Der TatA-Bedarf beim Tat-abhängigen Proteintransport über die Thylakoidmembran
Zusammenfassung der Projektergebnisse
The Tat pathway, which is found in chloroplasts, bacteria and archaea, is unique with respect to its property to translocate proteins in a fully folded conformation across ion-tight membranes. Transport is initiated by binding of the substrate precursor protein to the TatBC receptor complex. The actual membrane translocation of the passenger protein requires both the membrane potential as well as TatA which mediates the translocation step in a yet unknown manner. Three deviating working models are currently discussed. Despite distinct in detail, all three models address TatA as the key player in the actual membrane translocation step, i.e. being either a constitutent of an adaptable translocation pore, a membrane-weakening factor, or a coenzyme facilitating transport by the TatBC complex. As a first step to clarify this function, we had in our preliminary work exactly quantified the amount of TatA that is required to transport a model Tat substrate (16/23) across the thylakoid membrane. In the current project we have extended this quantitative analysis of TatA demand during thylakoid transport to a set of passenger proteins that differ from each other in terms of molecular mass, dimensions, and/or folding status. Among those were authentic Tat passengers, both from plants (OEC16, OEC33) and bacteria (SufI, GFOR), passengers that are normally transported in an unfolded conformation by the Sec pathway (OEC33, PC), and the cytosolic reporter protein EGFP (Enhanced Green Fluorescent Protein) from jellyfish. Quantification was performed with reconstitution experiments according to Hauer et al. (2013) that are based on antibody inhibition of the intrinsic TatA activity in isolated thylakoid vesicles and subsequent supplementation of the assays with defined amounts of purified, soluble TatA obtained from heterologous overexpression in E. coli. Thylakoid transport of the passengers was in all instances mediated by the Tat transport signal from the OEC16 precursor. In addition, the two bacterial Tat substrates were analysed also in combination with their respective authentic Tat signal peptides, in order to consider also the potential influence of the transport signal on the TatA demand. In these experiments, we could not observe any correlation of molecular mass or structural dimension of the passenger with its demand for TatA. Likewise, we could also not observe any correlation of the presumed folding status of the passenger and its TatA requirements. Instead, it seems that a TatA concentration of approximately 0.2 µM - 0.3 µM would be suitable for almost all passenger proteins if combined with the Tat transport signal of OEC16. The only exception is 16/SufI, which requires approximately twice the concentration of TatA (0.5 µM). However, in combination with its native Tat signal peptide the TatA demand of SufI drops down to the "normal" range (0.26 µM) demonstrating that, principally, also this passenger protein can be transported with "standard" TatA amounts. Taken together, our results do not support the model of an adaptable TatA translocation pore to achieve transport of passengers proteins of variable size or folding status, since at least some degree of direct correlation of passenger size and TatA demand would have been expected in this case. Instead, a largely substrate-independent catalytic activity of TatA appears more likely.
Projektbezogene Publikationen (Auswahl)
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(2018) Structure and dynamics of plant TatA in micelles and lipid bilayers studied by solution NMR. FEBS J. 285, 1886-1906
Pettersson, P., Ye, W., Jakob, M., Tannert, F., Klösgen, R.B., and Mäler, L.
