Mitochondrial processing peptidase regulates PINK1 processing, import and Parkin recruitment

Mutations in phosphatase and tensin homologue-induced kinase 1 (PINK1) cause recessively inherited Parkinson's disease (PD), a neurodegenerative disorder linked to mitochondrial dysfunction. In healthy mitochondria, PINK1 is rapidly degraded in a process involving both mitochondrial proteases and the proteasome. However, when mitochondrial import is compromised by depolarization, PINK1 accumulates on the mitochondrial surface where it recruits the PD-linked E3 ubiquitin ligase Parkin from the cytosol, which in turn mediates the autophagic destruction of the dysfunctional organelles. Using an unbiased RNA-mediated interference (RNAi)-based screen, we identified four mitochondrial proteases, mitochondrial processing peptidase (MPP), presenilin-associated rhomboid-like protease (PARL), m-AAA and ClpXP, involved in PINK1 degradation. We find that PINK1 turnover is particularly sensitive to even modest reductions in MPP levels. Moreover, PINK1 cleavage by MPP is coupled to import such that reducing MPP activity induces PINK1 accumulation at the mitochondrial surface, leading to Parkin recruitment and mitophagy. These results highlight a new role for MPP in PINK1 import and mitochondrial quality control via the PINK1–Parkin pathway.     

Mitochondrial protein import

Most mitochondrial proteins are synthesized as precursor proteins on cytosolic polysomes and are subsequently imported into mitochondria. Many precursors carry amino-terminal presequences which contain information for their targeting to mitochondria. In several cases, targeting and sorting information is also contained in non-amino-terminal portions of the precursor protein. Nucleoside triphosphates are required to keep precursors in an import-competent (unfolded) conformation. The precursors bind to specific receptor proteins on the mitochondrial surface and interact with a general insertion protein (GIP) in the outer membrane. The initial interaction of the precursor with the inner membrane requires the mitochondrial membrane potential (delta psi) and occurs at contact sites between outer and inner membranes. Completion of translocation into the inner membrane or matrix is independent of delta psi. The presequences are cleaved off by the processing peptidase in the mitochondrial matrix. In several cases, a second proteolytic processing event is performed in either the matrix or in the intermembrane space. Other modifications can occur such as the addition of prosthetic groups (e.g., heme or Fe/S clusters). Some precursors of proteins of the intermembrane space or the outer surface of the inner membrane are retranslocated from the matrix space across the inner membrane to their functional destination ('conservative sorting'). Finally, many proteins are assembled in multi-subunit complexes. Exceptions to this general import pathway are known. Precursors of outer membrane proteins are transported directly into the outer membrane in a receptor-dependent manner. The precursor of cytochrome c is directly translocated across the outer membrane and thereby reaches the intermembrane space. In addition to the general sequence of events which occurs during mitochondrial protein import, current research focuses on the molecules themselves that are involved in these processes.     

Mitochondrial protein import

Most polypeptides of mitochondria are imported from the cytosol. Precursor proteins contain targeting and sorting information, often in the form of amino-terminal presequences. Precursors first bind to receptors in the outer membrane. Two putative import receptors have been identified: a 19-kilodalton protein (MOM19) inNeurospora mitochondria, and a 70-kilodalton protein (MAS70) in yeast. Some precursors integrate directly into the outer membrane, but the majority are translocated through one or both membranes. This process requires an electrochemical potential across the inner membrane. Import appears to occur through a hydrophilic pore, although the inner and outer membranes may contain functionally separate translocation machineries. In yeast, a 42-kilodalton protein (ISP42) probably forms part of the outer membrane channel. After import, precursors interact with chaperonin ATPases in the matrix. Presequences then are removed by the matrix protease. Finally, some proteins are retranslocated across the inner membrane to the intermembrane space.     

Mitochondrial protein import: two membranes,three translocases

Most mitochondrial proteins are synthesised in the cytosol and must be translocated across one or two membranes to reach their functional destination inside mitochondria. Dynamic protein complexes in the outer and inner membranes function as specific machineries that recognise the various kinds of precursor proteins and promote their translocation through protein-conducting channels. At least three major translocase complexes with a high flexibility and versatility are needed to ensure the proper import of precursor proteins into mitochondria.     

