Uncovering targeting priority to peroxisomes using a new competition assay

Half of eukaryotic proteins are synthesized in the cytosol later to be targeted to organelles. Often multiple proteins compete for a single targeting pathway that may become restrictive. While much is known about the various targeting signals and pathways, it is unclear whether priority is encoded in targeting signals and if so, what establishes such priority. Here we developed a systematic tool to study targeting priority and used Pex5-mediated targeting to yeast peroxisomes as a model system. We expressed different levels of a Pex5-cargo competitor protein (mCherry-SKL) and examined how the localization of each peroxisomal protein is affected by the presence of the competitor. We found that while the majority of known Pex5 cargo proteins were competed out causing them to accumulate in the cytosol, five cargo proteins were not affected implying that they have high targeting priority. We dissected the mechanism of priority for these targets and suggest that targeting priority is governed by different parameters and can be altered upon metabolic conditions. Our approach can be modified to study targeting priority in various organelles, cell types and organisms.