Biochemical analysis of a cytosolic small heat shock protein, NtHSP18.3, from Nicotiana tabacum.
Small heat shock proteins (sHSPs) are widely distributed, and their function and diversity of structure have been much studied in the field of molecular chaperones. In plants, which frequently have to cope with hostile environments, sHSPs are much more abundant and diverse than in other forms of life. In response to high temperature stress, sHSPs of more than twenty kinds can make up more than 1% of soluble plant proteins. We isolated a genomic clone, NtHSP18.3, from Nicotiana tabacum that encodes the complete open reading frame of a cytosolic class I small heat shock protein. To investigate the function of NtHSP18.3 in vitro, it was overproduced in Escherichia coli and purified. The purified NtHSP18.3 had typical molecular chaperone activity as it protected citrate synthase and luciferase from high temperature-induced aggregation. When E. coli celluar proteins were incubated with NtHSP18.3, a large proportion of the proteins remained soluble at temperatures as high as 70 degrees . Native gel analysis suggested that NtHSP18.3 is a dodecameric oligomer as the form present and showing molecular chaperone activity at the condition tested. Binding of bis-ANS to the oligomers of NtHSP18.3 indicated that exposure of their hydrophobic surfaces increased as the temperature was raised. Taken together, our data suggested that NtHSP18.3 is a molecular chaperone that functions as a dodecameric complex and possibly in a temperature-induced manner.
Yu
Ji Hee
JH
Institute of Molecular Biology and Genetics, Seoul National University, Seoul 151-742, Korea.
Kim
Keun Pill
KP
Park
Soo Min
SM
Hong
Choo Bong
CB
D000817:Anilino Naphthalenesulfonates / Q000378:metabolism; D002950:Citrate (si)-Synthase / Q000187:drug effects; D003001:Cloning, Molecular; D003600:Cytosol / Q000737:chemistry; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000187:drug effects; D006360:Heat-Shock Proteins / Q000302:isolation & purification / Q000502:physiology*; D006358:Hot Temperature; D008156:Luciferases / Q000187:drug effects; D018832:Molecular Chaperones / Q000502:physiology; D011489:Protein Denaturation / Q000187:drug effects; D014026:Nicotiana / Q000737:chemistry* / Q000235:genetics
D000817:Anilino Naphthalenesulfonates; D029968:Escherichia coli Proteins; D006360:Heat-Shock Proteins; D018832:Molecular Chaperones; C035763:5,5'-bis(8-(phenylamino)-1-naphthalenesulfonate); D008156:Luciferases; D002950:Citrate (si)-Synthase