title: Conserved aromatic and basic amino acid residues in the pore region of Caenorhabditis elegans spastin play critical roles in microtubule severing.
abstract: Mutations of human spastin, an AAA (ATPases associated with diverse cellular activity) family protein, cause an autosomal dominant form of hereditary spastic paraplegia, which is characterized by weakness, spasticity and loss of the vibratory sense in the lower limbs. Recently, it has been reported that spastin displays microtubule-severing activity. We also previously reported that Caenorhabditis elegans spastin homologue SPAS-1 displays microtubule severing. However, the detailed molecular mechanism of microtubule severing remains unknown. Here, we describe that SPAS-1 forms a stable hexamer in a concentration-dependent manner and that ATPase activity of SPAS-1 is greatly stimulated by microtubules. Furthermore, MTBD (microtubule-binding domain) of SPAS-1 is essential for binding to microtubules. Taken these results together, we propose that MTBD of SPAS-1 plays a critical role in enrichment of SPAS-1 to microtubules, where SPAS-1 is concentrated and able to form a stable hexamer, subsequently its ATPase activity is stimulated. On the other hand, our mutational analyses revealed that the conserved aromatic and basic amino acid residues in the pore region are important for microtubule severing. We also detected the direct interaction of the extremely acidic C-terminal polypeptide of tubulin with SPAS-1. Consequently, we propose that the central pore residues are important for the recognition of substrates.
authors.affiliation: Department of Molecular Cell Biology, Institute of Molecular Embryology and Genetics, Kumamoto University, 2-2-1 Honjo, Kumamoto 860-0811, Japan.
authors.forename: Yuka
authors.initials: Y
authors.lastname: Matsushita-Ishiodori
authors.forename: Kunitoshi
authors.initials: K
authors.lastname: Yamanaka
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Hashimoto
authors.forename: Masatoshi
authors.initials: M
authors.lastname: Esaki
authors.forename: Teru
authors.initials: T
authors.lastname: Ogura
mesh_terms: D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D024322:Amino Acids, Aromatic / Q000737:chemistry; D024361:Amino Acids, Basic / Q000737:chemistry; D000818:Animals; D017173:Caenorhabditis elegans / Q000235:genetics / Q000378:metabolism*; D029742:Caenorhabditis elegans Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008870:Microtubules / Q000378:metabolism* / Q000648:ultrastructure; D008958:Models, Molecular; D009154:Mutation; D015419:Spastic Paraplegia, Hereditary / Q000378:metabolism; D000074181:Spastin; D020349:Surface Plasmon Resonance; D014404:Tubulin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D024322:Amino Acids, Aromatic; D024361:Amino Acids, Basic; D029742:Caenorhabditis elegans Proteins; D014404:Tubulin; D000251:Adenosine Triphosphatases; D000074181:Spastin; C400283:SPAST protein, human