System to quantify the import of peroxisomal matrix proteins by fluorescence intensity
System to quantify the import of peroxisomal matrix proteins by fluorescence intensity
复制标题
通过荧光强度量化过氧化物酶体基质蛋白输入的系统
作者：
Noguchi;M.;Okumoto;K.;and *Fujiki;Y
十四种不同的过氧化物酶对于哺乳动物的过氧化物酶体生物发生至关重要，其中十种参与将基质蛋白输入到过氧化物酶体中。过氧化物酶体基质蛋白的输入受多种细胞因素的调节；然而，人们对这一监管背后的机制知之甚少。这主要是因为没有高分辨率的定量检测方法可用于研究过氧化物酶体基质蛋白的输入。在这里，我们开发了一种监测系统，该系统使用荧光报告基因，该荧光报告基因在过氧化物酶体中稳定，但在细胞质中降解。 FK506 结合蛋白 12 变体称为去稳定结构域 (DD)，在哺乳动物细胞中表达时会被蛋白酶体快速组成型降解。通过添加特定的合成配体可逆地保护 DD。在没有配体的情况下，与 DD 和过氧化物酶体靶向信号 1 (DD-EGFP-PTS1) 融合的增强型 GFP (EGFP) 报告分子在胞质溶胶中大量降解。相比之下，在配体存在的情况下，报告分子被稳定并易位到过氧化物酶体中。撤回配体后，过氧化物酶体中的报告分子保持完整，而细胞质中的报告分子迅速降解。因此，通过测量全细胞的荧光强度可以容易地量化过氧化物酶体蛋白的输入。
Fourteen distinct peroxins are essential for peroxisome biogenesis in mammals, of which ten are involved in the import of matrix proteins into peroxisomes. Peroxisomal matrix protein import is regulated by various cellular factors; however, the mechanisms underlying this regulation are poorly understood. This is primarily because no quantitative detection method with high resolution is available to study the import of peroxisomal matrix proteins. Here, we developed a monitoring system that uses a fluorescent reporter that is stabilized in peroxisomes but is degraded in the cytosol. An FK506 binding protein 12 variant, termed destabilization domain (DD), is rapidly and constitutively degraded by proteasomes when expressed in mammalian cells. DD is reversibly protected by the addition of a specific synthetic ligand. In the absence of the ligand, a reporter molecule, enhanced GFP (EGFP) fused with DD and peroxisomal targeting signal 1 (DD‐EGFP‐PTS1), is largely degraded in the cytosol. By contrast, in the presence of the ligand, the reporter is stabilized and translocates into peroxisomes. Upon withdrawal of the ligand, the reporter in peroxisomes remains intact, whereas that in the cytosol is rapidly degraded. Thus, peroxisomal protein import can be readily quantified by measuring the fluorescence intensity of whole cells.