Glyoxysomal malate dehydrogenase in pumpkin: cloning of a cDNA and functional analysis of its presequence.
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 polypeptide 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 microbody 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 beta-glucuronidase and the N-terminal region of gMDH. Immunological and immunocytochemical studies revealed that the chimeric protein was imported into microbodies such as glyoxysomes 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.
Kato
A
A
Department of Cell Biology, National Institute for Basic Biology, Okazaki, Japan.
Takeda-Yoshikawa
Y
Y
Hayashi
M
M
Kondo
M
M
Hara-Nishimura
I
I
Nishimura
M
M
D000595:Amino Acid Sequence; D017360:Arabidopsis; D003001:Cloning, Molecular; D019662:Cucurbitaceae / Q000201:enzymology* / Q000648:ultrastructure; D018076:DNA, Complementary; D004792:Enzyme Precursors / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D008291:Malate Dehydrogenase / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D015388:Organelles / Q000201:enzymology* / Q000648:ultrastructure; D030821:Plants, Genetically Modified; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D012639:Seeds / Q000201:enzymology; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
D018076:DNA, Complementary; D004792:Enzyme Precursors; D011993:Recombinant Fusion Proteins; D008291:Malate Dehydrogenase