The Tat-dependent protein transport pathway promotes the translocation of folded proteins in or across the thylakoid membrane. This is enabled by a membrane-bound Tat-translocase consisting of TatA, TatB, and TatC. While TatB and TatC form a substrate binding receptor complex, TatA promotes translocation of the substrate. Using the chimeric Tat-substrate protein 16/23, the distinct steps of membrane interaction, receptor binding, and translocation can be examined separately of each other. It was the aim of this work to gain further knowledge about the underlying mechanisms of these processes. It turned out that, even though the 23 kDa passenger protein is involved in membrane binding, this step is not essential for Tat-dependent transport of the substrate protein. Furthermore, it was proven that C-terminal truncations of the passenger protein have effects on the receptor binding and thus also on the transport rate of the substrate protein. Moreover, for the first time the amount of TatA, which is required to promote the membrane translocation of the 16/23 protein, could be quantified.