Wouters F S, Bastiaens P I, Wirtz K W, Jovin T M
Centre for Biomembranes and Lipid Enzymology, Institute of Biomembranes, Utrecht University, Padualaan 8, NL-3584 CH, Utrecht, The Netherlands.
EMBO J. 1998 Dec 15;17(24):7179-89. doi: 10.1093/emboj/17.24.7179.
The fate of fluorescently labeled pre-nsL-TP (Cy3-pre-nsL-TP) microinjected into BALB/c 3T3 fibroblasts was investigated by confocal laser scanning microscopy. The protein exhibited a distinct punctate fluorescence pattern and colocalized to a high degree with the immunofluorescence pattern for the peroxisomal enzyme acyl-CoA oxidase. Proteolytic removal of the C-terminal leucine of the putative peroxisomal targeting sequence (AKL) resulted in a diffuse cytosolic fluorescence. These results indicate that microinjected Cy3-pre-nsL-TP is targeted to peroxisomes. The association of nsL-TP with peroxisomal enzymes was investigated in cells by measuring fluorescence resonance energy transfer (FRET) between the microinjected Cy3-pre-nsL-TP and Cy5-labeled antibodies against the peroxisomal enzymes acyl-CoA oxidase, 3-ketoacyl-CoA thiolase, bifunctional enzyme, PMP70 and catalase. The technique of photobleaching digital imaging microscopy (pbDIM), used to quantitate the FRET efficiency on a pixel-by-pixel basis, revealed a specific association of nsL-TP with acyl-CoA oxidase, 3-ketoacyl-CoA thiolase and bifunctional enzyme in the peroxisomes. These observations were corroborated by subjecting a peroxisomal matrix protein fraction to affinity chromatography on Sepharose-immobilized pre-nsL-TP. Acyl-CoA oxidase was retained. These studies provide strong evidence for a role of nsL-TP in the regulation of peroxisomal fatty acid beta-oxidation, e.g. by facilitating the presentation of substrates and/or stabilization of the enzymes.
通过共聚焦激光扫描显微镜研究了显微注射到BALB/c 3T3成纤维细胞中的荧光标记前体非甾体类脂质转运蛋白（Cy3-前体非甾体类脂质转运蛋白）的命运。该蛋白呈现出明显的点状荧光模式，并与过氧化物酶体酶酰基辅酶A氧化酶的免疫荧光模式高度共定位。对假定的过氧化物酶体靶向序列（AKL）的C末端亮氨酸进行蛋白水解去除后，导致了弥漫性的胞质荧光。这些结果表明，显微注射的Cy3-前体非甾体类脂质转运蛋白靶向于过氧化物酶体。通过测量显微注射的Cy3-前体非甾体类脂质转运蛋白与针对过氧化物酶体酶酰基辅酶A氧化酶、3-酮酰基辅酶A硫解酶、双功能酶、PMP70和过氧化氢酶的Cy5标记抗体之间的荧光共振能量转移（FRET），研究了非甾体类脂质转运蛋白与过氧化物酶体酶在细胞中的关联。用于逐像素定量FRET效率的光漂白数字成像显微镜技术（pbDIM）显示，非甾体类脂质转运蛋白与过氧化物酶体中的酰基辅酶A氧化酶、3-酮酰基辅酶A硫解酶和双功能酶存在特异性关联。通过使过氧化物酶体基质蛋白组分在固定于琼脂糖的前体非甾体类脂质转运蛋白上进行亲和层析，证实了这些观察结果。酰基辅酶A氧化酶被保留。这些研究为非甾体类脂质转运蛋白在过氧化物酶体脂肪酸β氧化调节中的作用提供了有力证据，例如通过促进底物的呈现和/或酶的稳定。
