Tailless keratins assemble into regular intermediate filaments in vitro.
To study the influence of the non alpha-helical tail domain of keratins in filament formation, we prepared a truncated keratin 8 mutant, K8/tailless. Using site-directed in vitro mutagenesis we introduced a stop codon in the position coding for amino acid number 417 of the K8/wild-type sequence, thereby deleting 86 amino acids of the non alpha-helical tail domain but leaving the consensus sequence at the end of the rod domain intact. Expression of the truncated keratin 8 in Escherichia coli allowed us to purify the protein by a two-step procedure. The filament-forming capacity of the truncated K8 with wild-type K18 and K19 was analyzed using in vitro reconstitution. The in vitro assembly studies with K8/tailless and K18 wild-type indicate that the C-terminal tail domain of a type II keratin, including the homologous subdomain H2, is not required for filament formation. Moreover, reconstitution experiments with K8/tailless and K19, a naturally occurring tailless keratin I, show that the tail domains of type I as well as type II keratins are not an essential requirement for in vitro filament formation. Our results suggest that in vitro filament elongation does not depend on interactions between head and tail domains, although the tail domain might have a role in stabilization of intermediate filaments arising from certain keratin pairs.
Hatzfeld
M
M
Max Planck Institute for Biophysical Chemistry, Goettingen, FRG.
Weber
K
K
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D004926:Escherichia coli; D007382:Intermediate Filaments / Q000737:chemistry*; D007633:Keratins / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000378:metabolism; D013329:Structure-Activity Relationship; D014982:Xenopus laevis
D011994:Recombinant Proteins; D007633:Keratins