cDNA cloning and expression of a gene for 3-ketoacyl-CoA thiolase in pumpkin cotyledons

Abstract

A cDNA clone for 3-ketoacyl-CoA thiolase (EC 2.3.1.16) was isolated from a lambda gt11 cDNA library constructed from the poly(A)+ RNA of etiolated pumpkin cotyledons. The cDNA insert contained 1682 nucleotides and encoded 461 amino acid residues. A study of the expression in vitro of the cDNA and analysis of the amino-terminal sequence of the protein indicated that pumpkin thiolase is synthesized as a precursor which has a cleavable amino-terminal presequence of 33 amino acids. The amino-terminal presequence was highly homologous to typical amino-terminal signals that target proteins to microbodies. Immunoblot analysis showed that the amount of thiolase increased markedly during germination but decreased dramatically during the light-inducible transition of microbodies from glyoxysomes to leaf peroxisomes. By contrast, the amount of mRNA increased temporarily during the early stage of germination. In senescing cotyledons, the levels of the thiolase mRNA and protein increased again with the reverse transition of microbodies from leaf peroxisomes to glyoxysomes, but the pattern of accumulation of the protein was slightly different from that of malate synthase. These results indicate that expression of the thiolase is regulated in a similar manner to that of other glyoxysomal enzymes, such as malate synthase and citrate synthase, during seed germination and post-germination growth. By contrast, during senescence, expression of the thiolase is regulated in a different manner from that of other glyoxysomal enzymes.

Molecular Characterization of an Arabidopsis Acyl-Coenzyme A Synthetase Localized on Glyoxysomal Membranes

“…1). It is important to note that the AtLACS6 is synthesized as larger precursor containing a cleavable aminoterminal presequence, as is the case for some other plant glyoxysomal proteins, such as malate dehydrogenase (Gietl, 1990;Kato et al, 1998), citrate synthase (Kato et al, 1995), thiolase (Preisig-Mü ller and Kindle, 1993; Kato et al, 1996b), and LACX (Hayashi et al, 1998). This further confirms the importance of the PTS2 signal for protein import in plant glyoxysomes.…”

“…B, The amino acid sequences represent the N terminus of AtLACS6 and other glyoxysomal proteins that are synthesized as larger precursors. PmLACX, pumpkin long-chain acylCoA oxidase (LACX; Hayashi et al, 1998); PmMDH, pumpkin malate dehydrogenase (Kato et al, 1998); PmTHI, pumpkin 3-keto-acyl-CoA thiolase (Kato et al, 1996b); PmCS, pumpkin citrate synthase (Kato et al, 1995). BOX I indicates the PTS2 consensus.…”

“…We have previously studied enzymes of ␤-oxidation and the glyoxylate cycle in pumpkin (Cucurbita sp. Kurokawa Amakuri) and Arabidopsis in detail (Mori and Nishimura, 1989;Kato et al, 1996bKato et al, , 1998Hayashi et al, 1998aHayashi et al, , 1998bHayashi et al, , 1999De Bellis et al, 1999Fukao et al, 2002). We extended our study to plant glyoxysomal ACSs.…”

ACX3, a Novel Medium-Chain Acyl-Coenzyme A Oxidase from Arabidopsis

“…Alignment of the N-terminal domains of Arabidopsis ACX3, Arabidopsis ACX2 (Hooks et al, 1999), pumpkin long-chain ACX (Hayashi et al, 1998a), Phalaenopsis sp. ACX (Do and Huang, 1997), pumpkin MDH (Kato et al, 1998), pumpkin CS (Kato et al, 1995), Arabidopsis PED1 (peroxisome defective) (Hayashi et al, 1998b), Arabidopsis PKT2 (peroxisomal thiolase) (Ferreira da Rocha et al, 1996), and pumpkin thiolase (Kato et al, 1996b) shows that these sequences share identical or conserved substitutions with the identified consensus sequence R-(I/Q/L)-X5-HL-X15-22-C (Fig. 2).…”

“…AtACX3, Arabidopsis ACX3; AtACX2, Arabidopsis ACX2 (Hooks et al, 1999); PumLACX, pumpkin long-chain ACX (Hayashi et al, 1998a); PhaACX, Phalaenopsis sp. ACX (Do and Huang, 1997); PumMDH, pumpkin glyoxysomal MDH (Kato et al, 1998); PumCS, pumpkin glyoxysomal CS (Kato et al, 1995); AtPED1, Arabidopsis thiolase (Hayashi et al, 1998b); AtPKT2, Arabidopsis thiolase (Ferreira da Rocha et al, 1996); PumTHI, pumpkin glyoxysomal thiolase (Kato et al, 1996b). Conserved amino acids, where five of the nine sequences are identical are shown in a black box.…”