Abstract
We showed previously that stable, detyrosinated (Glu) microtubules function to localize vimentin intermediate filaments in fibroblasts (Gurland, G., and Gundersen, G. G. (1995) J. Cell Biol. 131, 1275-1290). To identify candidate proteins that mediate the Glu microtubule-vimentin interaction, we incubated microtubules with microtubule-interacting proteins and saturating levels of antibodies to Glu or tyrosinated (Tyr) tubulin. Antibodies to Glu tubulin prevented the microtubule binding of kinesin obtained from fibroblast or brain extracts more effectively than antibodies to Tyr tubulin. Scatchard plot analysis showed that kinesin heads bound to Glu microtubules with an approximately 2.8-fold higher affinity than to Tyr microtubules. Purified brain kinesin cosedimented with vimentin, but not with neurofilaments, indicating that kinesin specifically associates with vimentin without accessory molecules. Kinesin binding to vimentin was not sensitive to ATP, and kinesin heads failed to bind to vimentin. By SDS-polyacrylamide gel electrophoresis, a kinesin heavy chain of approximately 120 kDa and a light chain of approximately 64 kDa were detected in vimentin/kinesin pellets. The light chain reacted with a general kinesin light chain antibody, but not with two other antibodies that recognize the two known isoforms of kinesin light chain in brain, suggesting that the kinesin involved in binding to vimentin may be a specific one. These results demonstrate a kinesin-based mechanism for the preferential interaction of vimentin with detyrosinated microtubules.
Highlights
We showed previously that stable, detyrosinated (Glu) microtubules function to localize vimentin intermediate filaments in fibroblasts
We found that antibodies to Glu tubulin reduced the binding of a kinesin to MTs more effectively than antibodies to Tyr tubulin
Kinesin Cosediments with Vimentin intermediate filaments (IFs)—We showed above that antibodies to Glu tubulin reduced the binding of a 110-kDa (3T3 cell) and a 120-kDa kinesin to MTs more effectively than antibodies to Tyr tubulin and that recombinant kinesin heads bound to Glu MTs with a higher affinity than to Tyr MTs
Summary
(Received for publication, December 2, 1997, and in revised form, February 10, 1998). The light chain reacted with a general kinesin light chain antibody, but not with two other antibodies that recognize the two known isoforms of kinesin light chain in brain, suggesting that the kinesin involved in binding to vimentin may be a specific one These results demonstrate a kinesin-based mechanism for the preferential interaction of vimentin with detyrosinated microtubules. In a more recent study, microinjection of nonpolymerizable Glu tubulin, but not Tyr tubulin, into the cytoplasm disrupted the distribution of IFs without affecting the level of stable MTs.. In a more recent study, microinjection of nonpolymerizable Glu tubulin, but not Tyr tubulin, into the cytoplasm disrupted the distribution of IFs without affecting the level of stable MTs.2 These results conclusively demonstrate that tubulin detyrosination, rather than increased MT stability, is responsible for the Glu MT-IF interaction. Our results show that kinesin can mediate the preferential interaction of vimentin IFs with Glu MTs
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