Central rod domain insertion and carboxy-terminal fusion mutants of human cytokeratin K19 are incorporated into endogenous keratin filaments.
Keratins comprise a multigene family of structural proteins that form the 10-nm filaments present in epithelial cells. Keratin filament formation requires the presence of stoichiometric quantities of type I and type II keratin peptides. Each keratin peptide contains an N-terminal "head" segment, a C-terminal "tail" segment, and a highly conserved, alpha-helical central rod domain. To investigate the importance of these domains in situ, we have altered the DNA coding sequence of human cytokeratin K19 and transiently expressed the mutants in PtK2 cells that contain an endogenous keratin filament system. Interestingly, K19 mutants containing 4, 8, 12, and 24 amino acid insertions in the non-alpha-helical L1 region of the central rod domain successfully integrate into the endogenous PtK2 keratin filaments. Another K19 mutant, K19-bGAL, that encodes bacterial beta-galactosidase (bGAL) fused in phase to the 3' end of the K19 central rod domain, also integrates into the endogenous PtK2 keratin filaments. Our results demonstrate 1) that the spacing between the highly conserved amino and carboxy terminal ends of the K19 central rod domain can be increased without significantly effecting K19's ability to interact with keratin filaments and 2) that addition of a highly soluble 66-kDa tail to K19 does not impede its interaction with the filament system.
Department of Physiology and Biophysics, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106.
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EA
Rorke
J
J
Crish
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RL
Eckert
D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D002460:Cell Line; D006224:Cricetinae; D005696:Galactosidases / Q000032:analysis; D006801:Humans; D007382:Intermediate Filaments / Q000737:chemistry*; D007633:Keratins / Q000032:analysis* / Q000235:genetics; D007614:Macropodidae; D008969:Molecular Sequence Data; D009154:Mutation; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000032:analysis; D014162:Transfection; D014746:Vimentin / Q000032:analysis
D011993:Recombinant Fusion Proteins; D014746:Vimentin; D007633:Keratins; D005696:Galactosidases