Mitochondrial processing peptidase: multiple-site recognition of precursor proteins

The Csk Homologous Kinase (CHK) has been shown to have an enzymatic activity similar to the tyrosine kinase Csk in that it down-regulates Src family kinase activity by causing phosphorylation of the Src C-terminal tyrosine residue. In megakaryocytic Mo7e cells, CHK associates with a specific phosphotyrosine juxtamembrane sequence of the SCF/KL-activated c-Kit receptor. Here, we show that in Mo7e cells, the major Src family kinase activity is p53/56(Lyn). Studies using immobilized c-Kit phosphopeptides show that Lyn is able to specifically associate with the tyrosine-phosphorylated juxtamembrane 568Y*VY*IDPT sequence of c-Kit which has previously been shown to associate with CHK. In cells over-expressing CHK by means of a recombinant vaccinia virus, we observed an elimination of the SCF/KL-stimulated Lyn kinase peak of activity observed at 2-5 minutes in cells infected with the helper T7-expressing vaccinia virus by itself. Examination of total tyrosine phosphorylation by Western blotting showed that over-expression of CHK resulted in a reduction in the levels of tyrosine phosphorylations in the range of 50-60 kDa, but had no apparent effect on c-Kit autophosphorylation. Taken together, these findings show that CHK is able to down-regulate SCF/KL-stimulated Lyn activity in megakaryocytes.     

Function of the stromal processing peptidase in the chloroplast import pathway

Chloroplast biogenesis depends on the import of a large diversity of proteins synthesized as precursors in the cytosol. The N-terminal targeting signal, the transit peptide, is proteolytically removed as proteins enter the organelle by a stromal processing peptidase (SPP) in a regulated series of steps. SPP contains a signature HXXEH zinc-binding motif found in members of the M16 metallopeptidase family, which includes, most notably, the mitochondrial processing peptidase. Here we discuss: (i) the broad range of substrates cleaved by SPP, yielding mature proteins for the numerous biosynthetic pathways of the organelle; (ii) the structural features that reside in both SPP and the transit peptide that determine the high specificity of precursor cleavage; (iii) the downregulation of SPP in vivo which shows that it is essential for plant survival; and (iv) the relationship between SPP from higher plants and proteases in several lower eukaryotes and the cyanobacteria.     

Mitochondrial protein import and human health and disease

The targeting and assembly of nuclear-encoded mitochondrial proteins are essential processes because the energy supply of humans is dependent upon the proper functioning of mitochondria. Defective import of mitochondrial proteins can arise from mutations in the targeting signals within precursor proteins, from mutations that disrupt the proper functioning of the import machinery, or from deficiencies in the chaperones involved in the proper folding and assembly of proteins once they are imported. Defects in these steps of import have been shown to lead to oxidative stress, neurodegenerative diseases, and metabolic disorders. In addition, protein import into mitochondria has been found to be a dynamically regulated process that varies in response to conditions such as oxidative stress, aging, drug treatment, and exercise. This review focuses on how mitochondrial protein import affects human health and disease.     

Mitochondrial protein import in trypanosomes: Expect the unexpected

Mitochondria have many different functions, the most important one of which is oxidative phosphorylation. They originated from an endosymbiotic event between a bacterium and an archaeal host cell. It was the evolution of a protein import system that marked the boundary between the endosymbiotic ancestor of the mitochondrion and a true organelle that is under the control of the nucleus. In present day mitochondria more than 95% of all proteins are imported from the cytosol in a proces mediated by hetero‐oligomeric protein complexes in the outer and inner mitochondrial membranes. In this review we compare mitochondrial protein import in the best studied model system yeast and the parasitic protozoan Trypanosoma brucei. The 2 organisms are phylogenetically only remotely related. Despite the fact that mitochondrial protein import has the same function in both species, only very few subunits of their import machineries are conserved. Moreover, while yeast has 2 inner membrane protein translocases, one specialized for presequence‐containing and one for mitochondrial carrier proteins, T. brucei has a single inner membrane translocase only, that mediates import of both types of substrates. The evolutionary implications of these findings are discussed.      

Mitochondrial protein import: convergent solutions for receptor structure

Complex machinery has evolved to recognise and import nuclear-encoded proteins into mitochondria. Recent work now shows that the plant Tom20 mitochondrial protein import receptor has a similar tertiary structure to animal Tom20, although the proteins are evolutionarily distinct, representing an elegant example of convergent evolution.     

