Molecular chaperone activity of tomato (Lycopersicon esculentum) endoplasmic reticulum-located small heat shock protein.
The gene encoding the small heat shock protein (sHSP), LeHSP21.5, has been previously cloned from tomato (GenBank accession no. AB026983). The deduced amino acid sequence of this tomato sHSP was most similar to that of other endoplasmic reticulum (ER)-localized sHSPs (ER-sHSP) and can be predicted to target the ER. We examined whether the gene product of LeHSP21.5 (probable ER-sHSP) can act as molecular chaperone. For functional analysis, LeHSP21.5 protein was expressed in Escherichia coli as His(6)-tagged protein in the C-terminal and purified. We confirmed that ER-sHSP could provide thermal protection of soluble proteins in vitro. We compared the thermal stability of E. coli strain BL21 (DE3) transformed with pET-ER-sHSP with the control E. coli strain BL21(DE3) transformed with only the pET vector under heat shock and IPTG-induced conditions. Most of the protein extracts from E. coli cells expressing ER-sHSP were protected from heat-induced denaturation, whereas extracts from cells not expressing ER-sHSP were very heat-sensitive under these conditions. A similar protective effect was observed when purified ER-sHSP was added to an E. coli cell extract. ER-sHSP prevented the thermal aggregation and inactivation of citrate synthase. These collective findings indicate that ER-sHSP can function as a molecular chaperone in vitro.
Mamedov
Tarlan G
TG
Tropical Agriculture Research Front, Japan International Research Center for Agricultural Sciences (JIRCAS), 1091-1 Maezato Kawarabaru, Ishigaki, Okinawa, 907-0002, Japan. tmammedov2@unlnotes.unl.edu
Shono
Mariko
M
D000595:Amino Acid Sequence; D002950:Citrate (si)-Synthase / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000378:metabolism; D050888:Heat-Shock Proteins, Small / Q000378:metabolism*; D006358:Hot Temperature; D018551:Solanum lycopersicum / Q000378:metabolism*; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000302:isolation & purification / Q000378:metabolism*; D011489:Protein Denaturation; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment
D029968:Escherichia coli Proteins; D050888:Heat-Shock Proteins, Small; D010940:Plant Proteins; D011994:Recombinant Proteins; D002950:Citrate (si)-Synthase