Plant Physiol. (1993) 103: 1471-1472

Plant Cene Register Sequence of a Cloned Tomato Ubiquitin Conjugating Enzyme’

Steve Picton, Julie E. Gray, Alex Lowe, Sarah 1. Barton, and Don Grierson* University of Nottingham, Agricultural and Food Research Council Research Group in Plant Gene Regulation, Department of Physiology and Environmental Science, Sutton Bonington Campus, Loughborough, LEI2 5RD United Kingdom

Ubiquitin is a small, abundant protein that is seemingly Table 1. Characteristics of the clone ERT 17 present in a11 eukaryotic cells and that is covalently ligated to specific protein substrates via an ATP-dependent reaction. Organism: Lycopersicon esculentum Mill. cv Ailsa Craig. This ubiquitinous has been demonstrated to target proteins Source: for subsequent cellular degradation. Briefly, the process in- cDNA library in XZAP 11, constructed from poly(A+)mRNA of volves activation of ubiquitin, catalyzed by ubiquitin-activat- early breaker tomato fruit percarp. ing enzymes, transfer to a family of ubiquitin-camer/conju- Techniques of Sequencing: gating proteins with different substrate specificities (UBCs or Double-stranded miniprep plasmid DNA sequencing with Se- E~s),and, finally, ubiquitin-protein ligation by either direct quenase 11: synthetic oligonucleotides to the known sequence transfer of ubiquitin from E2 or mediated transfer utilizing used as primers; sequencing of both strands. ubiquitin-protein ligases (Hershko and Ciechanover, 1992; Characteristics of cDNA: Jentsch, 1992). The 5’ flanking region is 12 bp long. The 3’ untranslated region In this paper we report the sequence of a full-length tomato is 212 bp long and is followed by 18 bp of poly(A) tail. There is no obvious polyadenylation signal sequence. The sequence cDNA clone (LeE2,6.5).The cloned mRNA encodes a derived has 77.7% identity in a 395-bp overlap with an A. thaliana 148-amino acid sequence of 16.5 kD with an isoelectric point clone YAP 161T7 (Z176921, 71.2% identity in a 410-bp over- of 7.95. The peptide has a conserved region containing a lap with the yeast UBC5 gene (P15732),78.5% identity in a putative active Cys residue at position 85 that is observed in 247-bp overlap with a A. thaliana clone TAYO5O (Z18473), other E2 sequences (Sullivan and Vierstra, 1989; Jentsch et and 70.7% identity in a 396-bp overlap with the yeast UBC4 al., 1990) and is thought to be required for the thiol ester gene (P15731). formation with ubiquitin (van Nocker and Vierstra, 1991). In Structural Features of the Deduced Protein: addition, a further Cys residue is present at position 108. The Open reading frame of 148 amino acids, predicted molecular presence of two Cys residues and two ubiquitin thiol ester mass of protein 16.5 kD, isoelectric point 7.95. Comparisons species has been observed for both wheat and Arabidopsis with sequenced and deduced protein sequences using FASTA (Pearson, 1990) show strong homology with yeast UBC4 (78% E2s, suggesting that E2s may interact with more than a single identity, 95% similarity, P15731), and UBC5 (75% identity, ubiquitin simultaneously (Sullivan and Vierstra, 1989; Sulli- 93% similarity, P15732) and the Drosophila UBC4 (78%iden- van et al., 1990). tity, 89% similarity, P25867). There is also at least 33% iden- The cDNA clone ERT 17 was isolated as part of a research tity (or 53% similarity) with a further 15 ubiquitin conjugating program to identify genes expressed at the onset of tomato enzyme sequences from yeast (P06104, P21734, P23566, fruit ripening (Picton et al., 1993). ERT 17 mRNA is detected P14682, P28263, P29340), Drosophila (P25153),Arabidopsis during fruit development and increases to a peak after the (P25865),Triticum (P25866, P25868, P16577), and mamma- onset of ripening. It is present both in leaves and at an lian (P27924, P23567) and vira1 (P27949, P25869) sources. increased leve1 in senescing and mechanically wounded leaf The sequence shows strong homology (but is not identical) to the recently reported multigene family AtUBCB-12 (214989, tissue. Although ripening, wounding, and foliar senescence Z1499O,Z14991,214992, and Z14993). The sequence con- are a11 ethylene-mediated processes, ERT 17 mRNA accu- tains the E2 putative active Cys residue (van Nocker and mulation in fruit is not increased by ethylene treatment, and Vierstra, 1991) at position 85. Method of Identificatior?: Complete DNA sequence and deduced amino acid sequences This work was supported by a grant from the Agricultura1 and were compared with CenBank, DDJB, and EMBL data bases. Food Research Council. Function: * Corresponding author; fax 44-602-516334. Ubiquitin conjugating enzyme (LeE2165). Abbreviation: E2, ubiquitin-camer/conjugating protein.

