了结构上或许还有功能上不同 的微管。
