Cleavage of the Carboxyl-Terminus of LEACS2, a Tomato 1-Aminocyclopropane-1-Carboxylic Acid Synthase Isomer, by a 64-kDa Tomato Metalloprotease Produces a Truncated but Active Enzyme

Abstract: 1‐Aminocyclopropane‐1‐carboxylic acid (ACC) synthase (ACS) is the principal enzyme in phytohormone ethylene biosynthesis. Previous studies have shown that the hypervariable C‐terminus of ACS is proteolytically processed in vivo. However, the protease responsible for this has not yet been identified. In the present study, we investigated the processing of the 55‐kDa full‐length tomato ACS (LeACS2) into 52‐, 50‐ and 49‐kDa truncated isoforms in ripening tomato (Lycopersicon esculentum Mill. cv. Cooperation 903) fruit using the sodium dodecyl sulfate‐boiling method. Meanwhile, an LeACS2‐processing protease was purified via multi‐step column chromatography from tomato fruit. Subsequent biochemical analysis of the 64‐kDa purified protease revealed that it is a metalloprotease active at multiple cleavage sites within the hypervariable C‐terminus of LeACS2. N‐terminal sequencing and matrix‐assisted laser desorption/ ionization time‐of‐flight analysis indicated that the LeACS2‐processing metalloprotease cleaves at the C‐terminal sites Lys⁴³⁸, Glu⁴⁴⁷, Lys⁴⁴⁸, Asn⁴⁵⁶, Ser⁴⁶⁰, Ser⁴⁶², Lys⁴⁶³, and Leu⁴⁷⁴, but does not cleave the N‐terminus of LeACS2. Four C‐terminus‐deleted (26—50 amino acids) LeACS2 fusion proteins were overproduced and subjected to proteolysis by this metalloprotease to identify the multiple cleavage sites located on the N‐terminal side of the phosphorylation site Ser⁴⁶⁰. The results indisputably confirmed the presence of cleavage sites within the region between the α‐helix domain (H14) and Ser⁴⁶⁰ for this metalloprotease. Furthermore, the resulting C‐terminally truncated LeACS2 isoforms were active enzymatically. Because this protease could produce LeACS2 isoforms in vitro similar to those detected in vivo, it is proposed that this metalloprotease may be involved in the proteolysis of LeACS2 in vivo. (Managing editor: Wei WANG)