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- Title
Glyoxysomal Malate Dehydrogenase in Pumpkin: Cloning of a cDNA and Functional Analysis of Its Presequence.
- Authors
Kato, Akira; Takeda-Yoshikawa, Yoko; Hayashi, Makoto; Kondo, Maki; Hara-Nishimura, Ikuko; Nishimura, Mikio
- Abstract
Glyoxysomal malate dehydrogenase (gMDH) is an enzyme of the glyoxylate cycle that participates in degradation of storage oil. We have cloned a cDNA for gMDH from etiolated pumpkin cotyledons that encodes a polypep-tide consisting of 356 amino acid residues. The nucleotide and N-terminal amino acid sequences revealed that gMDH is synthesized as a precursor with an N-terminal extrapeptide. The N-terminal presequence of 36 amino acid residues contains two regions homologous to those of other micro-body proteins, which are also synthesized as large precursors. To investigate the functions of the N-terminal presequence of gMDH, we generated transgenic Arabidopsis that expressed a chimeric protein consisting of rβ-glucuroni-dase and the N-terminal region of gMDH. Immunologi-cal and immunocytochemical studies revealed that the chimeric protein was imported into microbodies such as gly-oxysomes and leaf peroxisomes and was then subsequently processed. Site-directed mutagenesis studies showed that the conserved amino acids in the N-terminal presequence, Arg-10 and His-17, function as recognition sites for the targeting to plant microbodies, and Cys-36 in the presequence is responsible for its processing. These results correspond to those from the analyses of glyoxysomal citrate synthase (gCS), which was also synthesized as a large precursor, suggesting that common mechanisms that can recognize the targeting or the processing of gMDH and gCS function in higher plant cells.
- Subjects
PUMPKINS; MALATE dehydrogenase; PLANT enzymes; MOLECULAR cloning; ANTISENSE DNA; AMINO acid sequence; FUNCTIONAL analysis; GENE expression in plants
- Publication
Plant & Cell Physiology, 1998, Vol 39, Issue 2, p186
- ISSN
0032-0781
- Publication type
Article
- DOI
10.1093/oxfordjournals.pcp.a029356