Rapid diffusion of green fluorescent protein in the mitochondrial matrix.
Rapid diffusion of green fluorescent protein in the mitochondrial matrix.
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DOI：
10.1083/jcb.140.4.821
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发表时间：
1998-02-23
期刊：
影响因子：
--
通讯作者：
Verkman AS
中科院分区：
文献类型：
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作者：
Partikian A;Olveczky B;Swaminathan R;Li Y;Verkman AS
抽象。据认为，线粒体基质中的高蛋白质密度导致严重限制的溶质扩散和代谢物从一种酶到另一种酶的通道，而没有自由的水相扩散。为了验证这一假设，我们测量了绿色荧光蛋白（GFP）在成纤维细胞，肝脏，骨骼肌和上皮细胞系的线粒体基质中表达的扩散。用100×物镜（0.8 μm斑点直径）对GFP进行斑点光漂白，荧光恢复的半衰期为15-19 ms，其中>90%的GFP移动的。正如预测的水相扩散在一个封闭的隔间，荧光恢复减缓或取消增加激光光斑大小或漂白时间，并通过多聚甲醛固定。使用基质扩散的数学模型对漂白数据进行定量分析，得出GFP的扩散系数为2-3 × 10−7 cm 2/s，仅比GFP在水中的扩散系数小3 - 4倍。相比之下，对于与通常存在于基质中的脂肪酸β-氧化多酶复合物的亚基融合的GFP的漂白几乎没有发现恢复。通过时间分辨各向异性测量未缀合的GFP的旋转给出了23.3 ± 1 ns的旋转相关时间，类似于GFP在水中旋转的20 ns。一个快速的旋转相关时间为325 ps的一个小的荧光探针（BCECF，约0.5 kD）在基质中的分离的肝线粒体。线粒体基质中溶质的快速和不受限制的扩散表明，代谢物通道可能不需要克服扩散障碍。我们提出，在膜相关复合物的基质酶的集群可能有助于建立一个相对不拥挤的水的空间，溶质可以自由扩散。
Abstract. It is thought that the high protein density in the mitochondrial matrix results in severely restricted solute diffusion and metabolite channeling from one enzyme to another without free aqueous-phase diffusion. To test this hypothesis, we measured the diffusion of green fluorescent protein (GFP) expressed in the mitochondrial matrix of fibroblast, liver, skeletal muscle, and epithelial cell lines. Spot photobleaching of GFP with a 100× objective (0.8-μm spot diam) gave half-times for fluorescence recovery of 15–19 ms with >90% of the GFP mobile. As predicted for aqueous-phase diffusion in a confined compartment, fluorescence recovery was slowed or abolished by increased laser spot size or bleach time, and by paraformaldehyde fixation. Quantitative analysis of bleach data using a mathematical model of matrix diffusion gave GFP diffusion coefficients of 2–3 × 10−7 cm2/s, only three to fourfold less than that for GFP diffusion in water. In contrast, little recovery was found for bleaching of GFP in fusion with subunits of the fatty acid β-oxidation multienzyme complex that are normally present in the matrix. Measurement of the rotation of unconjugated GFP by time-resolved anisotropy gave a rotational correlation time of 23.3 ± 1 ns, similar to that of 20 ns for GFP rotation in water. A rapid rotational correlation time of 325 ps was also found for a small fluorescent probe (BCECF, ∼0.5 kD) in the matrix of isolated liver mitochondria. The rapid and unrestricted diffusion of solutes in the mitochondrial matrix suggests that metabolite channeling may not be required to overcome diffusive barriers. We propose that the clustering of matrix enzymes in membrane-associated complexes might serve to establish a relatively uncrowded aqueous space in which solutes can freely diffuse.