Characterization and Physiological Function of Class I Low-Molecular-Mass, Heat-Shock Protein Complex in Soybean

Abstract

Examination of an ammonium sulfate-enriched fraction (70-1 00% saturation) of heat-shock proteins (HSPs) by nondenaturing polyacrylamide gel electrophoresis revealed the presence of a high molecular m a s complex (280 kD) in soybean (Glycine max) seedlings. This complex cross-reacted with antibodies raised against soybean class I low-molecular-mass (LMW) HSPs. Dissociation of the complex by denaturing polyacrylamide gel electrophoresis showed the complex to contain at least 15 polypeptides of the 15-to 18-kD class I L M W HSPs that could be detected by staining, radiolabeling, and western blotting. A similar LMW-HSP complex was observed in mung bean (Vigna radiafa L.; 295 kD), in pea (Pisum safivum 1.; 270 kD), and in rice (Oryza sativa 1.; 310 kD). l h e complex was stable under high salt conditions (250 mM KCI), and the integrity was not affected by 1 % Nonidet P-40 and 3 & n L RNase treatment. l h e size of the isolated HSP complex in vitro was conserved to 55°C; however, starting at 37.5"C, it changed to higher molecular forms in the presence of soluble proteins. l h e isolated HSP complex was able to protect up to 75% of the soluble proteins from heat denaturation in vitro.The induction of HSP synthesis in response to thermal stress occurs in a11 organisms that have been examined, ranging from bacteria to humans (Schlesinger et al., 1982;Vierling, 1991). Whereas the physiological functions of HSPs have not been clearly established, these proteins are well correlated with the acquisition of thermotolerance in a time-and temperature-dependent manner. Under HS conditions the expression of the LMW HSPs in soybean (Glycine max) is increased, yielding final concentrations of up to 1% of the total cellular proteins (Hsieh et al., 1992). Based on this correlation, it has been hypothesized that accumulation of HSPs is a n essential component of a process that prevents heat damage (Lin et al., 1984;Key et al., 1985;Kimple et al., 1990). Some HSPs become selectively localized in cellular organelles in a temperature-dependent fashion and relocate to the cytoplasm after a 28°C incubation period (Lin et al., 1984). Isolated mitochondria with associated HSPs are functional in oxidative phosphorylation under thermal stress, suggesting that association of HSPs with organelles provide protection from heat damage (Chou et al., 1989 (Jinn et al., 1993). In soybean, depletion of the 15-to 18-kD HSPs in the HSP-enriched fraction resulted in the loss of the thermostabilizing ability, and this ability was again restored when these HSPs were added. The protection provided by these HSP-enriched fractions is effective mainly for membrane-associated proteins (Jinn et al., 1993).The LMW HSPs of plants and a11 other eukaryotes have a conserved carboxyl-terminal region homologous to the a-crystallin of the eye lens at the amino acid sequence leve1 (Ingolia and Craig, 1982; Augusteyn and Koretz, 1987;Lindquist and Craig, 1988). The LMW HSPs in vertebrates (Arrigo and Welch, 1987;Merck et al., 1993), Drosopkila (Arrigo and Pauli, 1988), ye...

Expression of a gene encoding a 16.9-kDa heat-shock protein, Oshsp16.9, in Escherichia coli enhances thermotolerance

“…The plant class I LMM HSPs have been shown to form multimeric complexes in vivo (22)(23)(24), similar to the LMM HSPs found in animal systems, although the mammalian complexes were much larger in size (6,34). The isolated GST-Oshsp16.9 fusion protein can also form a multimeric complex (data not shown), as does the excised 16.9-kDa HSP in vitro (24).…”

“…Thermostabilization of soluble proteins can be demonstrated in vitro by an HSP-enriched fraction (20); such HSP-enriched fractions are exchangeable among soybean, mung bean (Vigna radiata L.), and rice (Oryza sativa L.) in providing protein thermostability (21). In soybean and pea (Pisum sativum L.), the class I and class II LMM HSPs form multimeric complexes under native conditions (22,23), and such complexes function as molecular chaperones in thermostabilization by interacting with soluble proteins in vitro after heating (22,23).…”

Differentially expressed genes during malting and correlation with malting quality phenotypes in barley (Hordeum vulgare L.)

“…These proteins were identiWed as thermostable components of malt and beer (Perrocheau et al 2005). Other genes that were up-regulated from germination to kilning were those for heat shock proteins, which function to protect enzymes during heat stress (Jinn et al 1995;Waters et al 1996).…”

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

“…In our previous results, expression of a plant LMM HSP, Oshp16.9, allowed E. coli cells to survive at high temperature (Yeh et al, 1997). Basically, the chaperone behavior of plant LMM HSPs is ATP independent, different from the ATP-dependent HSP70, and lacks the specificity for substrate proteins (Jinn et al, 1995;Lee et al, 1995). However, the function of plant LMM HSPs in vivo remains unclear.…”

“…Recent studies showed that most LMM HSPs required intact N-terminal domains to form these multimeric complexes (Leroux et al, 1997;Berengian et al, 1999;Lambert et al, 1999). Plant LMM HSPs generally form multimeric protein complexes ranging in size from 200 to 310 kD (Jinn et al, 1995;Lee et al, 1995;Yeh et al, 1995), as shown in rice (Oryza sativa) Oshsp16.9 and pea HSP18.1 consisting of 15 to 18 and 12 subunits, respectively (Jinn et al, 1995;Lee et al, 1995;Yeh et al, 1995). In contrast, LMM HSP complexes from mammalian sources vary considerably and exhibit a larger size (300-800 kD) than their plant counterparts (Arrigo and Landry, 1994).…”