Functional Regions of Rice Heat Shock Protein, Oshsp16.9, Required for Conferring Thermotolerance inEscherichia coli 

Abstract

Rice (Oryza sativa) class I low-molecular mass (LMM) heat shock protein (HSP), Oshsp16.9, has been shown to be able to confer thermotolerance in Escherichia coli. To define the regions for this intriguing property, deletion mutants of this hsp have been constructed and overexpressed in E. coli XL1-blue cells after isopropyl ␤-d-thioglactopyranoside induction. The deletion of amino acid residues 30 through 36 (PATSDND) in the N-terminal domain or 73 through 78 (EEGNVL) in the consensus II domain of Oshsp16.9 led to the loss of chaperone activities and also rendered the E. coli incapable of surviving at 47.5°C. To further investigate the function of these two domains, we determined the light scattering changes of Oshsp16.9 mutant proteins at 320 nm under heat treatment either by themselves or in the presence of a thermosensitive enzyme, citrate synthase. It was observed that regions of amino acid residues 30 through 36 and 73 through 78 were responsible for stability of Oshsp16.9 and its interactions with other unfolded protein substrates, such as citrate synthase. Studies of two-point mutants of Oshsp16.9, GST-N74E73K and GST-N74E74K, indicate that amino acid residues 73 and 74 are an important part of the substrate-binding site of Oshsp16.9. Non-denaturing gel analysis of purified Oshsp16.9 revealed that oligomerization of Oshsp16.9 was necessary but not sufficient for its chaperone activity.Both eukaryotes and prokaryotes respond to high temperatures by synthesizing heat shock proteins (HSPs) when environmental temperatures were elevated 5°C to 10°C above normal growth temperatures (Parsell and Lindquist, 1993). The synthesis of HSPs occurs rapidly, whereas the expression of normally active genes is substantially repressed in responding to the onset of heat shock stress. Accumulation of HSPs is closely related to tolerance of extreme temperatures. HSPs are grouped into families based on sequence homology. Low-molecular mass (LMM) HSPs or small HSPs (sHSPs), ranging in size from 15 to 30 kD, are more abundant in higher plants than in other organisms.The conserved amino acid sequence within divergent LMM HSPs is generally restricted to the C-terminal region, which is termed "␣-crystallin domain" due to homologies to the ␣-crystallin of vertebrate eye lens. This domain can be further divided into two specific regions, consensus I and consensus II, separated by a varying length hydrophilic linker sequence (Yeh et al., 1994; Waters et al., 1996). Consensus I is highly conserved among all eukaryotic LMM HSPs discovered so far (Yeh et al., 1994). Some studies on ␣-crystallin and LMM HSPs have shown that this conserved ␣-crystallin domain is important for their distinct oligomerization as well as for chaperone activity (Merck et al., 1993;Andley et al., 1996;Lindner et al., 1998; Young et al., 1999).The term "molecular chaperone" is usually applied to describe the function of HSPs because they bind to and stabilize the unstable conformers of proteins and facilitate correct fate of substrate proteins in vivo (Parsell an...

Retarded germination of Nicotiana tabacum seeds following insertion of exogenous DNA mimics the seed persistent behavior

“…Interestingly, spot 1324 identified as 16.9 kDa class I heat shock protein 1-like was only detected in mutant seeds and not in WT. In rice, Oshsp16.9 gene is expressed during stress responses and transgenic plants have shown tolerance to salt, cold, heat and dehydration stresses [ 88 , 89 ]. As observed with other stress response proteins which increase in transgenic lines in parallel with storage proteins, it is possible that these proteins play a role in keeping proteins in a folding-competent state during seed desiccation and in preventing them from irreversible aggregation until ATP-dependent chaperones (such as Hsp70 and Hsp60 GroE) restore the refolding of denatured proteins to native physiological conditions [ 90 ].…”