Abstract
A large number of nuclear-encoded proteins are imported into chloroplasts after they are translated in the cytosol. Import is mediated by transit peptides (TPs) at the N termini of these proteins. TPs contain many small motifs, each of which is critical for a specific step in the process of chloroplast protein import; however, it remains unknown how these motifs are organized to give rise to TPs with diverse sequences. In this study, we generated various hybrid TPs by swapping domains between Rubisco small subunit (RbcS) and chlorophyll a/b-binding protein, which have highly divergent sequences, and examined the abilities of the resultant TPs to deliver proteins into chloroplasts. Subsequently, we compared the functionality of sequence motifs in the hybrid TPs with those of wild-type TPs. The sequence motifs in the hybrid TPs exhibited three different modes of functionality, depending on their domain composition, as follows: active in both wild-type and hybrid TPs, active in wild-type TPs but inactive in hybrid TPs, and inactive in wild-type TPs but active in hybrid TPs. Moreover, synthetic TPs, in which only three critical motifs from RbcS or chlorophyll a/b-binding protein TPs were incorporated into an unrelated sequence, were able to deliver clients to chloroplasts with a comparable efficiency to RbcS TP. Based on these results, we propose that diverse sequence motifs in TPs are independent functional units that interact with specific translocon components at various steps during protein import and can be transferred to new sequence contexts.
Highlights
A large number of nuclear-encoded proteins are imported into chloroplasts after they are translated in the cytosol
We fused the transit peptides (TPs) of Rubisco small subunit (RbcS-nt [N-terminal region]) and chlorophyll a/bbinding protein (Cab) (Cab-nt), which contain completely different sequence motifs (Fig. 1A), to GFP and examined whether they are imported into chloroplasts by the same general import pathway
In ppi2 and heat shock protein93-V (hsp93-V) mutant protoplasts, both RbcS-nt:GFP and Cab-nt:GFP localized in the cytosol as well as in chloroplasts (Fig. 1Bb, Bc, Be, and Bf), indicating that these mutant protoplasts show a defect in import of these reporter proteins
Summary
A large number of nuclear-encoded proteins are imported into chloroplasts after they are translated in the cytosol. The exact sequence is highly variable, the residues tend to be hydrophobic, making a high degree of hydrophobicity a common characteristic feature for both luminal and membrane proteins Despite their diversity in primary sequence, TPs share certain characteristics that serve as the basis for the software prediction of chloroplast proteins; these features include an amino acid composition with a high concentration of hydroxylated residues and a lack of acidic residues (Bruce, 2000; Bhushan et al, 2006), an unfolded and extended structure, an a-helix-containing secondary structure that may be induced by binding to the lipids of chloroplasts (Wienk et al, 1999; Bruce, 2000), and an abundance of Pro residues that may contribute to the unstructured nature of TPs (Pilon et al, 1995; Bruce, 2000; Zybailov et al, 2008). We demonstrated that functional synthetic transit peptides (SynTPs) can be generated by incorporating only a few sequence motifs from RbcS and Cab TPs into an unrelated sequence
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
