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

Abstract

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.

Determining the targeting specificity of the selective peroxisomal targeting factor Pex9

“…Our work exemplifies how changes in the binding cavity of two rather similar targeting factors lead to different cargo binding specificities. These findings expand the range of capabilities of how peroxisomes achieve exquisite targeting specificityfrom the presence of multiple differentially regulated parallel pathways for targeting, through affinity-tuning of various PTS1s to provide priority targeting (Rosenthal et al, 2020), to post-translational modifications of Pex5 or its cargo proteins that alter its binding specificity. Each cargo has a unique targeting propensity that can also be regulated to enable the dynamic rewiring of protein content in peroxisomes upon demand and shows the beauty and complexity of the peroxisomal targeting machinery.…”

Peroxisome-Derived Hydrogen Peroxide Modulates the Sulfenylation Profiles of Key Redox Signaling Proteins in Flp-In T-REx 293 Cells

“…However, it is also evident that, at least in some cells, varying portions of po-IBD-SBP-YAP1C and, to a certain extent, also mt-IBD-SBP-YAP1C still reside in the cytosol. Given that 1) the functionality of peroxisomal and mitochondrial targeting signals fused to a heterologous protein strongly depends on the protein context ( Yogev and Pines, 2011 ; Kunze, 2018 ), 2) the priority of protein import into mitochondria and peroxisomes is governed by competition for binding to limiting amounts of import receptor ( Weidberg and Amon, 2018 ; Rosenthal et al, 2020 ), and 3) expression of the YAP1C fusion proteins is driven by the cytomegalovirus promoter, one of the strongest naturally occurring promoters ( Even et al, 2016 ), this observation may not be that surprising. Although this may complicate the interpretation of downstream results, this knowledge also allows us to correctly anticipate this shortcoming.…”

BiFC Method Based on Intraorganellar Protein Crowding Detects Oleate-Dependent Peroxisomal Targeting of Pichia pastoris Malate Dehydrogenase

“…The localization of a protein to peroxisomes only in oleate is not unprecedented because, in S. cerevisiae , aspartate aminotransferase (Aat2) is cytosolic in glucose-grown cells, but peroxisomal in cells grown in oleate [ 28 ]. Indeed, recent studies show that several yeast proteins, particularly those involved in the β-oxidation of fatty acids, exhibit a priority for peroxisomal targeting in oleate, in comparison to glucose [ 29 ].…”