Muntau Ania C, Roscher Adelbert A, Kunau Wolf-H, Dodt Gabriele
Dr. von Hauner Children's Hospital, Research Center, Department of Biochemical Genetics and Molecular Biology, Ludwig-Maximilians-University, Munich, Germany.
Eur J Cell Biol. 2003 Jul;82(7):333-42. doi: 10.1078/0171-9335-00325.
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.
过氧化物酶体生物发生过程涉及多个PEX基因，这些基因编码组装该细胞器所需的机制。在相应的过氧化物酶体蛋白中，PEX3和PEX19之间的相互作用对于早期过氧化物酶体生物发生至关重要。然而，这种蛋白质相互作用在细胞内的位点仍不清楚。为了通过荧光共振能量转移（FRET）分析解决这个问题，我们将增强型黄色荧光蛋白（EYFP）工程化到PEX3的C末端，将增强型青色荧光蛋白（ECFP）工程化到PEX19的N末端。通过用带有标签的PEX3和PEX19版本转染来自泽尔韦格患者的人PEX3和PEX19缺陷成纤维细胞，证明了融合蛋白的功能。这导致在先前缺乏可检测过氧化物酶体膜结构的两种细胞系中重新形成具有导入能力的过氧化物酶体。通过FRET成像观察到在过氧化物酶体生物发生过程中，PEX3-EYFP与ECFP-PEX19在PEX3缺陷细胞系中的相互作用。尽管PEX19主要定位于细胞质，但过氧化物酶体被确定为PEX3-PEX19相互作用的主要细胞内位点。通过供体荧光漂白实验对结果进行了确认和定量。缺乏N末端过氧化物酶体靶向序列的PEX3缺失蛋白（PEX3 34-373-EYFP）或位于蛋白质C末端一半的PEX19结合结构域（PEX3 1-140-EYFP）没有显示出PEX3特有的过氧化物酶体定位，而是错误定位于细胞质（PEX3 34-373-EYFP）或线粒体（PEX3 1-140-EYFP），并且不与ECFP-PEX19相互作用。我们认为FRET是一种合适的工具，可用于获取有关单细胞过氧化物酶体生物发生过程中过氧化物酶体蛋白相互作用的定量空间信息。这些发现补充并扩展了传统体外蛋白质相互作用测定的数据，并支持PEX3作为PEX19在过氧化物酶体膜上的锚定物的假设。
