In Vitro Assembly and Structure of Trichocyte Keratin Intermediate Filaments: A Novel Role for Stabilization by Disulfide Bonding

J Cell Biol. 2000 Dec 25;151(7):1459-68. doi: 10.1083/jcb.151.7.1459.

Abstract

Intermediate filaments (IF) have been recognized as ubiquitous components of the cytoskeletons of eukaryotic cells for 25 yr. Historically, the first IF proteins to be characterized were those from wool in the 1960s, when they were defined as low sulfur keratins derived from "microfibrils." These proteins are now known as the type Ia/type IIa trichocyte keratins that constitute keratin IF of several hardened epithelial cell types. However, to date, of the entire class of >40 IF proteins, the trichocyte keratins remain the only ones for which efficient in vitro assembly remains unavailable. In this paper, we describe the assembly of expressed mouse type Ia and type IIa trichocyte keratins into IF in high yield. In cross-linking experiments, we document that the alignments of molecules within reduced trichocyte IF are the same as in type Ib/IIb cytokeratins. However, when oxidized in vitro, several intermolecular disulfide bonds form and the molecular alignments rearrange into the pattern shown earlier by x-ray diffraction analyses of intact wool. We suggest the realignments occur because the disulfide bonds confer substantially increased stability to trichocyte keratin IF. Our data suggest a novel role for disulfide bond cross linking in stabilization of these IF and the tissues containing them.

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Cell Differentiation
  • Chromatography, High Pressure Liquid
  • Disulfides / metabolism*
  • Humans
  • Intermediate Filaments / chemistry*
  • Intermediate Filaments / metabolism*
  • Intermediate Filaments / ultrastructure
  • Keratins / chemistry
  • Keratins / classification*
  • Keratins / metabolism*
  • Keratins / ultrastructure
  • Least-Squares Analysis
  • Mice
  • Microscopy, Electron
  • Molecular Sequence Data
  • Oxidation-Reduction
  • Protein Structure, Quaternary
  • Protein Structure, Tertiary
  • Sequence Alignment
  • Wool / chemistry*
  • Wool / cytology

Substances

  • Disulfides
  • Keratins