The ACC synthase TOE sequence is required for interaction with ETO1 family proteins and destabilization of target proteins

The Arabidopsis ETO1 protein is a negative regulator of ethylene biosynthesis. It specifically inhibits the enzyme activity of type 21-aminocyclopropane-1-carboxylate synthases (ACC synthases or ACS) and promotes their degradation by a proteasome-dependent pathway. To further understand the function of the ETO1 family in the plant kingdom, we cloned a cDNA of LeEOL1 (Lycopersicon esculentum ETO1-LIKE 1), an ETO1 homolog from tomato. LeEOL1 encodes a putative protein with domain architecture conserved in the Arabidopsis ETO1/EOL1/EOL2 proteins and in the predicted rice EOL proteins. LeEOL1 is expressed in leaf, stem, root, flower, and the full ripe stage of fruit, suggesting diverse regulatory roles in the development of tomato. Yeast two-hybrid analysis revealed specific interactions between LeEOL1 and type 2 ACC synthases. When the C-terminal 14 amino acids (TOE; target of ETO1) of LE-ACS3 specific to type 2 ACC synthases were fused to a type 1 ACS, LE-ACS2, at the corresponding position, it allowed LE-ACS2 to strongly interact with LeEOL1. A GFP-TOE LE-ACS3 fusion protein expressed in rice calli and in the roots of wild-type Arabidopsis showed reduced stability compared to native GFP. However, the fluorescence of GFP-TOE LE-ACS3 was comparable to that of the native GFP in Arabidopsis eto1-4 mutant. Furthermore, MG132 treatment significantly enhanced the fluorescence of GFP-TOE LE-ACS3 in the roots of wild-type Arabidopsis. These results suggest that the ETO1-family-mediated ACS protein degradation pathway is conserved in both monocots and dicots, and that TOE acts as a protein destabilization signal recognized by the ETO1 protein family.