Purification and analysis of authentic CLIP-170 and recombinant fragments

J Scheel, P Pierre, J E Rickard, G S Diamantopoulos, C Valetti, F G van der Goot, M Haner, U Aebi, T E Kreis

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Abstract

We have purified authentic CLIP-170 (cytoplasmic linker protein of 170 kDa) and fragments comprising functional domains of the protein to characterize the structural basis of the function of CLIP-170, Analysis of authentic CLIP-170 and the recombinant fragments by electron microscopy after glycerol spraying/low angle rotary metal shadowing reveals CLIP-170 as a thin, 135-nm-long molecule with two kinks in its central rod domain, which are approximately equally spaced from the two ends of the protein. The central domain consisting of heptad repeats, which is cu-helical in nature and forms a 2-stranded coiled-coil, mediates dimerization of CLIP-170, The rod domain harbors two kinks, each spaced similar to 37 nm from the corresponding end of the molecule, thus providing mechanical flexibility to the highly elongated molecule. The N-terminal domain of CLIP-170 binds to microtubules in vitro with a stoichiometry of one dimeric head domain per four tubulin heterodimers. Authentic CLIP-170 binds to microtubules with lower stoichiometry, indicating that the rod and tail domains affect microtubule binding of CLIP-170, These results document that CLIP-170 is a highly elongated polar molecule with the microtubule-binding domain and the organelle-interacting domains at opposite ends of the homodimer, thus providing a structural basis for the function of CLIP-170 as a microtubule-organelle linker protein.

Original languageEnglish
Pages (from-to)25883-25891
Number of pages9
JournalThe Journal of Biological Chemistry
Volume274
Publication statusPublished - 1999

Keywords

  • MICROTUBULE-ASSOCIATED PROTEIN
  • ACTIN-RELATED PROTEIN
  • BOVINE BRAIN KINESIN
  • ELECTRON-MICROSCOPY
  • DYNACTIN COMPLEX
  • BINDING PROTEIN
  • SMOOTH-MUSCLE
  • COILED-COIL
  • IDENTIFICATION
  • MYOSIN

Cite this

Scheel, J., Pierre, P., Rickard, J. E., Diamantopoulos, G. S., Valetti, C., van der Goot, F. G., Haner, M., Aebi, U., & Kreis, T. E. (1999). Purification and analysis of authentic CLIP-170 and recombinant fragments. The Journal of Biological Chemistry, 274, 25883-25891.