Prerequisites for Terminal Processing of Thylakoidal Tat Substrates

Abstract

In bacteria and chloroplasts, the Tat (twin arginine translocation) system is capable of translocating folded passenger proteins across the cytoplasmic and thylakoidal membranes, respectively. Transport depends on signal peptides that are characterized by a twin pair of arginine residues. The signal peptides are generally removed after transport by specific processing peptidases, namely the leader peptidase and the thylakoidal processing peptidase. To gain insight into the prerequisites for such signal peptide removal, we mutagenized the vicinity of thylakoidal processing peptidase cleavage sites in several thylakoidal Tat substrates. Analysis of these mutants in thylakoid transport experiments showed that the amino acid composition of both the C-terminal segment of the signal peptide and the N-terminal part of the mature protein plays an important role in the maturation process. Efficient removal of the signal peptide requires the presence of charged or polar residues within at least one of those regions, whereas increased hydrophobicity impairs the process. The relative extent of this effect varies to some degree depending on the nature of the precursor protein. Unprocessed transport intermediates with fully translocated passenger proteins are found in membrane complexes of high molecular mass, which presumably represent Tat complexes, as well as free in the lipid bilayer. This seems to indicate that the Tat substrates can be laterally released from the complexes prior to processing and that membrane transport and terminal processing of Tat substrates are independent processes.