Temperature interactions of the molecular chaperone Hsc70 from the eurythermal marine goby Gillichthys mirabilis.
Molecular chaperones participate in many aspects of protein biogenesis. Mechanistically, they recognize and bind to non-native proteins, prevent the aggregation of unfolded proteins and also, in some cases, facilitate refolding. Although a great deal is known about the cellular function of molecular chaperones in general, very little is known about the effect of temperature on molecular chaperones in non-model organisms, particularly in ectotherms that fold proteins under variable-temperature conditions in nature. To address this issue, we studied the temperature interactions of a major cytosolic molecular chaperone, Hsc70, from the eurythermal marine goby Gillichthys mirabilis. Using in vitro assays, we measured the intrinsic activity, unfolded-protein-stimulated activity, temperature sensitivity and heat stability of the ATPase activity of native Hsc70 purified from G. mirabilis white muscle. Similar to other chaperones in the 70kDa heat-shock protein family, G. mirabilis Hsc70 exhibited a low intrinsic ATPase activity that was stimulated in vitro by the addition of unfolded protein. Across the environmentally relevant temperature range (10-35 degrees C), the ATPase activity of G. mirabilis Hsc70 displayed differential thermal sensitivity, with the greatest sensitivity occurring between 10 and 15 degrees C and the least sensitivity between 15 and 25 degrees C. In addition, the activity of Hsc70 was not significantly different between the unstimulated and unfolded-protein-stimulated treatments, suggesting that the ATPase activity and the peptide-binding domain of Hsc70 have similar thermal sensitivities in vitro. Finally, the thermal stability of Hsc70 ATPase activity greatly exceeded environmental temperatures for G. mirabilis, with activity up to 62.5 degrees C. Overall, the biochemical characterization of the ATPase activity suggests that, although Hsc70 is not an extraordinarily thermally stable protein, it is capable of protein chaperoning cycles even at the extremes of environmental temperatures encountered by G. mirabilis in nature.
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Department of Biology, Arizona State University, Tempe, AZ 85287-1501, USA.
Hofmann
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D000251:Adenosine Triphosphatases / Q000378:metabolism; D000818:Animals; D004355:Drug Stability; D005399:Fishes / Q000378:metabolism*; D050883:HSC70 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D006358:Hot Temperature; D008156:Luciferases / Q000378:metabolism; D013696:Temperature
D050883:HSC70 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins; D008156:Luciferases; D000251:Adenosine Triphosphatases