Plant Molecular Biology 18: 1023-1025, 1992. © 1992 Kluwer Academic Publishers. Printed in Belgium.

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Sequence

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Isolation, characterization and nucleotide sequence of a full-length pea cDNA encoding thioredoxin-f

LoYc Lepiniec, Michael Hodges*, Pierre Gadal and Claude Cretin Physiologie Vdg~tale MolOculaire, Bat. 430, URA CNRS DI128, Universitd Paris-Sud, 9I 405 Orsay Cedex, France (* author for correspondence)

Received 7 January 1992; accepted 14 January 1992

Key words: thioredoxin-f, light regulation, pea, structure/function relations

Thioredoxins are ubiquitous, low-molecular- weight (12 kDa) and heat-stable proteins. They function via a reversible disulphide/dithiol reac- tion, and serve as redox carriers in a wide vari- ety of reactions [2]. In photosynthetic organisms, light regulates the activity of several chloroplast enzymes through the ferredoxin/thioredoxin sys- tem [3, 9]. Protein or nucleic acid sequences have been determined for different thioredoxins from prokaryotes and eukaryotes [4, 6, 13]. In plants three types of thioredoxins have been identified [ 14]. Thioredoxin-h is cytosolic and is involved in heterotrophic processes [7, 10]. The fand m types are encoded in the nucleus but are located in the chloroplast in their mature form. Thioredoxin-m preferentially activates NADP-MDH whereas thioredoxin-f activates specifically fructose 1,6- bisphosphatase [2]. Only one thioredoxin-f se- quence, namely the spinach leaf thioredoxin, has been published so far [12]. This paper reports a second sequence of thioredoxin-f.

A full-length cDNA of Pisum sativum, LTfP 1, has been isolated from a ,~-ZAP cDNA library of green pea leaves (Fig. 1). It consists of 804 nucleotides coding for 182 amino acids. When

compared to the spinach sequence, the pea DNA features a region coding for a putative leader pep- tide. The deduced sequence coding for the mature protein exhibits a high degree of identity both at the nucleotide (70 ~o) and amino acid (84 ~o) level.

The putative transit peptide has the character- istics of other peptides associated with proteins that have to be imported and processed in the chloroplast, with 20 serines and 8 threonines out of 73 residues [8]. It is however interesting to observe that this peptide is very different from the one of spinach. It has already been shown that the transit peptide of chloroplastic proteins can differ among species, while the sequences of the mature proteins are highly similar. This is the case for the transit peptide of the malic enzyme of maize compared to the one of Flaveria [1 ].

In pea thioredoxin-f, the highly conserved thioredoxin active site WCGPC is present. It is especially interesting to observe that the amino acid sequence MFT (residues 101-103, adjacent to the active site) found in the two f-type thiore- doxins is different from all the other thioredoxins. Other stretches which make thioredoxin-f struc- turally close to animal thioredoxins or thiore-

The nucleotide sequence data reported will appear in the EMBL, GenBank and DDBJ Nucleotide Sequence Databases under the accession number X63537.

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1 M A L N L C T S P K W I

i CTTCCCCACATCAATGGCTCTAAATCTCTC~CCTCCCCTAAATGC~TCG

13 G T T V F D S A S S S K P S L A S

51 GCACCACCGTCTTCGATAGTGCTTCTTCATCGAAGCCATCACTTGCTTCT

30 S F S T T S F S S S I L C S K R V

i01 TCTTTTTCTACTACTTCATTTTCTTCTTCCATTTTATGTTCC-C~GAGGGT

47 G L Q R L S L R R S I S V S V R

151 T GGTCTACAGAGGTTAAGCTTGAGGAGGAGTATCAGTGTCAGTGTAAGAT

63 S S L E T A G P T V T V* G K V T E

201 C CAGCTTCroAAACT GCGGGACCCACGGTGACGGTAGCrslAAGTAACCGAG

80 V N K D T F W P I V N A A G D K T

251 GTCP&CAAGC~TACGT TTTGGCCCATCGT TAATGCCGCCGGTGATAAAAC

97 V V L D M F T K W C G P C K V I

301 CGTCGTCCTCGATATGTTCACCAAATGGTGTGGTCCTTGCAAAGTGATAG

113 A P L Y E E L S Q K Y L D V V F L

351 CTCCATTATACGAAGAAT TATCTCAC~GTATT TGGATGTTGI~C TT TCTA

130 K L D C N 0 D N K S L A K E L G I

401 AAGCT TGAT TGCAACCAAGATAACAAGT CC TTGCCAAAAGAGC TAGGAhT

147 K V V P T F K I L K D N K I V K

451 TAAGGTGGTTCCCACTTT TAAAhTTCTGAAAGACAAC_~AGATTGTAAAAG

163 E V T G A K F D D L V A A I D T V 501 AAGTAACTGGTGC TAAATT TGATGATT TGGTCGCTGCCATTGACACTGTT

180 R S S

551 CGGTCTAGTTAGACAGTAT TATAACTAT TC TC TT TCTATGTAAAAATATC 601 ATGTTTGATACCCTCTGTAAGTATTTCTACTGTAATTGTGAGCTTGCTGA 651 GGG@hAGATGAC43AAATATCATAATGT TGTTGTAAGCTGTAATTGAT TGT 701 T CCCTATGTATA~GT TGCTCAAAATAATCTGT TACCAACTTGAT 751 CATGGACTATCTATA~AGGTGGC TCAGATTGCT ~ 801 A%AA

Fig. 1. Nucleotide sequence of LTfP 1 full length cDNA. The single letter amino acid code is shown above the middle base of each codon. The active site and other interesting regions are underlined. The asterisk indicates the putative end of transit peptide obtained by comparison with the mature spinach thioredoxin-f [ 11 ].

doxin-h are conserved, for example the VVFL- - D motif (residues 126-132) [6].

Our data confirm that thioredoxin-f has struc- tural features which sets it apart from all the other known thioredoxins and previous observations suggesting a phylogenetic tree where thioredoxin- f is closer to animal thioredoxins [2, 12].

Finally, a stretch of three amino acids seems to be typical for f-type thioredoxin, namely cysteine 60, asparagine 61 and glutamine 62 (numbering based on the Escherichia coli sequence, residue 133-135 in our sequence). Modification of cys- teine 60 changes the properties of the protein

(P. Sch/irmann, personal communication) and the importance of asparagine 61 in the specificity of thioredoxin-f for FBPase has been previously investigated by site-directed mutagenesis [5].

This work should permit to study structure/ function relationships in in vitro reconstituted sys- tems [5, 11].

Acknowledgements

We would like to acknowledge Drs P. Decottig- hies, J.P. Jacquot, and M. Miginiac-Maslow for critical reading of the manuscript and helpful dis- cussions.

References

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4. Decottignies P, Schmitter JM, Dutka S, Jacquot JP, Miginiac-Maslow M: Characterization and primary structure of a second thioredoxin from the green alga, Chlamydomonas reinhardtii. Eur J Biochem 198:505-512 (1991).

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Identification of an NADP/thioredoxin system in Chlamy- domonas reinhardtii. Planta, in press.

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12. Kamo M, Tsugita A, Wiessner C, Wedel N, Bartling D, Herrmann RG, Aguilar F, Gardet-Salvi L, Schurmann P: Primary structure of spinach-chloroplast thioredoxin f,

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protein sequencing and analysis of complete cDNA clones for spinach-chloroplast thioredoxin f. Eur J Biochem 182: 315-322 (1989).

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