The dynamics of protein transport

In bacteria, a variety of sophisticated membrane-bound complexes have evolved to transfer proteins across membranes. The majority of transported proteins cross membranes in an unfolded state. In contrast, the Twin Arginine Translocation (Tat) pathway is capable of transporting folded and even multimeric proteins across a membrane that maintains a proton gradient. Our aim is to elucidate the mechanism by which the E. coli Tat machine transports folded proteins across the cytoplasmic membrane. For this purpose we make use of sophisticated optical techniques.

We labeled various components of the Tat system by fusing them genetically with fluorescent proteins (eGFP, mCherry). We can control expression of these proteins, such that most bacteria contain only one or two fluorescently labeled Tat complexes. By tracking the motion of these fluorescent particles using a very sensitive multi-color widefield fluorescence microscope we can determine the diffusion coefficients of Tat complexes under various conditions.  This has given us new insights into the working mechanism of the Tat system. Our approach is also applicable to other dynamic membrane-bound processes, such as transport of nutrients or signaling.

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Tracking protein translocation complexes in living bacteria. (A) snapshots; (B) trajectory of a fluorescent Tat complex; (C) cumulative probability distribution (D) histogram of intensities (E) model for the Tat sytem’s activity cycle.

Contact: Yves Bollen. e-mail: yves.bollen@falw.vu.nl