Screening of Hsp105alpha-binding proteins using yeast and bacterial two-hybrid systems.
Hsp105alpha is a 105-kDa stress protein, which is expressed constitutively at especially high levels in the brain compared with other tissues in mammals, and is also induced by a variety of stressors. Recently, we have shown that Hsp105alpha binds to alpha-tubulin and prevents the heat-induced disaggregation of microtubules. To further elucidate the function of Hsp105alpha, we searched for Hsp105alpha-binding proteins by screening a mouse FM3A cell library and human and mouse brain cDNA libraries using the yeast and bacterial two-hybrid systems. We showed here that Hsp105alpha interacted with several cellular proteins, such as cofilin, dynein light chain 2A, alpha-adducin, ubiquitin activating enzyme E1, phosphoglycerate kinase 1, and platelet-activating factor acethylhydrolase alpha1-subunit. The interaction was validated by the results of a pull-down assay and indirect immunofluorescence analysis. The significance of Hsp105alpha and Hsp105alpha-binding proteins in cells was discussed.
Saito
Youhei
Y
Department of Biochemistry, Kyoto Pharmaceutical University, 607-8414 Kyoto, Japan.
Doi
Kazuya
K
Yamagishi
Nobuyuki
N
Ishihara
Keiichi
K
Hatayama
Takumi
T
D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry; D001921:Brain / Q000378:metabolism; D019556:COS Cells; D002148:Calmodulin-Binding Proteins / Q000378:metabolism; D003599:Cytoskeleton / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D019084:Fluorescent Antibody Technique, Indirect; D005656:Fungal Proteins / Q000737:chemistry; D015723:Gene Library; D050957:HSP110 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism*; D006801:Humans; D051379:Mice; D008856:Microscopy, Fluorescence; D008870:Microtubules / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D014176:Protein Biosynthesis; D011994:Recombinant Proteins / Q000378:metabolism; D014158:Transcription, Genetic; D014404:Tubulin / Q000378:metabolism; D020798:Two-Hybrid System Techniques; D000099291:Adducins
D001426:Bacterial Proteins; D002148:Calmodulin-Binding Proteins; D018076:DNA, Complementary; D005656:Fungal Proteins; D050957:HSP110 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins; D011994:Recombinant Proteins; D014404:Tubulin; D000099291:Adducins; C097205:HSPH1 protein, human; C490783:Hsp105 protein, mouse