A nuclear localization signal is required for the nuclear translocation of Fign and its microtubule-severing function

Abstract

It is commonly known that the specific function of a given ATPase associated with diverse cellular activities protein (i.e., a member of the AAA superfamily of proteins) depends primarily on its subcellular location. The microtubule-severing protein fidgetin (Fign) possesses a nuclear localization signal (NLS) that facilitates its translocation to the nucleus, where its assembly is finalized; here, Fign contributes to the regulation of microtubule configuration by cutting and trimming microtubule polymers. In the present study, Fign was found to be a nuclear protein, whose N-terminal sequence (SSLKRKAFYM; residues 314-323) acts as an NLS. Following substitution (KR to NN; 317-318) or deletion (NT; 314-323) mutations within the NLS, Fign, which is predominantly expressed in the nucleus, was found to reside in the cytoplasm of transfected cells. Furthermore, Fign was found to have an essential role in microtubule severing by preferentially targeting highly-tyrosinated microtubules (tyr-MTs). Mutation of the Fign NLS did not affect its microtubule-severing function or the cleavage of tyr-MTs, but did affect the cellular distribution of the Fign protein itself. Taken altogether, an NLS for Fign was identified, and it was demonstrated that the basic amino acids K317 and R318 are necessary for regulating its entry into the nucleus, whereas an increase in Fign in the cytosol due to mutations of the NLS did not affect its cleavage function.