The 90-kDa heat shock protein Hsp90 protects tubulin against thermal denaturation.
Hsp90 and tubulin are among the most abundant proteins in the cytosol of eukaryotic cells. Although Hsp90 plays key roles in maintaining its client proteins in their active state, tubulin is essential for fundamental processes such as cell morphogenesis and division. Several studies have suggested a possible connection between Hsp90 and the microtubule cytoskeleton. Because tubulin is a labile protein in its soluble form, we investigated whether Hsp90 protects it against thermal denaturation. Both proteins were purified from porcine brain, and their interaction was characterized in vitro by using spectrophotometry, sedimentation assays, video-enhanced differential interference contrast light microscopy, and native polyacrylamide gel electrophoresis. Our results show that Hsp90 protects tubulin against thermal denaturation and keeps it in a state compatible with microtubule polymerization. We demonstrate that Hsp90 cannot resolve tubulin aggregates but that it likely binds early unfolding intermediates, preventing their aggregation. Protection was maximal at a stoichiometry of two molecules of Hsp90 for one of tubulin. This protection does not require ATP binding and hydrolysis by Hsp90, but it is counteracted by geldanamycin, a specific inhibitor of Hsp90.
Weis
Felix
F
UMR-CNRS 6026, IFR 140-Génétique Fonctionnelle Agronomie et Santé, "Interactions Cellulaires et Moléculaires," Université de Rennes 1, Campus de Beaulieu, Bâtiment 13, 263 Avenue du Général Leclerc, 35042 Rennes, France.
Moullintraffort
Laura
L
UMR-CNRS 6026, IFR 140-Génétique Fonctionnelle Agronomie et Santé, "Interactions Cellulaires et Moléculaires," Université de Rennes 1, Campus de Beaulieu, Bâtiment 13, 263 Avenue du Général Leclerc, 35042 Rennes, France.
Heichette
Claire
C
UMR-CNRS 6026, IFR 140-Génétique Fonctionnelle Agronomie et Santé, "Interactions Cellulaires et Moléculaires," Université de Rennes 1, Campus de Beaulieu, Bâtiment 13, 263 Avenue du Général Leclerc, 35042 Rennes, France.
Chrétien
Denis
D
UMR-CNRS 6026, IFR 140-Génétique Fonctionnelle Agronomie et Santé, "Interactions Cellulaires et Moléculaires," Université de Rennes 1, Campus de Beaulieu, Bâtiment 13, 263 Avenue du Général Leclerc, 35042 Rennes, France.
Garnier
Cyrille
C
UMR-CNRS 6026, IFR 140-Génétique Fonctionnelle Agronomie et Santé, "Interactions Cellulaires et Moléculaires," Université de Rennes 1, Campus de Beaulieu, Bâtiment 13, 263 Avenue du Général Leclerc, 35042 Rennes, France. Electronic address: cyrille.garnier@univ-rennes1.fr.
D000255:Adenosine Triphosphate / Q000737:chemistry; D000818:Animals; D016227:Benzoquinones / Q000494:pharmacology; D001921:Brain / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D018841:HSP90 Heat-Shock Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D006358:Hot Temperature; D047029:Lactams, Macrocyclic / Q000494:pharmacology; D008027:Light; D008857:Microscopy, Interference / Q000379:methods; D008870:Microtubules / Q000378:metabolism; D011489:Protein Denaturation; D013053:Spectrophotometry / Q000379:methods; D013552:Swine; D014404:Tubulin / Q000737:chemistry*
D016227:Benzoquinones; D018841:HSP90 Heat-Shock Proteins; D047029:Lactams, Macrocyclic; D014404:Tubulin; D000255:Adenosine Triphosphate; C001277:geldanamycin