Experimental and Computational Analysis of Chloroplast Transit Peptide Domain Architecture and Function

The Majority of chloroplast proteins are nuclear-encoded and utilize an N-terminal transit peptide (TP) to target into chloroplasts via the general import pathway. Bioinformatic and proteomic analyses provide thousands of predicted TPs, which show low sequence similarity. How the common chloroplast translocon components recognize these diverse TPs is not well understood. Previous results support either sequence- or physicochemical-specific recognitions. To further address this question, a reverse sequence approach was utilized such that the reverse TP contains the same amino acid composition as wild-type TP but lack similar sequence motifs. Using both native and reverse TPs of the two well-studied precursors, we explored these two modes of recognition. We found that reverse TPs behaved similar to wild-type TPs during binding but failed to support protein translocation. We further showed the importance of the N-terminal domain of TPs in governing protein translocation into plastids. When the TP N-termini were replaced with unrelated peptides with varying Hsp70 affinities, we showed that a subset of TP N-termini functions as Hsp70-interacting domains. We proposed that these domains interact with the stromal motor Hsp70. We further identified a conserved spacer distance between these N-terminal Hsp70 domains to the translocon receptor Toc34 binding sites called FGLK motifs. Using mutants with varying spacer lengths, we observed that the most efficient translocation occurred only at an optimal spacer length of around 28 to 31 aa. These results led us to propose the bimodal interaction model of TP architecture and function where a TP contains an N-terminal stromal interacting domain that is linked to a Toc interacting domain via an optimal spacer length. This configuration permits a temporal and/or spatial coupling between a capturing step by a TOC receptor and a trapping/pulling step by a stromal ATP-dependent molecular motor that is required for productive translocatio