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Open AccessFeature PaperArticle

A Novel FRET Approach Quantifies the Interaction Strength of Peroxisomal Targeting Signals and Their Receptor in Living Cells

1
Center for Physiology and Pharmacology, Institute of Vascular Biology and Thrombosis Research, Medical University of Vienna, 1090 Vienna, Austria
2
Bioinformatics Institute (BII), Agency for Science, Technology and Research (A*STAR), Singapore 138671, Singapore
3
NUS Graduate School for Integrative Sciences and Engineering, National University of Singapore, Singapore 119077, Singapore
4
Department of Biochemistry and Cell Biology, Max Perutz Laboratories, University of Vienna, 1030 Vienna, Austria
5
Department of Biological Sciences, National University of Singapore, Singapore 117558, Singapore
6
Center for Brain Research, Department of Pathobiology of the Nervous System, Medical University of Vienna, 1090 Vienna, Austria
*
Authors to whom correspondence should be addressed.
Cells 2020, 9(11), 2381; https://doi.org/10.3390/cells9112381
Received: 21 September 2020 / Revised: 22 October 2020 / Accepted: 25 October 2020 / Published: 30 October 2020
(This article belongs to the Collection Feature Papers in Organelle Function)
Measuring Förster–resonance–energy–transfer (FRET) efficiency allows the investigation of protein–protein interactions (PPI), but extracting quantitative measures of affinity necessitates highly advanced technical equipment or isolated proteins. We demonstrate the validity of a recently suggested novel approach to quantitatively analyze FRET-based experiments in living mammalian cells using standard equipment using the interaction between different type-1 peroxisomal targeting signals (PTS1) and their soluble receptor peroxin 5 (PEX5) as a model system. Large data sets were obtained by flow cytometry coupled FRET measurements of cells expressing PTS1-tagged EGFP together with mCherry fused to the PTS1-binding domain of PEX5, and were subjected to a fitting algorithm extracting a quantitative measure of the interaction strength. This measure correlates with results obtained by in vitro techniques and a two-hybrid assay, but is unaffected by the distance between the fluorophores. Moreover, we introduce a live cell competition assay based on this approach, capable of depicting dose- and affinity-dependent modulation of the PPI. Using this system, we demonstrate the relevance of a sequence element next to the core tripeptide in PTS1 motifs for the interaction strength between PTS1 and PEX5, which is supported by a structure-based computational prediction of the binding energy indicating a direct involvement of this sequence in the interaction. View Full-Text
Keywords: FRET; live-cell measurements; flow cytometry; peroxisomes; PEX5; peroxisomal targeting signal FRET; live-cell measurements; flow cytometry; peroxisomes; PEX5; peroxisomal targeting signal
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MDPI and ACS Style

Hochreiter, B.; Chong, C.-S.; Hartig, A.; Maurer-Stroh, S.; Berger, J.; Schmid, J.A.; Kunze, M. A Novel FRET Approach Quantifies the Interaction Strength of Peroxisomal Targeting Signals and Their Receptor in Living Cells. Cells 2020, 9, 2381. https://doi.org/10.3390/cells9112381

AMA Style

Hochreiter B, Chong C-S, Hartig A, Maurer-Stroh S, Berger J, Schmid JA, Kunze M. A Novel FRET Approach Quantifies the Interaction Strength of Peroxisomal Targeting Signals and Their Receptor in Living Cells. Cells. 2020; 9(11):2381. https://doi.org/10.3390/cells9112381

Chicago/Turabian Style

Hochreiter, Bernhard; Chong, Cheng-Shoong; Hartig, Andreas; Maurer-Stroh, Sebastian; Berger, Johannes; Schmid, Johannes A.; Kunze, Markus. 2020. "A Novel FRET Approach Quantifies the Interaction Strength of Peroxisomal Targeting Signals and Their Receptor in Living Cells" Cells 9, no. 11: 2381. https://doi.org/10.3390/cells9112381

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