Intermediate filament (IF) assembly is remarkable, in that it appears to be self-driven by the primary sequence of IF proteins, a family (40-220 kd) with diverse sequences, but similar secondary structures. Each IF polypeptide has a central 310 amino acid residue alpha-helical rod domain, involved in coiled-coil dinner formation. Two short (approximately 10 amino acid residue) stretches at the ends of this rod are more highly conserved than the rest, although the molecular basis for this is unknown. In addition, the rod is segmented by three short nonhelical linkers of conserved location, but not sequence. To examine the degree to which different conserved helical and nonhelical rod sequences contribute to dimer, tetramer, and higher ordered interactions, we introduced proline mutations in residues throughout the rod of a type I keratin, and we removed existing proline residues from the linker regions. To further probe the role of the rod ends, we introduced more subtle mutations near the COOH-terminus. We examined the consequences of these mutations on (a) IF network formation in vivo, and (b) 10-nm filament assembly in vitro. Surprisingly, all proline mutations located deep in the coiled-coil rod segment showed rather modest effects on filament network formation and 10-nm filament assembly. In addition, removing the existing proline residues was without apparent effect in vivo, and in vitro, these mutants assembled into 10-nm filaments with a tendency to aggregate, but with otherwise normal appearance. The most striking effects on filament network formation and IF assembly were observed with mutations at the very ends of the rod. These data indicate that sequences throughout the rod are not equal with respect to their role in filament network formation and in 10-nm filament assembly. Specifically, while the internal rod segments seem able to tolerate considerable changes in alpha-helical conformation, the conserved ends seem to be essential for creating a very specific structure, in which even small perturbations can lead to loss of IF stability and disruption of normal cellular interactions. These findings have important implications for the disease Epidermolysis Bullosa Simplex, arising from point mutations in keratins K5 or K14.

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