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

Bernhard Hochreiter,Johannes Berger,Johannes A Schmid,Cheng-Shoong Chong,Sebastian Maurer-Stroh,Markus Kunze,Andreas Hartig

doi:10.3390/cells9112381

Abstract

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.

Highlights

Peroxisomes are ubiquitous, single membrane-bound organelles enclosing a variety of metabolic reactions such as the degradation of various types of fatty acids or hydrogen peroxide (H2 O2 ).In animals, they are involved in bile acid and ether-phospholipid biosynthesis and in plants in the glyoxylate cycle and photorespiration [1,2]
A Novel System to Investigate the Interaction between peroxin 5 (PEX5) and PTS1 by FRET
To study the interaction between PEX5 and diverse PTS1 sequences by FRET measurements, we generated a pair of fusion proteins, one consisting of the PTS1-binding TPR-domain of human

Summary

Introduction

Peroxisomes are ubiquitous, single membrane-bound organelles enclosing a variety of metabolic reactions such as the degradation of various types of fatty acids or hydrogen peroxide (H2 O2 ). In animals, they are involved in bile acid and ether-phospholipid biosynthesis and in plants in the glyoxylate cycle and photorespiration [1,2]. PEX5 consists of a C-terminal tetratricopeptide (TPR) domain binding PTS1-motifs and a long unstructured N-terminal part mediating the transport to the peroxisomal membrane [14,15]. Transferring the results of peptide binding assays to the in vivo situation can be problematic, because PTS1 peptides act embedded in full-length cargo proteins and specific properties of the cellular context such as molecular crowding can hardly be mimicked by in vitro systems [21], whereas predictions of this influence are rarely possible [22,23]

Methods

Results

Conclusion