Structural and functional analysis of a chloroplast transit peptide: Interactions with the chloroplast translocation apparatus

Protein targeting into organelles is a central cellular process that occurs in all-living organisms. Proper cellular targeting is essential for the functioning of most proteins within a cell, yet the mechanism by which this process is mediated is not clearly understood. Plastids from plants provide an excellent model system for studying protein targeting, as they are semi-autonomous organelles with a wide variety of structural and functional diversity. Although, plastids have their own genome they strongly rely on imported proteins that are encoded in the nuclear genome and translated in the cytoplasm. The proteins synthesized in the cytoplasm have an N-terminal extension called the transit peptide, which is considered necessary and sufficient for the import of proteins into plastids.; Binding of precursors to the plastid surface probably involves both proteins and lipids of the envelope membrane. Although, early reports have demonstrated that precursors directly interact with individual components of the two translocators, mechanistic details have been hampered by the inability to trap an early translocation intermediate. We have created a novel reagent that places a dual-epitope tag (His-S) at the N-terminus of the transit peptide (SStp) derived from the pea Rubisco small subunit precursor (prSSU). These two epitope tags permit facile purification of the fusion protein from E. coli via the His-tag, and a highly sensitive detection via the interaction of the S-protein with the S-tag. Subsequent to the successful binding of His-S-SStp to the chloroplast surface we can detect the transit peptide by far-western blotting, laser-scanning confocal microscopy (LSCM), flow cytometry and FRET analysis using S-protein conjugates. These methodologies in combination provide qualitative measurements like high-resolution spatial information about the distribution of the bound transit peptide and statistically significant quantitative measurements of the total number of transit peptides bound to the chloroplast. Moreover, label transfer cross-linking experiments show that His-S-SStp is bound to the chloroplast translocation apparatus.; Taken together, these approaches allow for sensitive and quantifiable evaluation of the in vitro binding of SStp to the chloroplast translocation apparatus, as well as the first direct visualization of a transit peptide bound to the chloroplast translocation apparatus.