Refining the Definition of Plant Mitochondrial Presequences through Analysis of Sorting Signals, N-Terminal Modifications, and Cleavage Motifs    

Abstract

Mitochondrial protein import is a complex multistep process from synthesis of proteins in the cytosol, recognition by receptors on the organelle surface, to translocation across one or both mitochondrial membranes and assembly after removal of the targeting signal, referred to as a presequence. In plants, import has to further discriminate between mitochondria and chloroplasts. In this study, we determined the precise cleavage sites in the presequences for Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa) mitochondrial proteins using mass spectrometry by comparing the precursor sequences with experimental evidence of the amino-terminal peptide from mature proteins. We validated this method by assessments of false-positive rates and comparisons with previous available data using Edman degradation. In total, the cleavable presequences of 62 proteins from Arabidopsis and 52 proteins from rice mitochondria were determined. None of these proteins contained amino-terminal acetylation, in contrast to recent findings for chloroplast stromal proteins. Furthermore, the classical matrix glutamate dehydrogenase was detected with intact and amino-terminal acetylated sequences, indicating that it is imported into mitochondria without a cleavable targeting signal. Arabidopsis and rice mitochondrial presequences had similar isoelectric points, hydrophobicity, and the predicted ability to form an amphiphilic a-helix at the amino-terminal region of the presequence, but variations in length, amino acid composition, and cleavage motifs for mitochondrial processing peptidase were observed. A combination of lower hydrophobicity and start point of the amino-terminal a-helix in mitochondrial presequences in both Arabidopsis and rice distinguished them (98%) from Arabidopsis chloroplast stroma transit peptides. Both Arabidopsis and rice mitochondrial cleavage sites could be grouped into three classes, with conserved 23R (class II) and 22R (class I) or without any conserved (class III) arginines. Class II was dominant in both Arabidopsis and rice (55%-58%), but in rice sequences there was much less frequently a phenylalanine (F) in the 21 position of the cleavage site than in Arabidopsis sequences. Our data also suggest a novel cleavage motif of (F/Y)Y(S/A) in plant class III sequences.

Organellar oligopeptidase (OOP) provides a complementary pathway for targeting peptide degradation in mitochondria and chloroplasts

“…Analysis of experimentally determined mitochondrial presequences in plants (A. thaliana and O. sativa), based on the identification of the N terminus of the mature mitochondrial proteins, showed ∼10% of presequences had a length ≤23 aa (16). From the group of mitochondrial presequences ≤23 aa, we tested pL29 (19 aa) and pACD1 (20 aa) as substrates for OOP.…”

“…In A. thaliana, mitochondrial presequences range from 19 to 109 aa (16); chloroplastic transit peptides usually are longer, ranging from 26 to 146 aa (9).…”

Mitochondrial Targeting of theArabidopsisF1-ATPase γ-Subunit via Multiple Compensatory and Synergistic Presequence Motifs

“…The N-terminal presequences (PSs) range in size from 15-to 70-amino acid residues and have been demonstrated to be both necessary and sufficient for protein import into MT (von Heijne, 1986;Rapaport, 2003). Although no primary sequence consensus for targeting to the MT has been derived, detailed analyses have revealed several characteristic features, including the enrichment of hydroxylated and hydrophobic amino acids, an abundance of Arg residues, a lack of acidic amino acids, and a propensity to form an a-helix (Glaser et al, 1998;Huang et al, 2009). The configuration of positively charged and hydrophobic amino acids has been demonstrated to form an amphipathic a-helix in solution, and NMR analysis of the central mitochondrial outer membrane (MOM) import receptor, Tom20, in complex with an N-terminal PS revealed that the hydrophobic surface of the amphipathic a-helix binds to the hydrophobic groove of Tom20 (Abe et al, 2000).…”

Molecular Mechanisms and Physiological Significance of Organelle Interactions and Cooperation

“…However, these sequences share preferences in physicochemical properties and the frequency of individual amino acids such as an overrepresentation of positively charged residues and, more specifically, of arginine, whereas negatively charged residues are nearly absent ( Figure 2B) (von Heijne et al, 1989;Huang et al, 2009). Accordingly, mitochondrial targeting signals can be generated quite easily de novo by mutations (Vassarotti et al, 1987) or insertion of arbitrary amino acid sequences at the N-terminus of a protein (Lemire et al, 1989).…”

“…These unfolded proteins can interact with membrane-bound receptors on the surface of mitochondria, chloroplasts, or the ER and the transfer to the organellar membrane can be accelerated by cytosolic factors such as mitochondrial import stimulatory factor (MSF) (Hachiya et al, 1993) or 14-3-3 proteins for chloroplasts (May and Soll, 2000). Properties that distinguish mitochondrial and chloroplast preproteins have been elucidated (Huang et al, 2009), but the import mechanisms do not suggest a hierarchical relation between these targeting signals ( Figure 3B; [3]). In contrast, peroxisomal, nuclear, and cytosolic proteins are folded in the cytoplasm with the help of folding chaperones.…”