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Journal of Biochemistry Advance Access originally published online on September 19, 2008
Journal of Biochemistry 2008 144(5):675-684; doi:10.1093/jb/mvn116
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© 2008 The Japanese Biochemical Society

Role of the Aromatic Residues in the Near-amino Terminal Motif of Vimentin in Intermediate Filament Assembly In Vitro

Rumi Gohara1, Sadakatsu Nishikawa2, Yozo Takasaki1 and Shoji Ando1,*

1Division of Biopolymer Research, Department of Biomolecular Sciences, Faculty of Medicine, Saga University, Saga 849-8501; and 2Department of Applied Chemistry, Faculty of Science and Engineering, Saga University, Saga 840-8502, Japan

*To whom correspondence should be addressed. Tel: +81-952-34-2192, Fax: +81-952-34-2418, E-mail: andohs{at}cc.saga-u.ac.jp

Received August 18, 2008; Accepted September 6, 2008


  Abstract

Type III and IV intermediate filament (IF) proteins share a conserved sequence motif of -Tyr-Arg-Arg-X-Phe- at the near-amino termini. To characterize significance of the aromatic residues in the motif, we prepared vimentin mutants in which Tyr-10 and Phe-14 are substituted with Asn and Ser (Vim[Y10N], Vim[F14S] and Vim[Y10N, F14S]), and examined assembly properties in vitro by electron microscopy and viscosity measurements. At 2 s after initiation of assembly reaction at pH 7.2 and 150 mM NaCl, all the vimentin mutants formed so-called unit-length filaments (ULFs) that were slightly larger than ULFs of wild-type vimentin. In following filament elongation, Vim[Y10N, F14S] and Vim[Y10N] performed longitudinal annealing of ULFs very rapidly and formed IFs within only 2.5 and 5 min, respectively, while Vim[F14S] and wild-type vimentin gave IFs by 40–60 min. The IFs of Vim[Y10N, F14S] and Vim[Y10N], however, tended to intertwine each other and formed bundles in parts of the specimens. The intertwinements decreased as the salt concentration decreased, and optimal salt concentration for the two mutants to form normal IFs was 50 mM. These results suggest that the aromatic residues, especially Tyr-10, in the motif have a role in controlling intermolecular interactions involved in IF assembly in vitro and suppress undesirable filament intertwinements at physiological ionic strength.

Key Words: electron microscopy, intermediate filament, site-directed mutagenesis, vimentin, viscosity

Abbreviations: EM, electron microscopy; IF, intermediate filament; MES, 2-(N-morpholino)ethanesulphonic acid; PCR, DNA polymerase chain reaction; PMSF, phenylmethylsulphonyl fluoride; ULF, unit-length filament


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