The N‐terminal β‐barrel structure of lipid body lipoxygenase mediates its binding to liposomes and lipid bodies

Phospholipase A 2 and a particular isoform of lipoxygenase are synthesized and transferred to lipid bodies during the stage of triacylglycerol mobilization in germinating cucumber seedlings. Lipid body lipoxygenase (LBLOX) is post‐translationally transported to lipid bodies without proteolytic modification. Fractionation of homogenates from cucumber cotyledons or transgenic tobacco leaves expressing LBLOX showed that a small but significant amount was detectable in the microsomal fraction. A β‐barrel‐forming N‐terminal domain in the structure of LBLOX, as deduced from sequence data, was shown to be crucial for selective intracellular transport from the cytosol to lipid bodies. Although a specific signal sequence for targeting protein domains to the lipid bodies could not be established, it was evident that the β‐barrel represents a membrane‐binding domain that is functionally comparable with the C2 domains of mammalian phospholipases. The intact β‐barrel of LBLOX was demonstrated to be sufficient to target in vitro a fusion protein of LBLOX β‐barrel with glutathione S‐transferase (GST) to lipid bodies. In addition, binding experiments on liposomes using lipoxygenase isoforms, LBLOX deletions and the GST‐fusion protein confirmed the role of the β‐barrel as the membrane‐targeting domain. In this respect, the cucumber LBLOX differs from cytosolic isoforms in cucumber and from the soybean LOX‐1. When the β‐barrel of LBLOX was destroyed by insertion of an additional peptide sequence, its ability to target proteins to membranes was abolished.