Differential Transit Peptide Recognition during Preprotein Binding and Translocation into Flowering Plant Plastids

Abstract

Despite the availability of thousands of transit peptide (TP) primary sequences, the structural and/or physicochemical properties that determine TP recognition by components of the chloroplast translocon are not well understood. By combining a series of in vitro and in vivo experiments, we reveal that TP recognition is determined by sequence-independent interactions and vectorial-specific recognition domains. Using both native and reversed TPs for two well-studied precursors, small subunit of ribulose-1,5-bis-phosphate carboxylase/oxygenase, and ferredoxin, we exposed these two modes of recognition. Toc34 receptor (34-kD subunit of the translocon of the outer envelope) recognition in vitro, preprotein binding in organellar, precursor binding in vivo, and the recognition of TPs by the major stromal molecular motor Hsp70 are specific for the physicochemical properties of the TP. However, translocation in organellar and in vivo demonstrates strong specificity to recognition domain organization. This organization specificity correlates with the N-terminal placement of a strong Hsp70 recognition element. These results are discussed in light of how individual translocon components sequentially interact with the precursor during binding and translocation and helps explain the apparent lack of sequence conservation in chloroplast TPs.

Non-native, N-terminal Hsp70 Molecular Motor Recognition Elements in Transit Peptides Support Plastid Protein Translocation

“…These synthetic TPs of SSU (SStp) and FD (FDtp) contain the reversed aa sequences from C-to N-ter. Together with a series of their N-ter mutants, these constructs have confirmed the TP N-ter as an essential determinant for protein translocation into plastid stroma (21).…”

“…The DNA sequences of nine peptides, pp38, pp9, PepG, V10, A6R, HbS, np09, DRC8, and HA, were added on the PCR primers to replace SSR10 and SSF10. The reversed and scrambled N-ter mutants were produced similarly to replace the N-ter of SSF and FDF in pBS-SSF-YFP and pBS-FDF-YFP (21), respectively.…”

“…To ease the generation of Escherichia coli expression vectors, an NheI site was added to a pET-30a-based pET-SSF-YFP (21), producing pET-NheI-YFP. The expression cassette in pBS was subcloned into pET-NheI-YFP to generate pET constructs.…”

“…The N-ter mutants were generated based on two previously generated constructs, pBS-SSR10-MtoA-SSF-YFP and pBS-SSF10-MtoA-SSR-YFP (21), for plant expression. The DNA sequences of nine peptides, pp38, pp9, PepG, V10, A6R, HbS, np09, DRC8, and HA, were added on the PCR primers to replace SSR10 and SSF10.…”

“…Based on the predictions, all of our import-competent mutants contain a strong H70BS at their N-ter, which the import-deficient mutants lack (21). Because the stromal Hsp70 was shown to act as a plastid translocation motor (25)(26)(27)(28)(29), we suspected that the N-ter H70BS in our import-competent constructs is required for the stromal Hsp70 interaction to trap and/or pull the precursor into plastids (21) similar to the proposed unfolding and pulling model of chloroplast protein import (30).…”