RAPID DIFFUSION OF GREEN FLUORESCENT PROTEIN IN THE MITOCHONDRIAL MATRIX

It is thought that the high protein density in the mitochondrial matri x results in severely restricted solute diffusion and metabolite chann eling from one enzyme to another without free aqueous-phase diffusion, To test this hypothesis, we measured the diffusion of green fluoresce nt protein (GFP) expressed in the mitochondrial matrix of fibroblast, liver, skeletal muscle, and epithelial cell lines. Spot photobleaching of GFP with a 100x objective (0.8-mu m spot diam) gave half-times for fluorescence recovery of 15-19 ms with >90% of the GFP mobile, As pre dicted for aqueous-phase diffusion in a confined compartment, fluoresc ence recovery was slowed or abolished by increased laser spot size or bleach time, and by paraformaldehyde fixation. Quantitative analysis o f bleach data using a mathematical model of matrix diffusion gave GFP diffusion coefficients of 2-3 x 10(-7) cm(2)/s, only three to fourfold less than that for GFP diffusion in water. In contrast, Little recove ry was found for bleaching of GFP in fusion with subunits of the fatty acid beta-oxidation multienzyme complex that are normally present in the matrix, Measurement of the rotation of unconjugated GFP by time-re solved anisotropy gave a rotational correlation time of 23.3 +/- 1 ns, similar to that of 20 ns for GFP rotation in water. A rapid rotationa l correlation time of 325 ps was also found for a small fluorescent pr obe (BCECF, similar to 0.5 kD) in the matrix of isolated liver mitocho ndria. The rapid and unrestricted diffusion of solutes in the mitochon drial matrix suggests that metabolite channeling may not be required t o overcome diffusive barriers. We propose that the clustering of matri x enzymes in membrane-associated complexes might serve to establish a relatively uncrowded aqueous space in which solutes can freely diffuse .