Towards the membrane topology of the early translocation intermediate Ti-1 during Tat-dependent protein transport
Final Report Abstract
The initial step during Tat-dependent thylakoid transport of the chimeric model substrate 16/23 is the unassisted binding of the precursor protein to the membrane which leads to the formation of an early translocation intermediate called Ti-1. Ti-1 is characterised by a protease-protected fragment of 14 kDa comprising the signal peptide plus the N-terminal half of the passenger polypeptide. It was the major goal of this project to determine in detail the membrane topology of Ti-1 in order to clarify if the 16/23 chimera assumes an integral loop topology within the membrane or if it is only partially inserted or even merely attached to the lipid bilayer in a conformation that prevents complete proteolytic degradation. Analysing a set of single-site cysteine substitution mutants of the 16/23 protein in liposome insertion and thylakoid transport experiments in the presence of cysteine modification compounds that were either added externally to the assays or encapsulated into the liposomes before incubation, we could essentially rule out an integral loop topology of the Tat substrate within the membrane. Instead, the Tat substrate is only partially inserted into the lipid phase and the observed protease protection of the 14 kDa domain is presumably caused by tight folding of this part of the passenger polypeptide rather than by its membrane insertion. In addition, we have studied the role of the C-terminal region of the passenger protein in membrane binding and transport. Analysing a set of C-terminal truncation mutants of the 16/23 chimera in thylakoid transport and liposome binding experiments it turned out that binding of this Tat substrate in Ti-1 topology requires the fully folded passenger protein, since it was not observed with 16/23 truncation derivatives lacking the C-terminal 20, 40, 60, or 88 residues. Remarkably, also thylakoid transport of these mutants is progressively reduced with increasing size of the truncated passenger polypeptide. The same holds true also for the interaction with the thylakoidal TatBC receptor complexes suggesting that in this case receptor binding, which is apparently impaired by extended unfolded or malfolded passenger polypeptides, is the rate limiting step in Tat-dependent membrane transport. Furthermore, we have studied the thylakoid transport properties and the membrane topology of a second chimeric Tat substrate (16/33), which like the 16/23 chimera shows a proteaseprotected fragment upon membrane transport. In this case, however, the protease-protected fragment turned out to represent an alternative end product of the membrane translocation process rather than a true translocation intermediate.
Publications
- (2014) Tat transport of a Sec passenger leads to both completely translocated as well as membrane-arrested passenger proteins. BBA - Molecular Cell Research 1843, 446-453
Dittmar, J., Schlesier, R., and Klösgen, R.B.
(See online at https://doi.org/10.1016/j.bbamcr.2013.11.025) - (2015) C-terminal truncation of a Tat passenger protein affects its membrane translocation by interfering with receptor binding. Biol. Chem. 396, 349-357
Schlesier, R. and Klösgen, R.B.
(See online at https://doi.org/10.1515/hsz-2014-0249)
