Protein Mobility in the Cytoplasm of <em>Escherichia coli</em>

M Elowitz

M Surette

P Wolf

J Stock

S Leibler

Departments of Physics^{and Molecular Biology,}^{Princeton University, Princeton, New Jersey 08544}

^{Corresponding author. Mailing address: Lewis Thomas Lab, Washington Rd., Princeton, NJ 08544. Phone: (609) 258-1574. Fax: (609) 258-6175. E-mail:}ude.notecnirp@ztiwolem.

^{Present address: Department of Microbiology and Infectious Diseases, University of Calgary, Calgary, Alberta, Canada T2N 4N1.}

^{Present address: Centre de Recherches sur les Très Basses Températures, CNRS, Laboratoire associé à l’Université Joseph Fourier, F-38042 Grenoble Cedex 9, France.}

Received 1998 Aug 7; Accepted 1998 Oct 21.

Abstract

The rate of protein diffusion in bacterial cytoplasm may constrain a variety of cellular functions and limit the rates of many biochemical reactions in vivo. In this paper, we report noninvasive measurements of the apparent diffusion coefficient of green fluorescent protein (GFP) in the cytoplasm of Escherichia coli. These measurements were made in two ways: by photobleaching of GFP fluorescence and by photoactivation of a red-emitting fluorescent state of GFP (M. B. Elowitz, M. G. Surette, P. E. Wolf, J. Stock, and S. Leibler, Curr. Biol. 7:809–812, 1997). The apparent diffusion coefficient, D_a, of GFP in E. coli DH5α was found to be 7.7 ± 2.5 μm^{/s. A 72-kDa fusion protein composed of GFP and a cytoplasmically localized maltose binding protein domain moves more slowly, with}D_a of 2.5 ± 0.6 μm^{/s. In addition, GFP mobility can depend strongly on at least two factors: first,}D_a is reduced to 3.6 ± 0.7 μm^{/s at high levels of GFP expression; second, the addition to GFP of a small tag consisting of six histidine residues reduces}D_a to 4.0 ± 2.0 μm^{/s. Thus, a single effective cytoplasmic viscosity cannot explain all values of}D_a reported here. These measurements have implications for the understanding of intracellular biochemical networks.

Abstract

Response times and reaction rates in Escherichia coli often depend on the movement of proteins from one location to another in the cell. These proteins may have regulatory or signaling functions, or they may act as enzymes or substrates for cellular reactions. How do such molecules reach their destinations? In eukaryotic cells, cytoskeletal networks and motor proteins facilitate active transport of molecules (1). In some cases, including Drosophila oocytes, mixing of cytoplasm can also be achieved by the cytoskeleton-dependent process of cytoplasmic streaming (27). However, such structures and processes have not been observed in bacteria. Therefore, in bacteria, diffusion may be the primary means of intracellular movement. The diffusional mobility of cytoplasmic proteins may constrain the rates of some cellular reactions. The in vivo diffusive properties of proteins are therefore of general interest for understanding a variety of processes in the bacterial cell.

The interior of a bacterial cell is an environment crowded with a heterogeneous collection of macromolecules. In E. coli, the concentrations of protein, RNA, and DNA are 200 to 320 mg/ml, 75 to 120 mg/ml, and 11 to 18 mg/ml, respectively (6, 30). These high macromolecular concentrations imply large excluded volume effects, which strongly affect the activities of cytoplasmic molecules (14, 30). The validity of extrapolations from the values of physical or biochemical constants measured at lower macromolecular concentrations in cell-free in vitro systems to their effective values in actual cytoplasm is therefore uncertain (12). In particular, the rate of protein diffusion inside the cell cannot necessarily be inferred from in vitro measurements.

One of the most useful techniques for studying cytoplasmic diffusion in eukaryotic cells and cell membranes has been the method of “fluorescence recovery after photobleaching” (FRAP) (3, 16). By this method, fluorescent tracer molecules are introduced into the cell. Those tracers located in a small region are photobleached by a laser. The size, L, of the bleached area, and the characteristic time, τ, over which unbleached tracer molecules return to it, determine an apparent diffusion coefficient, D_a, which is proportional to L^{/τ. In spot photobleaching experiments,}L is the diameter of the spot, whereas in the experiments described here, it is the length of the cell. For diffusive particles, D_a is independent of L and equal to the diffusion coefficient, D. In a nonideal medium, on the other hand, particles may behave nondiffusively or exhibit anomalous diffusion, in which case the apparent diffusion coefficient, D_a, will depend on L (21). Because the cytoplasm may be such an environment, mobility measurements are reported here in terms of an apparent diffusion coefficient, D_a, valid specifically at the scale of the cell length.

Until now, it has been difficult to apply FRAP to E. coli cells. These bacteria are smaller than the eukaryotic cells previously studied by FRAP, and it is difficult to introduce fluorescently labeled molecules into them. Here, we have used the Aequorea victoria green fluorescent protein (GFP) as a tracer molecule to measure cytoplasmic protein diffusion. Like fluorochromes used in previous FRAP experiments, GFP can be irreversibly photobleached with sufficiently intense illumination (25). Unlike traditional FRAP fluorophores, however, GFP can be expressed endogenously. Further, it was recently shown that under conditions of low oxygen concentration, a short pulse of blue light converts the normally green-emitting GFP to a red-emitting state (10). Thus, apparent diffusion coefficients can also be measured by photoactivating GFP molecules at one pole and observing their subsequent propagation through the cell. Together, photobleaching and photoactivation techniques permit us to make direct in vivo measurements of protein diffusion in bacterial cytoplasm.

ACKNOWLEDGMENTS

We thank B. Aguera y Arcas, U. Alon, T. Holy, and A. C. Maggs for help with data analysis and software. We also thank U. Alon, P. Cluzel, L. Frisen, P. Lopez, and T. Surrey for critical reading of the manuscript. We are grateful to B. P. Cormack for providing GFP mutants.

ACKNOWLEDGMENTS

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