Identification of alpha-tubulin as an hsp105alpha-binding protein by the yeast two-hybrid system.
Hsp105alpha is a mammalian stress protein that belongs to the HSP105/110 family. Hsp105alpha prevents stress-induced apoptosis in neuronal cells and binds to Hsp70/Hsc70 and suppresses the Hsp70 chaperone activity in vitro. In this study, to further elucidate the function of Hsp105alpha, we searched for Hsp105alpha-binding proteins by screening a mouse FM3A cell cDNA library with full-length Hsp105alpha using the yeast two-hybrid system and obtained alpha-tubulin as an Hsp105alpha-binding protein. Hsp105alpha bound directly to alpha-tubulin both in vitro and in vivo. Indirect immunofluorescence analysis with anti-Hsp105 and anti-alpha-tubulin antibodies indicated that Hsp105alpha was colocalized with microtubules. Furthermore, the disorganization of microtubules induced by heat shock was prevented in Hsp105alpha-overexpressing COS-7 cells. These findings suggested that Hsp105alpha associates with alpha-tubulin and microtubules in cells and plays a role in protection of microtubules under conditions of stress.
Department of Biochemistry, Kyoto Pharmaceutical University, 5 Nakauchicho, Misasagi, Yamashina-ku, Kyoto 607-8414, Japan.
Youhei
Y
Saito
Nobuyuki
N
Yamagishi
Keiichi
K
Ishihara
Takumi
T
Hatayama
D000818:Animals; D017209:Apoptosis / Q000235:genetics; D019556:COS Cells; D018076:DNA, Complementary / Q000032:analysis / Q000235:genetics; D005057:Eukaryotic Cells / Q000378:metabolism*; D050957:HSP110 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism*; D006367:HeLa Cells; D018869:Heat-Shock Response / Q000235:genetics; D006801:Humans; D051379:Mice; D008870:Microtubules / Q000378:metabolism*; D011485:Protein Binding / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics; D020127:Recovery of Function / Q000235:genetics; D013312:Stress, Physiological / Q000235:genetics / Q000378:metabolism*; D014404:Tubulin / Q000235:genetics / Q000378:metabolism*; D020798:Two-Hybrid System Techniques
D018076:DNA, Complementary; D050957:HSP110 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins; C097205:HSPH1 protein, human; C490783:Hsp105 protein, mouse; D014404:Tubulin