Sequence Motifs in Transit Peptides Act as Independent Functional Units and Can Be Transferred to New Sequence Contexts  

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/bbinding 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.

Arabidopsis BAG1 Functions as a Cofactor in Hsc70-Mediated Proteasomal Degradation of Unimported Plastid Proteins

“…We transformed the wild-type construct RbcS-nt:GFP with or without T7-Hsc70-4 or T7-AtBAG1 into hsp93-V protoplasts and examined the precursor level of RbcS-nt-GFP. In hsp93-V plants, import of preproteins is significantly delayed and M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT 4 precursors remain in the cytosol (Lee et al, 2015). Unlike in wild-type protoplasts ( Figure 1F and 1G), in hsp93-V protoplasts, the precursors of RbcS-nt:GFP were degraded in a proteasome-dependent manner even when introduced into protoplasts alone, and they were degraded at higher levels in the presence of T7-Hsc70-4 or T7-AtBAG1 ( Figure 1I;…”

“…The construct O2-GFP contains the minimal sequence motif directly recognized by Hsc70-4 and is strongly degraded in protoplasts overexpressing Hsc70-4 (Lee et al, 2009). RbcS-nt∆T6,7-GFP, a construct in which the T6 and T7 segments in the RbcS transit peptide were deleted, primarily remains in the cytosol even in the presence of critical sequence motifs required for efficient chloroplast targeting, such as the MLM, FNGLK, and FP/RK motifs ( Figure 1E) (Lee et al, 2006(Lee et al, , 2015.…”

“…Therefore, in the current study, we tested three types of constructs as the substrates of Hsc70-4 and AtBAG1 ( Figure 1E). RbcS-nt:GFP has been used as a model substrate to monitor preprotein targeting to chloroplasts, and the overexpression of Hsc70-4 has little effect on this construct (Lee et al, 2006(Lee et al, , 2009(Lee et al, , 2015. The construct O2-GFP contains the minimal sequence motif directly recognized by Hsc70-4 and is strongly degraded in protoplasts overexpressing Hsc70-4 (Lee et al, 2009).…”

Transit peptide elements mediate selective protein targeting to two different types of chloroplasts in the single-cell C4 species Bienertia sinuspersici

“…To characterize the specific import pathway in Bienertia, we used extensive alanine scanning, a method that has previously been utilized successfully to identify specific motifs in the TPs of Arabidopsis34353637. Interestingly, our mutation experiments indicate that the identified elements on the TPs of P-specific precursors prevent import into the CC rather than facilitating the specific import into the P-chloroplasts (Fig.…”

Chloroplast Hsp93 Directly Binds to Transit Peptides at an Early Stage of the Preprotein Import Process

“…In Arabidopsis (Arabidopsis thaliana), Hsp93 exists as two isoforms encoded by the genes HSP93III and HSP93V. Removal of the more abundant Hsp93V results in protein import defects, while double knockout of the two genes causes lethality (Constan et al, 2004;Kovacheva et al, 2007;Chu and Li, 2012;Lee et al, 2015). Purified recombinant Hsp93III can bind to the transit peptide of pea (Pisum sativum) ferredoxin-NADP+ reductase in vitro (Rosano et al, 2011).…”