BPS2026 – The N-terminal hydrophobic (Hp) domain regulates the activity of microtubule severing protein spastin

Spastin is an AAA-ATPase that severs microtubules and is frequently mutated in hereditary spastic paraplegia (HSP). It exists as two major isoforms, the full length M1 and the short M87, generated via alternative translation initiation. While M87 has been extensively studied, the biochemical activities of M1 remain poorly understood, largely due to difficulties in purification. Here, we report the first successful purification of full-length M1 spastin and demonstrate that purified protein both severs microtubules and promotes microtubule growth in vitro, providing the first direct demonstration of its enzymatic activity. In contrast, when expressed in cells, M1 spastin does not sever microtubules, suggesting that its activity is regulated in vivo. In cells, M1 predominantly localizes to endoplasmic reticulum (ER) and lipid droplet membranes via its N-terminal hydrophobic (Hp) domain; when purified M1 is incubated with artificial liposomes it does not to sever microtubules, implicating the role of N-terminal Hp domain in regulating its severing activity. Deletion of the Hp domain (spastinΔHp) abolishes membrane binding, resulting in diffuse cytoplasmic distribution and robust microtubule severing activity in cells, revealing a critical regulatory role for the hydrophobic domain in modulating enzymatic activity. Together, these findings support a model in which membrane binding via the hydrophobic domain suppresses microtubule severing, allowing M1 spastin to perform alternative functions in membrane remodeling. Ongoing studies aim to elucidate how membrane interactions control M1 spastin activity at the molecular and structural levels.