NtHSP70-2 and NtHSP17.6 from Nicotiana tabacum Could Function Together in Chaperone Network

Abstract

Heat shock protein 70 (HSP70) is a highly conserved protein family that shows molecular chaperone activity in protecting proteins from aggregation and folding of polypeptides. Small heat shock proteins (sHSPs) that are abundant in plants under abiotic stress conditions are also known to function as molecular chaperones, however, active function of sHSPs in refolding their substrates has not been detected in plants. Thus, possible composite function of sHSPs with other molecular chaperones has been suggested and, in many cases, proven. NtHSP70-2, a HSP70 from Nicotiana tabacum, was overexpressed and purified from Escherichia coli. Firstly, purified NtHSP70-2 was examined for its chaperone activity in vitro and was confirmed as a typical and strong molecular chaperone. Then composite function of NtHSP70-2 and NtHSP17.6, a cytosolic sHSP from N. tabacum, was tested for the activity to refold firefly luciferase in vitro. Luciferase refolding assay showed that luciferase associated with NtHSP17.6 could be reactivated by the components of rabbit reticulocyte lysate and the activity of reticulocyte lysate was proportional to the concentration of NtHSP17.6. NtHSP70-2 could not replace the reticulocyte lysate but dramatically enhanced refolding of luciferase. These data suggest that NtHSP70-2 and NtHSP17.6 function together to refold heat-denatured substrates with the help of other chaperones.