Audebert S, Desbruyères E, Gruszczynski C, Koulakoff A, Gros F, Denoulet P, Eddé B
Laboratoire de Biochimie Cellulaire, Collège de France, Paris.
Mol Biol Cell. 1993 Jun;4(6):615-26. doi: 10.1091/mbc.4.6.615.
The relationship between microtubule dynamics and polyglutamylation of tubulin was investigated in young differentiating mouse brain neurons. Selective posttranslational labeling with [3H]glutamate and immunoblotting with a specific monoclonal antibody (GT335) enabled us to analyze polyglutamylation of both alpha and beta subunits. Nocodazole markedly inhibited incorporation of [3H]glutamate into alpha- and beta-tubulin, whereas taxol had no effect for alpha-tubulin and a stimulating effect for beta-tubulin. These results strongly suggest that microtubule polymers are the preferred substrate for polyglutamylation. Chase experiments revealed the existence of a reversal reaction that, in the case of alpha-tubulin, was not affected by microtubule drugs, suggesting that deglutamylation of this subunit can occur on both polymers and soluble tubulin. Evidence was obtained that deglutamylation of alpha-tubulin operates following two distinct rates depending on the length of the polyglutamyl chain, the distal units (4th-6th) being removed rapidly whereas the proximal ones (1st-3rd) appearing much more resistant to deglutamylation. Partition of glutamylated alpha-tubulin isoforms was also correlated with the length of the polyglutamyl chain. Forms bearing four to six units were recovered specifically in the polymeric fraction, whereas those bearing one to three units were distributed evenly between polymeric and soluble fractions. It thus appears that the slow rate component of the deglutamylation reaction offers to neurons the possibility to maintain a basal level of glutamylated alpha-tubulin in the soluble pool independently of microtubule dynamics. Finally, some differences observed in the glutamylation of alpha- and beta-tubulin suggest that distinct enzymes are involved.
在年轻的正在分化的小鼠脑神经元中研究了微管动力学与微管蛋白多聚谷氨酰胺化之间的关系。用[3H]谷氨酸进行选择性翻译后标记以及用特异性单克隆抗体（GT335）进行免疫印迹，使我们能够分析α和β亚基的多聚谷氨酰胺化。诺考达唑显著抑制[3H]谷氨酸掺入α-和β-微管蛋白，而紫杉醇对α-微管蛋白没有影响，对β-微管蛋白有刺激作用。这些结果强烈表明微管聚合物是多聚谷氨酰胺化的优选底物。追踪实验揭示了一种逆转反应的存在，就α-微管蛋白而言，该反应不受微管药物影响，这表明该亚基的去谷氨酰胺化可在聚合物和可溶性微管蛋白上发生。有证据表明，α-微管蛋白的去谷氨酰胺化以两种不同的速率进行，这取决于多聚谷氨酰链的长度，远端单元（第4 - 6个）被快速去除，而近端单元（第1 - 3个）对去谷氨酰胺化的抗性要强得多。谷氨酰化α-微管蛋白同工型的分配也与多聚谷氨酰链的长度相关。带有四到六个单元的形式特异性地在聚合物部分中回收，而带有一到三个单元的形式则均匀分布在聚合物和可溶性部分之间。因此，去谷氨酰胺化反应的慢速成分似乎使神经元有可能在独立于微管动力学的情况下，在可溶性池中维持谷氨酰化α-微管蛋白的基础水平。最后，在α-和β-微管蛋白的谷氨酰胺化中观察到的一些差异表明涉及不同的酶。
