Glyoxysomal Malate Dehydrogenase in Pumpkin: Cloning of a cDNA and Functional Analysis of Its Presequence

Glyoxysomal malate dehydrogenase (gMDH) is an enzyme of theglyoxylate cycle that participates in degradation of storageoil. We have cloned a cDNA for gMDH from etiolated pumpkin cotyledonsthat encodes a polypep-tide consisting of 356 amino acid residues.The nucleotide and N-terminal amino acid sequences revealedthat gMDH is synthesized as a precursor with an N-terminal extrapeptide.The N-terminal presequence of 36 amino acid residues containstwo regions homologous to those of other micro-body proteins,which are also synthesized as large precursors. To investigatethe functions of the N-terminal presequence of gMDH, we generatedtransgenic Arabidopsis that expressed a chimeric protein consistingof rß-glucuroni-dase and the N-terminal region ofgMDH. Immunologi-cal and immunocytochemical studies revealedthat the chimeric protein was imported into microbodies suchas gly-oxysomes and leaf peroxisomes and was then subsequentlyprocessed. Site-directed mutagenesis studies showed that theconserved amino acids in the N-terminal presequence, Arg-10and His-17, function as recognition sites for the targetingto plant microbodies, and Cys-36 in the presequence is responsiblefor its processing. These results correspond to those from theanalyses of glyoxysomal citrate synthase (gCS), which was alsosynthesized as a large precursor, suggesting that common mechanismsthat can recognize the targeting or the processing of gMDH andgCS function in higher plant cells. (Received July 10, 1997; Accepted November 22, 1997)