Purification and analysis of authentic CLIP-170 and recombinant fragments.
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 alpha-helical in nature and forms a 2-stranded coiled-coil, mediates dimerization of CLIP-170. The rod domain harbors two kinks, each spaced approximately 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.
Scheel
J
J
Department of Cell Biology, Sciences III, Sciences II, University of Geneva, CH-1211 Geneva 4, Switzerland.
Pierre
P
P
Rickard
J E
JE
Diamantopoulos
G S
GS
Valetti
C
C
van der Goot
F G
FG
Häner
M
M
Aebi
U
U
Kreis
T E
TE
D001665:Binding Sites; D006801:Humans; D008869:Microtubule-Associated Proteins / Q000032:analysis* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D008870:Microtubules / Q000378:metabolism*; D009363:Neoplasm Proteins; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
D008869:Microtubule-Associated Proteins; D009363:Neoplasm Proteins; D010446:Peptide Fragments; D011994:Recombinant Proteins; C076744:cytoplasmic linker protein 170