Uncovering targeting priority to yeast peroxisomes using an in-cell competition assay

Mira Rosenthal, Eyal Metzl-Raz, Jerome Buergi, Eden Yifrach, Layla Drwesh, Amir Fadel, Yoav Peleg, Doron Rapaport, Matthias Wilmanns, Naama Barkai, Maya Schuldiner, Einat Zalckvar

Research output: Contribution to journalArticlepeer-review


Approximately half of eukaryotic proteins reside in organelles. To reach their correct destination, such proteins harbor targeting signals recognized by dedicated targeting pathways. It has been shown that differences in targeting signals alter the efficiency in which proteins are recognized and targeted. Since multiple proteins compete for any single pathway, such differences can affect the priority for which a protein is catered. However, to date the entire repertoire of proteins with targeting priority, and the mechanisms underlying it, have not been explored for any pathway. Here we developed a systematic tool to study targeting priority and used the Pex5-mediated targeting to yeast peroxisomes as a model. We titrated Pex5 out by expressing high levels of a Pex5-cargo protein and examined how the localization of each peroxisomal protein is affected. We found that while most known Pex5 cargo proteins were outcompeted, several cargo proteins were not affected, implying that they have high targeting priority. This priority group was dependent on metabolic conditions. We dissected the mechanism of priority for these proteins and suggest that targeting priority is governed by different parameters, including binding affinity of the targeting signal to the cargo factor, the number of binding interfaces to the cargo factor, and more. This approach can be modified to study targeting priority in various organelles, cell types, and organisms.

Original languageEnglish
Pages (from-to)21432-21440
Number of pages9
JournalProceedings of the National Academy of Sciences of the United States of America
Issue number35
Early online date17 Aug 2020
StatePublished - 1 Sep 2020

All Science Journal Classification (ASJC) codes

  • General


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