Unfolded proteins stimulate molecular chaperone Hsc70 ATPase by accelerating ADP/ATP exchange.
The mammalian 70-kilodalton heat shock cognate protein (Hsc70) is an abundant, cytosolic molecular chaperone whose interactions with protein substrates are regulated by ATP hydrolysis. In vitro, purified Hsc70 was found to have a slow, intrinsic ATPase activity in the absence of protein substrates. The addition of an unfolded protein such as apocytochrome c stimulated ATP hydrolysis 2-3-fold. In contrast, the native holoprotein, cytochrome c, did not stimulate the ATPase rate, in accord with recent observations that 70-kilodalton heat shock proteins interact selectively with unfolded proteins. Stimulation of ATP hydrolysis by apocytochrome c was due to an increase in the Vmax, with no effect on the Km for ATP. Following hydrolysis of [3H]ATP, a relatively stable [3H]ADP.Hsc70 complex was formed. Release of [3H]ADP from Hsc70 was most efficient in the presence of other nucleotides such as ADP or ATP, suggesting that ADP release occurs as an ADP/ATP exchange reaction. The loss of radiolabeled ADP from Hsc70 in the presence of exogenous nucleotides followed first-order kinetics. In the presence of nucleotides, apocytochrome c induced a 2-fold increase in the rate of ADP release from Hsc70. Moreover, rate constants of the nucleotide exchange reaction measured in the absence and presence of apocytochrome c (0.16 and 0.34 min-1, respectively) closely matched the kcat values derived from ATP hydrolysis measurements (0.15 and 0.38 min-1, respectively). The results suggest that ADP release in a rate-limiting step in the Hsc70 ATPase reaction and that unfolded proteins stimulate ATP hydrolysis by accelerating the rate of ADP/ATP exchange.
Sadis
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Department of Molecular and Cell Biology, University of Connecticut, Storrs 06269-3044.
Hightower
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D000244:Adenosine Diphosphate / Q000378:metabolism*; D000251:Adenosine Triphosphatases / Q000187:drug effects / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism*; D001059:Apoproteins / Q000378:metabolism; D002352:Carrier Proteins / Q000187:drug effects / Q000378:metabolism*; D018833:Chaperonins; D003574:Cytochrome c Group / Q000378:metabolism; D045304:Cytochromes c; D050883:HSC70 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins; D006360:Heat-Shock Proteins / Q000187:drug effects / Q000378:metabolism; D007700:Kinetics; D011189:Potassium Chloride / Q000494:pharmacology; D017510:Protein Folding; D011506:Proteins / Q000187:drug effects / Q000378:metabolism*
D001059:Apoproteins; D002352:Carrier Proteins; D003574:Cytochrome c Group; D050883:HSC70 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins; D006360:Heat-Shock Proteins; D011506:Proteins; D000244:Adenosine Diphosphate; D011189:Potassium Chloride; D000255:Adenosine Triphosphate; D045304:Cytochromes c; D000251:Adenosine Triphosphatases; D018833:Chaperonins