Nucleotide binding and hydrolysis properties of Neurospora crassa cytosolic molecular chaperones, Hsp70 and Hsp80, heat-inducible members of the eukaryotic stress-70 and stress-90 families.
Formation of a hetero-oligomeric complex between Hsp70 and Hsp80 of Neurospora crassa was observed previously by means of chemical crosslinking and enzyme-linked immunosorbent assays (ELISA). The present study documents the effect of nucleotides on the subunit structure of Hsp70 and Hsp80 by crosslinking with bifunctional reagents: glutaraldehyde, dimethyl adipimidate (DMA), and dimethyl suberimidate (DMS). The inter-protomer crosslinking of Hsp80 with DMA and DMS was suppressed by ATP and to a lesser extent by ADP, CTP, and NAD. Crosslinking of purified Hsp70 by glutaraldehyde yielded dimers and higher order oligomers. Binding of ATP, ADP, CTP, and NAD, but not NADH, led to a marked reduction in the yield of oligomers. Similarly, crosslinking by DMA and DMS was suppressed by ADP, ATP, and CTP. Both Hsp70 and Hsp80 exhibited intrinsic ATPase activity. Interestingly, ATP levels exceeding 25 microM resulted in pronounced inhibition of the ATPase activity of Hsp80 and 0.5 mM and 0.25 mM ATP led to a prolonged lag in the reaction. Addition of NAD resulted in the abolition of the lag period. The binding of 2-p-toluidinylnapthalene-6-sulfonate (TNS) to Hsp70 and its displacement by ATP and other nucleotides demonstrated the hydrophobic nature of the nucleotide-binding region.
Ouimet
P M
PM
Department of Biological Sciences, University of Calgary, Canada.
Kapoor
M
M
D000251:Adenosine Triphosphatases / Q000378:metabolism; D003432:Cross-Linking Reagents; D003600:Cytosol / Q000378:metabolism; D004119:Dimethyl Adipimidate; D004120:Dimethyl Suberimidate; D005456:Fluorescent Dyes / Q000378:metabolism; D005656:Fungal Proteins / Q000378:metabolism*; D005976:Glutaral; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism*; D006360:Heat-Shock Proteins / Q000378:metabolism*; D006361:Heating; D006868:Hydrolysis; D009282:Naphthalenesulfonates / Q000378:metabolism; D009492:Neurospora crassa / Q000378:metabolism*; D009711:Nucleotides / Q000378:metabolism*; D013379:Substrate Specificity
D003432:Cross-Linking Reagents; D005456:Fluorescent Dyes; D005656:Fungal Proteins; D018840:HSP70 Heat-Shock Proteins; D006360:Heat-Shock Proteins; D009282:Naphthalenesulfonates; D009711:Nucleotides; D004119:Dimethyl Adipimidate; D004120:Dimethyl Suberimidate; C009497:2-(4-toluidino)-6-naphthalenesulfonic acid; D000251:Adenosine Triphosphatases; D005976:Glutaral