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 proteins, which are obligate heteropolymers, at least in rodents. These IF proteins therefore provide good systems for elucidating the mechanism of intermediate filament assembly. To analyze the roles of the head domains of these proteins in contributing to their differential assembly properties, we generated chimeric proteins by swapping the head domains between rat alpha-internexin and either rat NF-L or NF-M and examined their assembly properties in transfected cells that lack their own cytoplasmic intermediate filament network. Lalphaalpha and Malphaalpha, the chimeric proteins generated by 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, alphaLL, 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 filaments, whereas MLL, a chimeric NF-L protein containing the NF-M head domain, was 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 Lalphaalpha with NF-M and coassembly of Malphaalpha with NF-L resulted in formation of filaments, coassembly of Lalphaalpha with NF-L and coassembly of Malphaalpha with NF-M yielded punctate patterns. These coassembly results show that heteropolymeric filament formation requires 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 important roles in determining the obligate heteropolymeric nature of filament formation. The data obtained from these self-assembly and coassembly studies provide some new insights into the mechanism of intermediate filament assembly.
Ching
G Y
GY
Department of Pathology, Columbia University College of Physicians and Surgeons, New York, New York 10032, USA.
Liem
R K
RK
D000595:Amino Acid Sequence; D000818:Animals; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D006801:Humans; D007381:Intermediate Filament Proteins; D046911:Macromolecular Substances; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D016900:Neurofilament Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D009474:Neurons / Q000378:metabolism*; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014162:Transfection
D002352:Carrier Proteins; D007381:Intermediate Filament Proteins; D046911:Macromolecular Substances; D016900:Neurofilament Proteins; D011993:Recombinant Fusion Proteins; C046643:alpha-internexin; C044882:neurofilament protein L; C061195:neurofilament protein M