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.