Approaches to defining dual-targeted proteins in Arabidopsis

A variety of approaches were used to predict dual-targeted proteins in Arabidopsis thaliana. These predictionswere experimentally tested using GFP fusions. Twelve new dual-targeted proteins were identified: five thatwere dual-targeted to mitochondria and plastids, six that were dual-targeted to mitochondria andperoxisomes, and one that was dual-targeted to mitochondria and the nucleus. Two methods to predictdual-targeted proteins had a high success rate: (1) combining the AraPerox database with a variety ofsubcellular prediction programs to identify mitochondrial- and peroxisomal-targeted proteins, and (2) using avariety of prediction programs on a biochemical pathway or process known to contain at least one dualtargetedprotein. Several technical parameters need to be taken into account before assigning subcellularlocalization using GFP fusion proteins. The position of GFP with respect to the tagged polypeptide, the tissueor cells used to detect subcellular localization, and the portion of a candidate protein fused to GFP are allrelevant to the expression and targeting of a fusion protein. Testing all gene models for a chromosomal locus isrequired if more than one model exists.