The interaction between human PEX3 and PEX19 characterized by fluorescence resonance energy transfer (FRET) analysis

Abstract

The process of peroxisome biogenesis involves several PEX genes that encode the machinery required to assemble the organelle. Among the corresponding peroxins the interaction between PEX3 and PEX19 is essential for early peroxisome biogenesis. However, the intracellular site of this protein interaction is still unclear. To address this question by fluorescence resonance energy transfer (FRET) analysis, we engineered the enhanced yellow fluorescent protein (EYFP) to the C-terminus of PEX3 and the enhanced cyan fluorescent protein (ECFP) to the N-terminus of PEX19. Functionality of the fusion proteins was shown by transfection of human PEX3- and PEX19-deficient fibroblasts from Zellweger patients with tagged versions of PEX3 and PEX19. This led to reformation of import-competent peroxisomes in both cell lines previously lacking detectable peroxisomal membrane structures. The interaction of PEX3-EYFP with ECFP-PEX19 in a PEX3-deficient cell line during peroxisome biogenesis was visualized by FRET imaging. Although PEX19 was predominantly localized to the cytoplasma, the peroxisome was identified to be the main intracellular site of the PEX3-PEX19 interaction. Results were confirmed and quantified by donor fluorescence photobleaching experiments. PEX3 deletion proteins lacking the N-terminal peroxisomal targeting sequence (PEX3 34-373-EYFP) or the PEX19-binding domain located in the C-terminal half of the protein (PEX3 1-140-EYFP) did not show the characteristic peroxisomal localization of PEX3, but were mislocalized to the cytoplasm (PEX3 34-373-EYFP) or to the mitochondria (PEX3 1-140-EYFP) and did not interact with ECFP-PEX19. We suggest that FRET is a suitable tool to gain quantitative spatial information about the interaction of peroxins during the process of peroxisome biogenesis in single cells. These findings complement and extend data from conventional in vitro protein interaction assays and support the hypothesis of PEX3 being an anchor for PEX19 at the peroxisomal membrane.