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© The Rockefeller University Press, 0021-9525/1998//821 $5.00
The Journal of Cell Biology, Volume 140, Number 4, , 1998 821-829

Article

Rapid Diffusion of Green Fluorescent Protein in the Mitochondrial Matrix



Arthur Partikian, Bence Ölveczky, R. Swaminathan, Yuxin Li, and A.S. Verkman

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 100x 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 x 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.

1. Abbreviations used in this paper: {alpha}MFAB, {alpha}-subunit of rat mitochondrial fatty acid β-oxidation multienzyme complex; βMFAB, β-subunit of rat mitochondrial fatty acid β-oxidation multienzyme complex; BCECF, 2,7-bis-(2-carboxyethyl)-5-(and 6-)-carboxyfluorescein; CF, carboxyfluorescein; COX8, targeting presequence of subunit VIII of human cytochrome C oxidase; D, diffusion coefficient; FRAP, fluorescence recovery after photobleaching; GFP, green fluorescent protein (humanized with red-shifted excitation); t1/2, half-time for fluorescence recovery after photobleaching.

We thank Katherine Chen for cell culture and transfections, Drs. Javier Farinas, Tonghui Ma, and N. Periasamy for advice in design of the GFP constructs and in the computational analysis.

This work was supported by grants DK43840, HL42368, DK35124, and DK16095 from the National Institutes of Health. A. Partikian was the recipient of a summer student stipend from the American Heart Association, California Affiliate.

Address correspondence to Alan S. Verkman, M.D., Ph.D., 1246 Health Sciences East Tower, Cardiovascular Research Institute, University of California, San Francisco, CA 94143-0521. Tel.: (415) 476-8530. Fax: (415) 665-3847. E-mail: verkman{at}itsa.ucsf.edu


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