Import Determinants of Organelle-Specific and Dual Targeting Peptides of Mitochondria and Chloroplasts in Arabidopsis thaliana

Abstract

Most of the mitochondrial and chloroplastic proteins are synthesized in the cytosol as precursor proteins carrying an N-terminal targeting peptide (TP) directing them specifically to a correct organelle. However, there is a group of proteins that are dually targeted to mitochondria and chloroplasts using an ambiguous N-terminal dual targeting peptide (dTP). Here, we have investigated pattern properties of import determinants of organelle-specific TPs and dTPs combining mathematical multivariate data analysis (MVDA) with in vitro organellar import studies. We have used large datasets of mitochondrial and chloroplastic proteins found in organellar proteomes as well as manually selected data sets of experimentally confirmed organelle-specific TPs and dTPs from Arabidopsis thaliana. Two classes of organelle-specific TPs could be distinguished by MVDA and potential patterns or periodicity in the amino acid sequence contributing to the separation were revealed. dTPs were found to have intermediate sequence features between the organelle-specific TPs. Interestingly, introducing positively charged residues to the dTPs showed clustering towards the mitochondrial TPs in silico and resulted in inhibition of chloroplast, but not mitochondrial import in in vitro organellar import studies. These findings suggest that positive charges in the N-terminal region of TPs may function as an 'avoidance signal' for the chloroplast import.

Identification of cleavage sites and substrate proteins for two mitochondrial intermediate peptidases in Arabidopsis thaliana

“…Therefore, it is possible that more dual-targeted proteins have slightly different cleavage sites within the two organelles. However, in vitro import experiments suggest that most dual-targeted proteins are cleaved to the same size, indicating either identical cleavage sites or cleavage sites that are very close together ( Chew et al , 2003 ; Berglund et al , 2009 ; Ge et al , 2014 ).…”

“…In previous work looking at the presequences of dual-targeted proteins, it has been suggested that they fall somewhere in between plastid transit peptides and mitochondrial presequences ( Berglund et al , 2009 ; Mitschke et al , 2009 ; Ge et al , 2014 ). However, most of this work has been performed on arbitrary lengths of ~70 amino acids, not using the actual presequences from known dual-targeted proteins.…”

Interaction of the dual targeting peptide of Thr‐tRNA synthetase with the chloroplastic receptor Toc34 inArabidopsis thaliana

“…In previous work, the dual targeting peptide of AtThr-tRNA synthetase was shown to be unstructured in buffer but had a propensity to form an amphiphilic helix in the N-terminal part in membrane mimetic media [17]. Moreover, in a recent study, it has been suggested that the enrichment of arginines in mTPs may function as an ''avoidance signal'' for chloroplast import [30]. We now turn to a comparison of the binding of AtThrRS-dTP (2-60) to the two receptors.…”

Divergent evolution of the M3A family of metallopeptidases in plants

“…A, Table S1), with the OOP‐like orthologs predicted to localize either to the mitochondria or the chloroplasts (Table S1). As observed in Arabidopsis, it is likely that plant OOP orthologs are in fact dually targeted to mitochondria and chloroplasts, a feature that is difficult to predict due to the intermediate sequence composition of these targeting sequences (Carrie and Small , Carrie and Whelan , Ge et al ). The high sequence similarity between OOP and CyOP (92%) suggests that they arose from a recent gene duplication event, followed by the acquisition of organellar localization by OOP‐like peptidases.…”