title: Modulation of keratin intermediate filament assembly by single amino acid exchanges in the consensus sequence at the C-terminal end of the rod domain.
abstract: All known intermediate filament (IF) proteins display -8 -4 -1 a consensus sequence TYRKLLEGE at the carboxyl end of the rod domain. To analyse the contribution of this sequence to the formation of IF we have changed two of the invariant positions of this motif by site-directed mutagenesis. We produced three mutant keratins, each containing a single point mutation. Tyrosine at position -8 was changed to alanine in keratin K8 (K8Y----A-8) and keratin K18 (K18Y----A-8) and leucine at position -4 was changed to glycine in keratin K18 (K18L----G-4). Mutant keratins were expressed in Escherichia coli, purified and analysed for their filament-forming capacity in vitro using either the complementary wild-type keratin or the corresponding mixture of mutant keratins. In standard filament buffer (50 mM Tris-HCl, pH7.5), assembly involving any of the mutants leads to large electron-dense aggregates instead of normal IF. In order to explain this effect, we studied the process of filament formation in more detail. Whereas the formation of tetramers in buffers containing 4M urea is unaffected, the elongation process seems slowed down. In buffer of lower ionic strength (10 mM Tris-HCl, pH7.5) mutant keratins K8Y----A-8 plus K18Y----A-8 become able to form long filaments, although short filaments and protofilamentous material are still detected. The filaments formed differ from normal keratin IF by their remarkable tendency to aggregate into thick cables. Assemblies involving K18L----G-4 can only form short IF lengths. The dense aggregates formed in standard filament buffer are able to dissociate into IF and their fragments upon dialysis into 10 mM Tris-HCl, pH7.5. The results show that the consensus sequence is needed for IF formation under normal conditions and that already one mutation per heterodimer affects the assembly.
authors.affiliation: Max Planck Institute for Biophysical Chemistry, Department of Biochemistry, Goettingen, FRG.
authors.forename: M
authors.initials: M
authors.lastname: Hatzfeld
authors.forename: K
authors.initials: K
authors.lastname: Weber
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D016384:Consensus Sequence; D004247:DNA; D003956:Dialysis; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007382:Intermediate Filaments / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure; D007633:Keratins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009994:Osmolar Concentration; D016415:Sequence Alignment; D012995:Solubility; D014508:Urea
chemical_list: D007633:Keratins; D014508:Urea; D004247:DNA