In vitro assembly and structure of trichocyte keratin intermediate filaments: A novel role for stabilization by disulfide bonding

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