Heat shock protein 80 of Neurospora crassa, a cytosolic molecular chaperone of the eukaryotic stress 90 family, interacts directly with heat shock protein 70.
The subunit structure of Hsp80, the most abundant heat-shock protein of Neurospora crassa, was examined by chemical cross-linking of the purified protein in vitro. Resolution of glutaraldehyde-treated Hsp80 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis SDS-PAGE suggests that the native state of this protein is a tetramer; the relative proportion of cross-linked species, estimated by the fraction of protein recovered in each category, is consistent with a dimer-of-dimer structure. Upon interaction with nucleotides, higher order cross-linked oligomers were detected, indicating ligand-induced conformational changes. The effect of nucleotides was also monitored by following tryptophan fluorescence: CTP, UTP, and NAD led to fluorescence quenching, the effect of CTP being the most pronounced. As individual molecular chaperones often act in concert with cochaperones, interaction between the two major cytosolic stress proteins--Hsp80 and Hsp70--was examined. Purified Hsp70 was immobilized on ATP-agarose and purified Hsp80 was applied to the Hsp70-saturated matrix; while Hsp80 did not bind to ATP-agarose by itself, it was bound strongly by immobilized Hsp70. The [Hsp70-Hsp80] complex was eluted with ATP and coelution of both proteins was confirmed by Western blot analysis, using specific polyclonal antibodies raised against each protein. The physical association of stress-inducible Hsp70 and Hsp80 was verified by interprotein cross-linking in vitro followed by immunoblot analysis and by immunoprecipitation.
Freitag
D G
DG
Department of Biological Sciences, University of Calgary, Calgary, Alberta, Canada T2N 1N4.
Ouimet
P M
PM
Girvitz
T L
TL
Kapoor
M
M
D003432:Cross-Linking Reagents; D003600:Cytosol / Q000378:metabolism*; D005656:Fungal Proteins; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism*; D006360:Heat-Shock Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D009492:Neurospora crassa / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence
D003432:Cross-Linking Reagents; D005656:Fungal Proteins; D018840:HSP70 Heat-Shock Proteins; D006360:Heat-Shock Proteins