The roles of K5 and K14 head, tail, and R/K L L E G E domains in keratin filament assembly in vitro

A. K. Wilson, P. A. Coulombe, E. Fuchs

Research output: Contribution to journalArticlepeer-review

Abstract

Type I and type II keratins form obligatory heterodimers, which self- assemble into 10-nm intermediate filaments (IFs). Like all IF proteins, they have a central α-helical rod domain, flanked by nonhelical head and tail domains. The IF rod is more highly conserved than head and tail, and within the rod, the carboxy R/K L L E G E sequence is more highly conserved than most other regions. Mutagenesis studies have shed some light on the roles of the head, tail, and R/K L L E G E sequence in 10-nm filament structure. However, interpretations have often been complicated in part because many of these studies have focused on transfected cells, where filament structure cannot be evaluated. Of the few in vitro assembly studies thus far conducted, comparison of keratin mutants with other IF mutants have often been difficult, due to the obligatory heteropolymeric nature of keratin IFs. In this report, we describe in vitro filament assembly studies on headless, tailless, headless/tailless, and R/K L L E G E truncated mutants of keratin 5 and its partner keratin 14. Using varying conditions of ionic strength and pH, we examine effects of analogous K5 and K14 mutations on the stability of 10-nm filament structure. Using EM, we examine effects of mutations on the ability of subunits/protofibrils to (a) elongate and (b) laterally associate. Our results demonstrate that (a) tails of K5 and K14 are required for filament stabilization; (b) the head of K5, but not of K14, is required for filament elongation and lateral alignments; and (c) the R/K L L E G E domains are required for lateral alignments, but not for filament elongation.

Original languageEnglish (US)
Pages (from-to)401-414
Number of pages14
JournalJournal of Cell Biology
Volume119
Issue number2
DOIs
StatePublished - 1992

ASJC Scopus subject areas

  • Cell Biology

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