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Dual targeting of a nuclearly encoded protein into two different cell organelles is an exceptional event in eukaryotic cells. Yet, the frequency of such dual targeting is remarkably high in case of mitochondria and chloroplasts, the two endosymbiotic organelles of plant cells. In most instances, it is mediated by “ambiguous” transit peptides, which recognize both organelles as the target. A number of different approaches including in silico, in organello as well as both transient and stable in vivo assays are established to determine the targeting specificity of such transit peptides. In this review, we will describe and compare these approaches and discuss the potential role of this unusual targeting process. Furthermore, we will present a hypothetical scenario how dual targeting might have arisen during evolution.
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