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J. Cell Biol.,
Volume 140, Number 4, February 23, 1998 821-829
Departments of Medicine and Physiology, Cardiovascular Research Institute, University of California, San Francisco, California,
94143-0521
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 
-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.
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