The cochaperone murine stress-inducible protein 1: overexpression, purification, and characterization.
Murine stress-inducible protein 1 (mSTI1) is a cochaperone that is homologous with the human heat shock cognate protein 70 (Hsc70)/heat shock protein 90 (Hsp90)-organizing protein (Hop). To analyze the biochemical properties of mSTI1 and the stoichiometry of the Hsc70.mSTI1.Hsp90 association, recombinant mSTI1 was produced in untagged, histidine (His)-tagged, and glutathione S-transferase (GST)-tagged forms. His-mSTI1 was detected either as a dimer during size-exclusion-high-performance liquid chromatography (SE-HPLC) or as a monomer during Superdex 200 gel filtration chromatography. SE-HPLC on GST-mSTI1 and untagged mSTI1 suggested that mSTI1 existed as a monomer. Cross-linking of His-mSTI1 detected a compact monomeric species and a dimeric species. Gel filtration on the association of bovine STI1 or His-mSTI1 with Hsc70 detected species of molecular mass consistent with a dimeric STI1 species or a 1:1 complex of STI1 and Hsc70. Our data and that of others suggest that mSTI1 and its homologues exist as either a monomer or a dimer and that this facilitates its proposed function as an Hsc70/Hsp90 organizing protein.
van der Spuy
J
J
Protein Structure-Function Research Programme, Department of Biochemistry, University of the Witwatersrand, PO WITS, Johannesburg 2050, South Africa.
Cheetham
M E
ME
Dirr
H W
HW
Blatch
G L
GL
D000818:Animals; D015153:Blotting, Western; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D003432:Cross-Linking Reagents / Q000378:metabolism; D019281:Dimerization; D004926:Escherichia coli; D005976:Glutaral / Q000378:metabolism; D050883:HSC70 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism; D018841:HSP90 Heat-Shock Proteins / Q000378:metabolism; D006360:Heat-Shock Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D051379:Mice; D018832:Molecular Chaperones / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D008970:Molecular Weight; D011485:Protein Binding; D020836:Protein Structure, Quaternary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism
D003432:Cross-Linking Reagents; D050883:HSC70 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins; D018841:HSP90 Heat-Shock Proteins; D006360:Heat-Shock Proteins; C494372:Hspa8 protein, mouse; D018832:Molecular Chaperones; D011993:Recombinant Fusion Proteins; C489791:Stip1 protein, mouse; D005976:Glutaral