Nucleotide sequence of a cDNA encoding a tobacco thioredoxin

Abstract

Thioredoxins are small (about 12 kDa) proteins which reduce disulphide bridges on target enzymes [6]. Protein or nucleic acid sequences have been established for different thioredoxins from vertebrates [7, 8, 15, 16], algae [3, 4, 12] and bacteria [6, 10]. The active site shows a highly conserved sequence -C-G-P-C-. Recently a larger consensus thioredoxin signature (AT)-x-W-C(AG)-(PH)-C has been defined in the PROSITE database [ 1 ]. It matches all thioredoxins but no other protein sequences. In higher plants a spinach f-type cDNA [9], an m-type cDNA [ 14] and an m-type protein [ 11 ] have been sequenced. The f-type has a signal peptide typical of chloroplast targeting. Both enzymes are encoded in the nucleus, but are localized and active in the chloroplast in their mature form. M-type activates NADP-malate dehydrogenase wild f-type activates enzymes of the photosynthetic carbon cycle. In addition, cytosolic thioredoxin activities have been characterized in spinach. The proteins have been purified but not yet sequenced [5]. We have isolated a complete or nearly complete clone from a cDNA library from Nicotiana tabacum cells cultivated in vitro. This 699 nucleotide insert hybridizes on northern blots with a 0.9 kb mRNA. It contains an ORF of 378 bp (Fig. 1). The 3' untranslated region is 242bp long, including 16bp of poly(A) ÷ tail. Two AT-rich regions could serve as polyadenylation signals. The ORF codes for a protein of 126 amino acids. At positions 43-49 it contains a sequence which perfectly matches the thioredoxin signature. In addition, the protein sequence shows 40-50 ~o homologies with Chlamydomonas Chl assumed to be a cytoplasmic protein [4] and vertebrate thioredoxins using FASTA [13]. In contrast, the homology with m and f spinach chloroplast thioredoxins is only 30~o. Figure2 presents a multiple alignment [2] with some of the published thioredoxin sequences sorted by decreasing homology with the tobacco sequence. Chlamydomonas [3, 4], rabbit [7], Anacystis [ 12], Corynebacterium [ 10] and m-type spinach 2 [ 11] sequences were obtained by protein sequencing. Human [16], rat [15], chicken [8], Escherichia coli [6], f-type [9] and m-type 1 [14] spinach sequences are derived from cDNA sequencing, showing the presence of a large signal peptide in the spinach thioredoxins. The tobacco sequence matches the mature protein sequences and has no signal peptide, suggesting that the protein encoded by the clone we have isolated is cytoplasmic.