Cytoplasmic linker protein (CLIP)-170, CLIP-115, and the dynactin subunit p150Glued are structurally related proteins, which associate specifically with the ends of growing microtubules (MTs). Here, we show that down-regulation of CLIP-170 by RNA interference results in a strongly reduced accumulation of dynactin at the MT tips. The NH2 terminus of p150Glued binds directly to the COOH terminus of CLIP-170 through its second metal-binding motif. p150Glued and LIS1, a dynein-associating protein, compete for the interaction with the CLIP-170 COOH terminus, suggesting that LIS1 can act to release dynactin from the MT tips. We also show that the NH2-terminal part of CLIP-170 itself associates with the CLIP-170 COOH terminus through its first metal-binding motif. By using scanning force microscopy and fluorescence resonance energy transfer-based experiments we provide evidence for an intramolecular interaction between the NH2 and COOH termini of CLIP-170. This interaction interferes with the binding of the CLIP-170 to MTs. We propose that conformational changes in CLIP-170 are important for binding to dynactin, LIS1, and the MT tips.
Conformational changes in CLIP-170 regulate its binding to microtubules and dynactin localization
C.C. Hoogenraad's present address is The Picower Center for Learning and Memory, Massachusetts Institute of Technology, Cambridge, MA 02139.
Abbreviations used in this paper: CLIP, cytoplasmic linker protein; FRET, fluorescence resonance energy transfer; HIS, 6X histidine; IP, immunoprecipitation; MT, microtubule; RNAi, RNA interference; siRNA, small interfering RNA; SFM, scanning force microscopy; +TIP, plus end–tracking protein.
Gideon Lansbergen, Yulia Komarova, Mauro Modesti, Claire Wyman, Casper C. Hoogenraad, Holly V. Goodson, Régis P. Lemaitre, David N. Drechsel, Erik van Munster, Theodorus W.J. Gadella, Frank Grosveld, Niels Galjart, Gary G. Borisy, Anna Akhmanova; Conformational changes in CLIP-170 regulate its binding to microtubules and dynactin localization . J Cell Biol 27 September 2004; 166 (7): 1003–1014. doi: https://doi.org/10.1083/jcb.200402082
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