PEP A9, a new,unstable variant in the peptidase a system

Summary A rare peptidase A variant, tentatively designated PEP A⁹, was observed in six members of a German family, indicating autosomal codominant inheritance. The electrophoretic mobility is similar to that of PEP A 3-1, but it has very low in vivo stability. There is no apparent association with a disease state. A simple and sensitive staining reagent for PEP A was found in o-phthalaldehyde.     

Mitochondrial protein import regulates cytosolic protein homeostasis and neuronal integrity

Neurodegeneration is characterized by protein aggregate deposits and mitochondrial malfunction. Reduction in Tom40 (translocase of outer membrane 40) expression, a key subunit of the translocase of the outer mitochondrial membrane complex, led to accumulation of ubiquitin (Ub)-positive protein aggregates engulfed by Atg8a-positive membranes. Other macroautophagy markers were also abnormally accumulated. Autophagy was induced but the majority of autophagosomes failed to fuse with lysosomes when Tom40 was downregulated. In Tom40 RNAi tissues, autophagosome-like (AL) structures, often not sealed, were 10 times larger than starvation induced autophagosomes. Atg5 downregulation abolished Tom40 RNAi induced AL structure formation, but the Ub-positive aggregates remained, whereas knock down of Syx17, a gene required for autophagosome-lysosome fusion, led to the disappearance of giant AL structures and accumulation of small autophagosomes and phagophores near the Ub-positive aggregates. The protein aggregates contained many mitochondrial preproteins, cytosolic proteins, and proteasome subunits. Proteasome activity and ATP levels were reduced and the ROS levels was increased in Tom40 RNAi tissues. The simultaneous inhibition of proteasome activity, reduction in ATP production, and increase in ROS, but none of these conditions alone, can mimic the imbalanced proteostasis phenotypes observed in Tom40 RNAi cells. Knockdown of ref(2)P or ectopic expression of Pink1 and park greatly reduced aggregate formation in Tom40 RNAi tissues. In nerve tissues, reduction in Tom40 activity leads to aggregate formation and neurodegeneration. Rather than diminishing the neurodegenerative phenotypes, overexpression of Pink1 enhanced them. We proposed that defects in mitochondrial protein import may be the key to linking imbalanced proteostasis and mitochondrial defects.

Abbreviations: AL: autophagosome-like; Atg12: Autophagy-related 12; Atg14: Autophagy-related 14; Atg16: Autophagy-related 16; Atg5: Autophagy-related 5; Atg6: Autophagy-related 6; Atg8a: Autophagy-related 8a; Atg9: Autophagy-related 9; ATP: adenosine triphosphate; Cas9: CRISPR associated protein 9; cDNA: complementary DNA; COX4: Cytochrome c oxidase subunit 4; CRISPR: clustered regularly interspaced short palindromic repeats; Cyt-c1: Cytochrome c1; DAPI: 4,6-diamidino-2-phenylindole dihydrochloride; Dcr-2: Dicer-2; FLP: Flippase recombination enzyme; FRT: FLP recombination target; GFP: green fluorescent protein; GO: gene ontology; gRNA: guide RNA; Hsp60: Heat shock protein 60A; HDAC6: Histone deacetylase 6; htt: huntingtin; Idh: Isocitrate dehydrogenase; IFA: immunofluorescence assay; Irp-1A: Iron regulatory protein 1A; kdn: knockdown; Marf: Mitochondrial assembly regulatory factor; MitoGFP: Mitochondrial-GFP; MS: mass spectrometry; MTPAP: mitochondrial poly(A) polymerase; Nmnat: Nicotinamide mononucleotide adenylyltransferase; OE: overexpression; Pink1/PINK1: PTEN-induced putative kinase 1; polyQ: polyglutamine; PRKN: parkin RBR E3 ubiquitin protein ligase; Prosα4: proteasome α4 subunit; Prosβ1: proteasome β1 subunit; Prosβ5: proteasome β5 subunit; Prosβ7: proteasome β7 subunit; ref(2)P: refractory to sigma P; RFP: red fluorescent protein; RNAi: RNA interference; ROS: reactive oxygen species; Rpn11: Regulatory particle non-ATPase 11; Rpt2: Regulatory particle triple-A ATPase 2; scu: scully; sicily: severe impairment of CI with lengthened youth; sesB: stress-sensitive B; Syx17: Syntaxin17; TEM: transmission electron microscopy; ttm50: tiny tim 50; Tom: translocase of the outer membrane; Tom20: translocase of outer membrane 20; Tom40: translocase of outer membrane 40; Tom70: translocase of outer membrane 70; UAS: upstream active sequence; Ub: ubiquitin; VNC: ventral nerve cord; ZFYVE1: zinc finger FYVE-type containing 1     

