Molecular characterization of a glyoxysomal citrate synthase that is synthesized as a precursor of higher molecular mass in pumpkin.
A cDNA clone for glyoxysomal citrate synthase (gCS) was isolated from a lambda gt11 cDNA library prepared from etiolated pumpkin cotyledons. The cDNA of 1989 bp consisted of a 1548 bp open reading frame that encoded 516 amino acid residues. The deduced amino acid sequence of gCS did not have a typical peroxisomal targeting signal at its carboxyl terminal. A study of expression in vitro of the cDNA and an analysis of the amino-terminal sequence of the citrate synthase indicated that gCS is synthesized as a larger precursor that has a cleavable amino-terminal presequence of 43 amino acids. The predicted amino-terminal sequence of pumpkin gCS was highly homologous to those of other microbody enzymes, such as 3-ketoacyl-CoA thiolase of rat and malate dehydrogenase of watermelon that are also synthesized as precursors of higher molecular mass. Immunoblot analysis showed that the level of gCS protein increased markedly during germination and decreased rapidly during the light-induced transition of microbodies from glyoxysomes to leaf peroxisomes. By contrast, the level of mRNA for gCS reached a maximum earlier than that of the protein and declined even in darkness. The level of the mRNA was low during the microbody transition. These results indicate that the accumulation of the gCS protein does not correspond to that of the mRNA and that degradation of gCS is induced during the microbody transition, suggesting that post-transcriptional regulation plays an important role in the microbody transition.
Kato
A
A
Department of Cell Biology, National Institute for Basic Biology, Okazaki, Japan.
Hayashi
M
M
Mori
H
H
Nishimura
M
M
D000818:Animals; D001483:Base Sequence; D002950:Citrate (si)-Synthase / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D003001:Cloning, Molecular; D018548:Cotyledon / Q000201:enzymology / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D018506:Gene Expression Regulation, Plant; D017343:Genes, Plant / Q000235:genetics*; D008027:Light; D008830:Microbodies / Q000201:enzymology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011498:Protein Precursors / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D012333:RNA, Messenger / Q000096:biosynthesis; D018749:RNA, Plant / Q000096:biosynthesis; D051381:Rats; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D014675:Vegetables / Q000201:enzymology / Q000235:genetics*
D018076:DNA, Complementary; D011498:Protein Precursors; D012333:RNA, Messenger; D018749:RNA, Plant; D002950:Citrate (si)-Synthase