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 disag...

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Bibliographic Details
Published in:Biochemical and biophysical research communications 2004-02, Vol.314 (2), p.396-402
Main Authors: Saito, Youhei, Doi, Kazuya, Yamagishi, Nobuyuki, Ishihara, Keiichi, Hatayama, Takumi
Format: Article
Language:English
Subjects:
Animals
Bacterial Proteins - chemistry
Brain - metabolism
Calmodulin-Binding Proteins - metabolism
COS Cells
Cytoskeleton - metabolism
DNA, Complementary - metabolism
Fluorescent Antibody Technique, Indirect
Fungal Proteins - chemistry
Gene Library
HSP110 Heat-Shock Proteins
HSP70 Heat-Shock Proteins - metabolism
Humans
Mice
Microscopy, Fluorescence
Microtubules - metabolism
Plasmids - metabolism
Protein Binding
Protein Biosynthesis
Recombinant Proteins - metabolism
Transcription, Genetic
Tubulin - metabolism
Two-Hybrid System Techniques
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Summary: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.
ISSN:0006-291X