Sequence of a cloned tomato ubiquitin conjugating enzyme.

Abstract

Ubiquitin is a small, abundant protein that is seemingly present in a11 eukaryotic cells and that is covalently ligated to specific protein substrates via an ATP-dependent reaction. This ubiquitinous has been demonstrated to target proteins for subsequent cellular degradation. Briefly, the process involves activation of ubiquitin, catalyzed by ubiquitin-activating enzymes, transfer to a family of ubiquitin-camer/conjugating proteins with different substrate specificities (UBCs or E~s) , and, finally, ubiquitin-protein ligation by either direct transfer of ubiquitin from E2 or mediated transfer utilizing ubiquitin-protein ligases (Hershko and Ciechanover, 1992; Jentsch, 1992). In this paper we report the sequence of a full-length tomato cDNA clone (LeE2,6.5). The cloned mRNA encodes a derived 148-amino acid sequence of 16.5 kD with an isoelectric point of 7.95. The peptide has a conserved region containing a putative active Cys residue at position 85 that is observed in other E2 sequences (Sullivan and Vierstra, 1989; Jentsch et al., 1990) and is thought to be required for the thiol ester formation with ubiquitin (van Nocker and Vierstra, 1991). In addition, a further Cys residue is present at position 108. The presence of two Cys residues and two ubiquitin thiol ester species has been observed for both wheat and Arabidopsis E2s, suggesting that E2s may interact with more than a single ubiquitin simultaneously (Sullivan and Vierstra, 1989; Sullivan et al., 1990). The cDNA clone ERT 17 was isolated as part of a research program to identify genes expressed at the onset of tomato fruit ripening (Picton et al., 1993). ERT 17 mRNA is detected during fruit development and increases to a peak after the onset of ripening. It is present both in leaves and at an increased leve1 in senescing and mechanically wounded leaf tissue. Although ripening, wounding, and foliar senescence are a11 ethylene-mediated processes, ERT 17 mRNA accumulation in fruit is not increased by ethylene treatment, and