Analysis of the roles of the head domains of type IV rat neuronal intermediate filament proteins in filament assembly using domain-swapped chimeric proteins

Type IV neuronal intermediate filament proteins consist of alpha-internexin , which can self-assemble into filaments and the neurofilament triplet prot eins, which are obligate heteropolymers, at least in rodents. These IF prot eins therefore provide good systems for elucidating the mechanism of interm ediate filament assembly. To analyze the roles of the head domains of these proteins in contributing to their differential assembly properties, we gen erated chimeric proteins by swapping the head domains between rat alpha-int ernexin and either rat NF-L or NF-M and examined their assembly properties in transfected cells that lack their own cytoplasmic intermediate filament network. L alpha alpha and M alpha alpha, the chimeric proteins generated b y replacing the head domain of alpha-internexin with those of NF-L and NF-M , respectively, were unable to self-assemble into filaments. In contrast, a lpha LL, a chimeric NF-L protein generated by replacing the head domain of NF-L with that of alpha-internexin, was able to self-assemble into filament s, whereas MLL, a chimeric NF-L protein containing the NF-M head domain, wa s unable to do so. These results demonstrate that the alpha-internexin head domain is essential for alpha-internexin's ability to self-assemble. While coassembly of L alpha alpha with NF-M and coassembly of M alpha alpha with NF-L resulted in formation of filaments, coassembly of L alpha alpha with NF-L and coassembly of M alpha alpha with NF-M yielded punctate patterns. T hese coassembly results show that heteropolymeric filament formation requir es that one partner has the NF-L head domain and the other partner has the NF-M head domain. Thus, the head domains of rat NF-L and NF-M play importan t roles in determining the obligate heteropolymeric nature of filament form ation. The data obtained from these self-assembly and coassembly studies pr ovide some new insights into the mechanism of intermediate filament assembl y.