1471 1472 Picton et al. Plant Physiol. Vol. 103, 1993 thus its interrelationship with ethylene evolution appears LITERATURE ClTED casual rather than causal. The terminal differentiation of Girod P-A, Carpenter TB, Van Nocker S, Sullivan ML, Vierstra these ripening, senescent, and wounded tissues suggests a RD (1993) Homologs of the essential ubiquitin conjugating en- possible role for LeE216.5enzyme in selective protein degra- zymes UBC1,4, and 5 in yeast are encoded by a multigene family in Arabidopsis thaliana. Plant J 3 545-552 dation that occurs during plant cell or organ senescence. Hershko A, Ciechanover A (1992) The ubiquitin system for protein The other three plant E2s reported (Sullivan and Vierstra, degradation. Annu Rev Biochem 61: 761-807 1989,1991) a11 show high homology with an E2 encoded by Jentsch S (1992) The ubiquitin-conjugation system. Annu Rev Genet the Saccharomyces cerevisiae DNA repair gene, RAD6. The 26 179-207 sequence reported here shows greatest homology with the Jentsch S, Seufert W, Sommer T, Rains H-A (1990) Ubiquitin- conjugating enzymes-nove1 regulators of eukaryotic cells. Trends yeast class I UBC4/UBC5 type E2 enzymes, implicated spe- Biochem Sci 15 195-198 cifically in the ubiquitination and breakdown of very short- Pearson WR (1990) Rapid and sensitive sequence comparison with lived and abnormal proteins (Jentsch, 1992) and is the first FASTAP and FASTA. Methods Enzymol183: 63-93 E2 of this class to be identified from tomato. Recently, a Picton S, Gray J, Barton S, AbuBakar U, Lowe A, Grierson D (1993) cDNA cloning and characterisation of nove1 ripening-re- multigene family of similar E2s from Arabidopsis (At UBC8- lated mRNAs with altered pattems of accumulation in the ripening 12) has been reported (Girod et al., 1993). inhibitor (rin) tomato ripening mutant. Plant Mo1 Biol23 193-207 Sullivan ML,'Callis J, Vierstra RD (1990) High performance liquid ACKNOWLEDGMENT chromatography resolution of ubiquitin pathway enzymes from . wheat germ. Plant Physiol94 710-716 Sequence analysis benefited from the use of the Science and Sullivan ML, Vierstra RD (1989) A ubiquitin carrier protein from Engineering Research Council SEQNET facility, Daresbury Labora- wheat germ is structurally and functionally similar to the yeast tory, DNA repair enzyme encoded by RAD6. Proc Natl Acad Sci USA U.K. 86 9861-9865 Received July 6, 1993; accepted August 24, 1993. Sullivan ML, Vierstra RD (1991) Cloning of a 16-kDa ubiquitin carrier protein from wheat and Arabidopsis thaliana. J Biol Chem Copyright Clearance Center: 0032-0889/93/103/1471/02. 266 23878-23885 The nucleotide sequence data reported in this paper has been sub- van Nocker S, Vierstra RD (1991) Cloning and characterization of mitted to the EMBL, GenBank, and DDBJ data bases under the a 20-kDa ubiquitin camer protein from wheat that catalyzes mul- accession number X73419. tiubiquitin chain formation in vitro. Biochemistry 88: 10297-10301