Dissecting the molecular mechanisms of mitochondrial import and maturation of peroxiredoxins from yeast and mammalian cells

Abstract

Peroxiredoxins (Prxs) are cysteine-based peroxidases that play a central role in keeping the H₂O₂ at physiological levels. Eukaryotic cells express different Prxs isoforms, which differ in their subcellular locations and substrate specificities. Mitochondrial Prxs are synthesized in the cytosol as precursor proteins containing N-terminal cleavable presequences that act as mitochondrial targeting signals. Due to the fact that presequence controls the import of the vast majority of mitochondrial matrix proteins, the mitochondrial Prxs were initially predicted to be localized exclusively in the matrix. However, recent studies showed that mitochondrial Prxs are also targeted to the intermembrane space by mechanisms that remain poorly understood. While in yeast the IMP complex can translocate Prx1 to the intermembrane space, the maturation of yeast Prx1 and mammalian Prdx3 and Prdx5 in the matrix has been associated with sequential cleavages of the presequence by MPP and Oct1/MIP proteases. In this review, we describe the state of the art of the molecular mechanisms that control the mitochondrial import and maturation of Prxs of yeast and human cells. Once mitochondria are considered the major intracellular source of H₂O₂, understanding the mitochondrial Prx biogenesis pathways is essential to increase our knowledge about the H₂O₂-dependent cellular signaling, which is relevant to the pathophysiology of some human diseases.

Keywords: H2O2; Intermembrane space; Matrix; Mitochondria; Peroxiredoxin; Presequence.

Fig. 1

Overview of the protein import machineries of mitochondria. The figure depicts only the protein import routes that use the translocase of the outer mitochondrial membrane (TOM complex) as the mitochondrial gateway. The cytosolic precursors are recognized by the TOM complex and targeted to their dedicated mitochondrial subcompartments by the corresponding protein sorting pathways. Presequence-carrying precursors are targeted to the matrix by the translocase of the inner membrane (TIM23) and presequence translocase-associated motor (PAM). Upon arrival in the matrix, the presequence is removed by the mitochondrial processing protease (MPP). Moreover, some matrix proteins are further cleaved by the intermediate cleaving peptidase (Icp55) or by the octapeptidyl aminopeptidase (Oct1) proteases, which remove a single or eight amino acids from the MPP-cleaved N-termini, respectively. Some presequence-carrying precursors contain an additional hydrophobic sorting signal that causes the lateral release of the precursor into the lipid phase of the inner mitochondrial membrane. Cysteine-rich proteins destined to the intermembrane space (IMS) are imported by the mitochondrial intermembrane space assembly (MIA) machinery, which promotes the insertion of disulfide bonds into the imported proteins. Hydrophobic metabolite carriers of the inner mitochondrial membrane are imported by the small TIM chaperones of the IMS and the carrier translocase of the inner membrane (TIM22). Finally, the precursors of β-barrel proteins of the outer membrane are imported by the small TIM chaperones and by the sorting and assembly machinery (SAM) complex

Fig. 2

Proposed biogenesis pathway of yeast peroxiredoxin Prx1. Prx1 is synthesized on cytosolic ribosomes as a precursor protein with an N-terminal cleavable presequence which directs the import of Prx1 into two distinct mitochondrial subcompartments: matrix and IMS. During its import through mitochondria, the Prx1 precursor is initially translocated across the outer membrane via the TOM complex. The precursor is then handed over to the TIM23 complex, which mediates the protein translocation across the inner membrane. At the TIM23 level, the Prx1 precursor can be sorted into the inner membrane or into the matrix. The import into the matrix is performed with the help of the PAM complex. After import, Prx1 presequence is removed by the MPP protease, generating an intermediate form of the Prx1 that is further cleaved by Oct1, which takes out an additional octapeptide from the Prx1 N-terminus, leaving Lys39 as the N-terminus of the mature Prx1. Alternatively, Prx1 import into the inner membrane probably involves a stop-transfer pathway triggered by an abridge hydrophobic sorting signal located immediately downstream of the Prx1 presequence, which induces the lateral release of the precursor into the lipid phase of the inner membrane. Therefore, Prx1 is cleaved by the IMP complex and released as a mature protein into the IMS with Ala32 in its N-terminus. The upper panel denotes the Prx1 precursor N-terminal amino acid sequence. Arrows indicate the cleavage sites of the proteases involved in Prx1 maturation

Fig. 3

Presequence analysis of the human Prdx3 and Prdx5. The amino acid frequency blots of the human MIP cleavage site recognition motif were generated with the WebLogo program using a list of 32 predicted substrates of human MIP protease. The R-10 motif contains three main features: a highly conserved arginine at position − 10, the bulky hydrophobic residues at position − 8, and the small hydroxylated residues at position − 5 relative to the mature N-termini of the proteins. The red bars indicate cleavage by MPP and MIP, respectively. The 14 residues depicted by negative numbers (including the octapeptide) constitute the C-terminal segment of the presequence and the 4 residues highlighted by positive numbers represent the N-terminal of the mature proteins

Fig. 4

Proposed biogenesis pathway of human peroxiredoxins Prdx3 and Prdx5. Both Prxs are synthesized in the cytosol as precursor proteins containing N-terminal cleavable presequences, which direct the precursors to the mitochondrial matrix. In Prx precursor import into mitochondria, proteins are translocated across the TOM and TIM23 complexes. The complete translocation into the matrix further requires the motor PAM. After reaching the mitochondrial matrix, the presequences are cleaved by the MPP protease. Based on experimental data with the mouse Prdx5, human Prdx5 probably undergoes a second cleavage catalyzed by the MIP protease, which removes an octapeptide from the MPP-cleaved N-termini. Similarly, human Prdx3 probably is also cleaved by MIP as it displays the R-10 cleavage site motif (see Fig. 3), and it is cleaved by the yeast MIP homologue (Oct1 protease). Besides being directed to the matrix, Prdx3 is also sorted to the IMS. However, the molecular mechanism involved in the sorting of the Prdx3 to the IMS remains unknown. The upper panel denotes the human Prdx3 and Prdx5 N-terminal amino acid sequences. Arrows indicate the cleavage sites of the proteases involved in the maturation of human Prxs