Plant glyoxysomal but not mitochondrial malate dehydrogenase can fold without chaperone assistance

Abstract

Glyoxysomal (gMDH) and mitochondrial malate dehydrogenase (mMDH) from watermelon are synthesized as higher molecular weight precursor proteins. By overexpressing the precursor forms as well as the mature subunits with a histidine arm at the carboxy-terminus, it has been possible to purify relatively large amounts especially of the glyoxysomal precursor protein for studies of their refolding capacities after denaturation with guanidinium hydrochloride, heat or low pH. Glyoxysomal MDH and its precursor is capable of its spontaneous folding over a wide range of temperature conditions. Refolding can be enhanced by inclusion of BSA and ATP as stabilisers in the folding buffer. The N-terminal transit peptide of gMDH facilitates folding, but does not function as an intramolecular chaperon. Chemically denatured mitochondrial MDH requires chaperones for refolding. GroEL/GroES/ATP increase the yield and rate of watermelon mMDH folding dramatically while GroEL and Mg-ATP alone are not sufficient to provide folding assistance similar to the results with hydrophobic mammalian mMDH. The watermelon glyoxysomal MDH interacts with GroEL-like hydrophilic mammalian cytoplasmic MDH, a binding which has to be released by Mg-ATP before spontaneous folding can ensue. Interestingly, watermelon mMDH exhibited a much higher heat stability than gMDH or mammalian mMDH in the presence of BSA/ATP as well as GroEL/GroES/ATP. The differences between glyoxysomal and chaperone-assisted mitochondrial folding patterns are discussed.