Arabidopsis Cor15am is a chloroplast stromal protein that has cryoprotective activity and forms oligomers.

Many plants acquire increased freezing tolerance when they are exposed to nonfreezing temperatures of a certain duration. This process is known as cold acclimation and allows plants to protect themselves from freezing injury. A wide variety of polypeptides are induced during cold acclimation, among which is one encoded by COR15A in Arabidopsis (Arabidopsis thaliana). Previous studies showed that the COR15A gene encodes a small, plastid-targeted polypeptide that is processed to a mature form called Cor15am. In this study, we examined the biochemical properties and activities of Cor15am in more detail. We provide evidence that Cor15am localizes almost exclusively to the chloroplast stroma. In addition, the cold-regulated accumulation of Cor15am is affected by chloroplast functionality. Both gel-filtration chromatography and protein cross-linking reveal that Cor15am forms oligomers in the stroma of chloroplasts. Although Cor15am accumulates in response to low temperature, cold acclimation is not a prerequisite for oligomerization of Cor15am. Structural analysis suggests that Cor15am is composed of both ordered and random structures, and can stay soluble with small structural change after boiling and freeze-thaw treatments. Recombinant Cor15am exhibits in vitro cryoprotection of a freeze-labile enzyme, l-lactate dehydrogenase. Furthermore, Cor15am is capable of associating with l-lactate dehydrogenase in vitro and with potential stromal substrates in vivo. On the basis of these results, we propose that Arabidopsis Cor15am is a cryoprotective protein that forms oligomers in the chloroplast stroma, and that direct association of Cor15am with its substrates is part of its cryoprotective mechanism.

73-kDa molecular chaperone HSP73 is a direct target of antibiotic gentamicin.

Although gentamicin (GM) has been used widely as an antibiotic, the specific binding protein of the drug has not yet been understood sufficiently. Here we show that GM specifically associates with the 73-kDa molecular chaperone HSP73 and reduces its chaperone activity in vitro. In the present study, we investigated GM-specific binding proteins using a GM-affinity column and porcine kidney cytosol. After washing the column, only the 73-kDa protein was eluted from the column by the addition of 10 mm GM. None of the other proteins were found in the eluant. Upon immunoblotting, the protein was identical to HSP73. Upon CD spectrum analysis, the binding of GM to HSP73 resulted in a conformational change in the protein. Although HSP73 prevents aggregation of unfolded rhodanese in vitro, the chaperone activity of HSP73 was suppressed in the presence of GM. Using limited proteolysis of HSP73 by TPCK-trypsin, the GM binding site is a COOH-terminal for one third of the protein known to be a peptide-binding domain. During immunohistochemistry, HSP73 and GM were co-localized in enlarged lysosomes of rat kidneys with GM-induced acute tubular injury in vivo. Our results suggest that the specific association between HSP73 and GM may reduce the chaperone activity of HSP73 in vitro and/or in vivo, and this may have an interaction with GM toxicity in kidneys with GM-induced acute tubular injury.