The plastid is a unique organelle found in land plants and algae. Of the four main types of plastids, chloroplasts are responsible for photosynthesis, amino acid and fatty acid synthesis, and the plant immune response. Chloroplasts are thought to contain more than 3,000 different types of proteins; however, the chloroplast genome only encodes approximately 100 proteins. Most proteins in the chloroplast proteome are encoded by the nuclear genome and post-translationally imported into the chloroplast.
The import of nuclear-encoded proteins into chloroplasts occurs via a complex process with distinct steps that occur in a sequential manner.
It is thought that two to three thousand different proteins are targeted to the chloroplast, and the ‘transit peptides’ that act as chloroplast targeting sequences are usually the largest class of targeting sequences in plants. At a primary structural level, transit peptide sequences are highly divergent in length, composition and organization. An emerging concept suggests that transit peptides have multiple domains that provide either distinct or overlapping functions. These functions include direct interaction with envelope lipids, chloroplast receptors and the stromal processing peptidase. The genomic organization of transit peptides suggests that these domains might have originated from distinct exons, which were shuffled and streamlined throughout evolution to produce a modern, multifunctional transit peptide. The plasticity of transit peptide design is consistent with the diverse biological functions of chloroplast proteins.
Chloroplasts import many pre proteins that can be classified based on their physicochemical properties. The cleavable N-terminal transit peptide (TP) of chloroplast pre proteins contains all the information required for import into chloroplasts through Toc/Tic translocons. It has presented that Pro residues in TP mediate efficient translocation through the chloroplast envelope membranes for proteins containing transmembrane domains (TMDs) or proteins prone to aggregation. By contrast, the translocation of soluble proteins through the chloroplast envelope membranes is less dependent on TP prolines. Proless TPs failed to mediate protein translocation into chloroplasts; instead, these mutant TPs led to protein mis-targeting to the chloroplast envelope membranes or nonspecific protein aggregation during import into chloroplasts.
The precise mechanism that efficiently translocates pre proteins through the import channel after binding to the receptor complexes at the chloroplast surface has not been elucidated. Chloroplast pre proteins are in a largely unfolded state before reaching their final destination, which may allow efficient translocation across the outer/inner envelopes and increase the chances of interacting with molecular chaperones involved in delivering pre proteins to chloroplasts. The N-terminal transit peptide (TP) of unfolded chloroplast pre proteins is inserted into the import channel at the outer envelope membrane and then passed through channels at inner envelope membranes to finally emerge into the stroma. Many complexes are present at the stromal side of the inner envelope membrane that bind to and pull the TP through the envelope.
ChloroP, version 1.1 is a neural network method for identifying probable chloroplast transit peptide sequences and predicting the proteolytic cleavage site of each transit peptide. ChloroP presents its prediction of chloroplast targeting as a “Y” or “N” output based upon the predicted presence of a chloroplast transit peptide.
TargetP, version 1.01 is a layered neural network method for predicting subcellular targeting based upon the type of targeting/transit peptide predicted to be at the amino terminus of each protein. TargetP predicts whether the protein in question is trafficked to the chloroplast, mitochondria, secretory pathway, or “other” subcellular location.
PSORT is an expert system using a knowledge-based setup as an “if-then” cascade. PSORT predicts subcellular localization with much finer resolution than any of the other programs examined. PSORT includes a hydrophobic moment analysis for chloroplast proteins as one of its expert analysis programs. The usefulness of this calculation is based on the assumption that all chloroplast proteins have a similar stromal targeting domain in the amino-terminal targeting peptide. The hydrophobic moment analysis in PSORT distinguishes chloroplast protein status as negative, positive, or undetermined.
Predotar, version 0.5 is a program still under development designed to be a Web-based method for distinguishing chloroplast- from mitochondria-targeting sequences. Predotar predicts localization to the chloroplast, mitochondria, both organelles, or neither organelle.
MitoProt II is a computational method for predicting mitochondrial targeting sequences and for predicting the proteolytic cleavage sites of the targeting peptides.