Characterization of periplastidal compartment-targeting signals in chlorarachniophytes

Secondary plastids are acquired by the engulfment and retention of eukaryotic algae, which results in an additional surrounding membrane or pair of membranes relative to the more familiar primary plastids of land plants. In most cases, the endocytosed alga loses its eukaryotic genome as it becomes integrated, but in two algal groups, the cryptophytes and chlorarachniophytes, the secondary plastids retain a vestigial nucleus in the periplastidal compartment (PPC), the remnant eukaryotic cytoplasm between the inner and the outer membrane pairs. Many essential housekeeping genes are missing from these reduced genomes, suggesting that they are now encoded in the host nucleus and their products are targeted to the PPC. One such nucleus-encoded, PPC-targeted protein, the translation elongation factor like (EFL) was recently identified in chlorarachniophytes. It bears an N-terminal-targeting sequence comprising a signal peptide and a transit peptide-like sequence (TPL) similar to the plastid-targeted proteins of chlorarachniophytes as well as a hydrophilic C-terminal extension rich in lysine and aspartic acid. Here, we characterize the function of the N-and C-terminal extensions of PPC-targeted EFL in transformed chlorarachniophyte cells. Using green fluorescent protein as a reporter molecule, we demonstrate that several negatively charged amino acids within the TPL are essential for accurate targeting to the PPC. Our findings further reveal that the C-terminal extension functions as a PPC retention signal in combination with an N-terminal plastid-targeting peptide, which suggests that plastid and PPC proteins may be sorted in the PPC.