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.
Pubmed ID: 14733918 RIS Download
Animals | Bacterial Proteins | Brain | COS Cells | Calmodulin-Binding Proteins | Cytoskeleton | DNA, Complementary | Fluorescent Antibody Technique, Indirect | Fungal Proteins | Gene Library | HSP110 Heat-Shock Proteins | HSP70 Heat-Shock Proteins | Humans | Mice | Microscopy, Fluorescence | Microtubules | Plasmids | Protein Binding | Protein Biosynthesis | Recombinant Proteins | Transcription, Genetic | Tubulin | Two-Hybrid System Techniques