Subcellular organization of the Tat translocation machinery

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The twin-arginine translocation (Tat) machinery can translocate folded proteins across biological membranes. Components of the Tat system have been identified and their structure and function are subject of current international research. By fusing the green fluorescent protein (GFP) to the Tat components from Escherichia coli, we recently revealed that TatB and TatC, two essential components of the translocon, are mainly polarly localized within the cells (Berthelmann and Brüser, 2004). In contrast, TatA distribution is less focussed at the poles. We now plan to study the structural basis and functional consequences for this subcellular organization of the translocon components. The localization of different combinations of Tat system components will be studied in detail by GFP-fusion techniques and electron microscopy. As we expect from preliminary results, observed polar structures might contribute significantly to the understanding of Tat system functionality. The electron micros-copical analyses will be carried out in the microscopy unit at the neighbouring Biocenter. The role of known polarity determining factors - such as the Min system or bacterial proteins of the actin-family - will be addressed. Sorting factors will be analyzed by in vivo and in vitro protein interaction analyses and their functionality will be tested in vivo.