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.

Spastin是一种微管切断AAA ATPase，可调节微管动力学，并在细胞分裂和神经发生中起重要作用。Spastin编码基因SPAST中的突变导致神经退行性疾病并引起痉挛性截瘫4型。Spastin具有两个主要的同工型M1和M87，它们仅在存在或不存在86个N末端氨基酸时有所不同，并且具有缺少exon4的其他剪接变体。M1的N端区域包含一个疏水域，核定位信号（NLS）和核输出信号（NES），部分解释了这两种同工型定位的差异。但是，尚不完全了解调节同工型本地化的机制，以及本地化对spastin功能的影响。我们发现内源性M1和M87在细胞周期中穿梭在细胞核和细胞质之间。我们确定了一个NES（氨基酸195-204），它跨越了微管相互作用域和内体运输域和exon4区域。此外，NES序列同时包含spastin亚型的卷曲螺旋区和exon4区。外显子3中高度保守的亮氨酸195和外显子4中的两个残基对于预测卷曲螺旋形成至关重要。NES或瘦霉素B处理中的突变减少了M87而不是M1的胞质定位和微管碎裂。与NLS相邻的苏氨酸306的拟磷酸盐突变（氨基酸309-312）抑制了M87的核转运。我们的结果表明，在含有exon4的spastin亚型中新发现的NES与受spastin磷酸化状态控制的NLS协同调节spastin的亚细胞定位，并参与微管切割。外显子3中高度保守的亮氨酸195和外显子4中的两个残基对于预测卷曲螺旋形成至关重要。NES或瘦霉素B处理中的突变减少了M87而不是M1的胞质定位和微管碎裂。与NLS相邻的苏氨酸306的拟磷酸盐突变（氨基酸309-312）抑制了M87的核转运。我们的结果表明，在含有exon4的spastin亚型中新发现的NES与受spastin磷酸化状态控制的NLS协同调节spastin的亚细胞定位，并参与微管切割。外显子3中高度保守的亮氨酸195和外显子4中的两个残基对于预测卷曲螺旋形成至关重要。NES或瘦霉素B处理中的突变减少了M87而不是M1的胞质定位和微管碎裂。与NLS相邻的苏氨酸306的拟磷酸盐突变（氨基酸309-312）抑制了M87的核转运。我们的结果表明，在含有exon4的spastin亚型中新发现的NES与受spastin磷酸化状态控制的NLS协同调节spastin的亚细胞定位，并参与微管切割。与NLS相邻的苏氨酸306的拟磷酸盐突变（氨基酸309-312）抑制了M87的核转运。我们的结果表明，在含有exon4的spastin亚型中新发现的NES与受spastin磷酸化状态控制的NLS协同调节spastin的亚细胞定位，并参与微管切割。与NLS相邻的苏氨酸306的拟磷酸盐突变（氨基酸309-312）抑制了M87的核转运。我们的结果表明，在含有exon4的spastin亚型中新发现的NES与受spastin磷酸化状态控制的NLS协同调节spastin的亚细胞定位，并参与微管切割。