Molecular Mechanism of the Specificity of Protein Import into Chloroplasts and Mitochondria in Plant Cells

Plants possess both types of endosymbiotic organelles, chloroplasts and mitochondria. Transit peptides and presequences function as signal sequences for specific import into chloroplasts and mitochondria, respectively. However, how these highly similar signal sequences confer the protein import specificity remains elusive. Here, we show that mitochondrial- or chloroplast-specific import involves two distinct steps, specificity determination and translocation across envelopes, which are mediated by the N-terminal regions and functionally interchangeable C-terminal regions, respectively, of transit peptides and presequences. A domain harboring multiple-arginine and hydrophobic sequence motifs in the N-terminal regions of presequences was identified as the mitochondrial specificity factor. The presence of this domain and the absence of arginine residues in the N-terminal regions of otherwise common targeting signals confers specificity of protein import into mitochondria and chloroplasts, respectively. AtToc159, a chloroplast import receptor, also contributes to determining chloroplast import specificity. We propose that common ancestral sequences were functionalized into mitochondrial- and chloroplast-specific signal sequences by the presence and absence, respectively, of multiple-arginine and hydrophobic sequence motifs in the N-terminal region.

Alternative Transcription Initiation and the AUG Context Configuration Control Dual-Organellar Targeting and Functional Competence of Arabidopsis Lon 1 Protease

Cellular homeostasis relies on components of protein quality control including chaperones and proteases. In bacteria and eukaryotic organelles, Lon proteases play a critical role in removing irreparably damaged proteins and thereby preventing the accumulation of deleterious degradation-resistant aggregates. Gene expression, live-cell imaging, immunobiochemical, and functional complementation approaches provide conclusive evidence for Lonl dual-targeting to chloroplasts and mitochondria. Dual-organellar deposition of Lonl isoforms depends on both transcriptional regula- tion and alternative translation initiation via leaky ribosome scanning from the first AUG sequence context that deviates extensively from the optimum Kozak consensus. Organelle-specific Lonl targeting results in partial complementation of Arabidopsis Ion1-1 mutants, whereas full complementation is solely accomplished by dual-organellar targeting. Both the optimal and non-optimal AUG sequence contexts are functional in yeast and facilitate leaky ribosome scanning com- plementing the piml phenotype when the mitochondrial presequence is used. Bioinformatic search identified a limited number of Arabidopsis genes with Lonl-type dual-targeting sequence organization. Lon4, the paralog of Lonl, has an ambiguous presequence likely evolved from the twin presequences of an ancestral Lonl-like gene, generating a single dual-targeted protein isoform. We postulate that Lonl and its subfunctional paralog Lon4 evolved complementary sub- sets of transcriptional and posttranscriptional regulatory components responsive to environmental cues for dual-organel- lar targeting.

Converting antimicrobial into targeting peptides reveals key features governing protein import into mitochondria and chloroplasts

We asked what peptide features govern targeting to the mitochondria versus the chloroplast,using antimicrobial peptides as a starting point.This approach was inspired by the endosymbiotic hypothesis that organelle-targeting peptides derive from antimicrobial amphipathic peptides delivered by the host cell,to which organelle progenitors became resistant.To explore the molecular changes required to convert antimicrobial into targeting peptides,we expressed a set of 13 antimicrobial peptides in Chlamydomonas reinhardtii.Peptides were systematically modified to test distinctive features of mitochondrion-and chloroplast-targeting peptides,and we assessed their targeting potential by following the intracellular localization and maturation of a Venus fluorescent reporter used as a cargo protein.Mitochondrial targeting can be achieved by some unmodified antimicrobial peptide sequences.Targeting to both organelles is improved by replacing lysines with arginines.Chloroplast targeting is enabled by the presence of flanking unstructured sequences,additional constraints consistent with chloroplast endosymbiosis having occurred in a cell that already contained mitochondria.If indeed targeting peptides evolved from antimicrobial peptides,then required modifications imply a temporal evolutionary scenario with an early exchange of cationic residues and a late acquisition of chloroplast-specific motifs.