A newly identified NES sequence present in spastin regulates its subcellular localization and microtubule severing activity
A newly identified NES sequence present in spastin regulates its subcellular localization and microtubule severing activity
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DOI：
10.1016/j.bbamcr.2020.118862
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发表时间：
2021-01-01
影响因子：
5.1
通讯作者：
Matsuura, Tohru
中科院分区：
文献类型：
--
作者：
Sakoe, Kumi;Shioda, Norifumi;Matsuura, Tohru
Spastin是一种切断微管的AAA-ATPase，调节微管动力学，在细胞分裂和神经发生中发挥重要作用。Spastin编码基因spast的突变会导致神经退行性疾病，并导致4型痉挛截瘫。spastin有两种主要的亚型，M1和M87，它们只有86个N末端氨基酸的存在或不存在的不同，并且有不同的剪接变体，缺少外显子4。M1的N-末端含有一个疏水结构域、核定位信号(NLS)和核输出信号(NES)，这部分解释了这两种异构体定位的差异。然而，调控异构体定位的机制以及定位对spastin功能的影响还不完全清楚。我们发现内源性M1和M87在细胞周期中穿梭于胞核和胞浆之间。我们鉴定了一个NES(氨基酸195-204)，它跨越微管相互作用和内体转运结构域和外显子4区域。此外，NES序列还包含spastin亚型的卷曲卷曲和外显子4区域。外显子3高度保守的亮氨酸195和外显子4的两个残基对预测的卷曲形成至关重要。NES或软霉素B处理中的突变减少了M87的细胞质定位和微管碎裂，而不是M1。邻近NLS(氨基酸309-312)的苏氨酸306的拟磷突变抑制了M87的核转运。我们的结果表明，新发现的含有外显子4的spastin异构体中的NES与由spastin的磷酸化状态控制的NLS协同调节spastin的亚细胞定位，并参与微管的切断。
Spastin, a microtubule-severing AAA ATPase, regulates microtubule dynamics and plays important roles in cell division and neurogenesis. Mutations in the spastin-coding gene SPAST lead to neurodegenerative disorders and cause spastic paraplegia type 4. Spastin has two main isoforms, M1 and M87, that differ only in the presence or absence of 86 N-terminal amino acids and have alternative splicing variants that lack exon4. The N-terminal region of M1 contains a hydrophobic domain, nuclear localization signal (NLS), and nuclear export signal (NES), which partly explains the differences in the two isoforms' localization. However, the mechanisms involved in regulating isoform localization, and the effects of localization on spastin functions are not fully understood. We found endogenous M1 and M87 shuttled between the nucleus and cytoplasm during the cell cycle. We identified a NES (amino acids 195-204) that spans the microtubule-interacting and endosomal-trafficking domain and exon4 region. Furthermore, the NES sequence contains both the coiled-coil and exon4 region of spastin isoforms. Highly conserved leucine 195 in exon3 and the two residues in exon4 are crucial for predicted coiled-coil formation. Mutations in NES or leptomycin B treatment reduced cytoplasmic localization and microtubule fragmentation in M87 rather than in M1. Phosphomimetic mutation of threonine 306 adjacent to the NLS (amino acids 309-312) inhibited nuclear transport of M87. Our results indicate that the newly identified NES in the spastin isoforms containing exon4 regulates the subcellular localization of spastin in coordination with NLS controlled by the phosphorylation state of spastin, and is involved in microtubule severing.