Purification and analysis of authentic CLIP-170 and recombinant fragments

Article Article scientifique Article Purification and analysis of authentic CLIP-170 and recombinant fragments eng 1999-09-03 author Scheel Jochen Konrad 28724 author Pierre Philippe 670067 author Rickard Janet Elizabeth 28855 author Diamantropoulos Georgios author Valetti Caterina 62866 author Van Der Goot Grunberg Françoise G. 24466 author Häner Markus author Aebi Ueli author Kreis Thomas 22803 25883-25891 10.1074/jbc.274.36.25883 0021-9258 <p>We have purified authentic CLIP-170 (cytoplasmic linker protein of 170 kDa) and fragments comprising functional domains of the proteins 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 tis central rod domain, which are approximately equally spaced from the two ends of the protein. The central domain consisting of heptad repeats, which is α-helical in nature and forms a 2-stranded coiled-coil, mediates dimerization of CLIP-170. The rod domain harbors two kinks, each spaced ~37 nm from the corresponding end of the molecule, thus providing mechanical flexibility to the highly elongated melocule. The N-terminal domain of CLIP-170 binds to microtubules <i>in vitro </i>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 microtubules 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.</p> Dewey 540 Dewey 570 39 Département de biochimie 144 46 Département de biologie cellulaire 165 false The Journal of biological chemistry 274 36 facad6af-a427-471c-b637-c4d31b30634b Article (Published version) Public 1-s2.0-S0021925819553558-main.pdf 1759750 application/pdf CC BY openAccess Faculté des sciences / Section de chimie et biochimie / Département de biochimie Faculté des sciences / Section de biologie / Département de biologie cellulaire

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Purification and analysis of authentic CLIP-170 and recombinant fragments	The Journal of biological chemistry			1999	146	239

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Scheel, Jochen Konrad