Mitochondrial protein import: modification of sulfhydryl groups of the inner mitochondrial membrane import machinery in Solanum tuberosum inhibits protein import

Protein import into mitochondria involves several components of the mitochondrial outer and inner membranes as well as molecular chaperones located inside mitochondria. Here, we have investigated the effect of sulfhydryl group reagents on import of the in vitro transcribed/translated precursor of the F₁ subunit of the ATP synthase (pF₁) into Solanum tuberosum mitochondria. We have used a reducing agent, dithiothreitol (DTT), a membrane-permeant alkylating agent, N-ethylmaleimide (NEM), a non-permeant alkylating agent, 3-(N-maleimidopropionyl)biocytin (MPB), an SH-group specific agent and cross-linker 5,5-dithiobis-(2-nitrobenzoic acid) (DTNB) as well as an oxidizing cross-linker, copper sulfate. DTT stimulated the mitochondrial protein import, whereas NEM, MPB, DTNB and Cu²⁺ were inhibitory. Inhibition by Cu²⁺ could be reversed by addition of DTT. The efficiency of inhibition was higher in energized mitochondria than in non-energized. We have dissected the effect of the SH-group reagents on binding, unfolding and transport of the precursor into mitochondria. Our results demonstrated that the inhibitory effect of NEM, DTNB and Cu²⁺ on the efficiency of import was not due to the interaction of the SH-group reagents with import receptors. Modification of pF₁ with NEM prior to the import resulted in stimulation of import, whereas DTNB and Cu²⁺ were inhibitory. NEM, MPB, DTNB and Cu²⁺ inhibited import of the NEM-modified pF₁ into intact mitochondria. Import of pF₁ through a receptor-independent bypass-route as well as import into mitoplasts were sensitive to DTT, NEM, MPB, DTNB and Cu²⁺ in a similar manner as import into mitochondria. As MPB does not cross the inner membrane, these results indicated that redox and conformational status of SH groups located on the outer surface of the inner mitochondrial membrane were essential for protein import.     

The processing peptidase of yeast mitochondria: the two co-operating components MPP and PEP are structurally related.

Two proteins co-operate in the proteolytic cleavage of mitochondrial precursor proteins: the mitochondrial processing peptidase (MPP) and the processing enhancing protein (PEP). In order to understand the structure and function of this novel peptidase, we have isolated mutants of Saccharomyces cerevisiae which were temperature sensitive in the processing of mitochondrial precursor proteins. Here we report on the mif2 mutation which is deficient in MPP. Mitochondria from the mif2 mutant were able to import precursor proteins, but not to cleave the presequences. The MPP gene was isolated. MPP is a hydrophilic protein consisting of 482 amino acids. Notably, MPP exhibits remarkable sequence similarity to PEP. We speculate that PEP and MPP have a common origin and have evolved into two components with different but mutually complementing functions in processing of precursor proteins.     

Mitochondrial protein import: recognition of internal import signals of BCS1 by the TOM complex

BCS1, a component of the inner membrane of mitochondria, belongs to the group of proteins with internal, noncleavable import signals. Import and intramitochondrial sorting of BCS1 are encoded in the N-terminal 126 amino acid residues. Three sequence elements were identified in this region, namely, the transmembrane domain (amino acid residues 51 to 68), a presequence type helix (residues 69 to 83), and an import auxiliary region (residues 84 to 126). The transmembrane domain is not required for stable binding to the TOM complex. The Tom receptors (Tom70, Tom22 and Tom20), as determined by peptide scan analysis, interact with the presequence-like helix, yet the highest binding was to the third sequence element. We propose that the initial recognition of BCS1 precursor at the surface of the organelle mainly depends on the auxiliary region and does not require the transmembrane domain. This essential region represents a novel type of signal with targeting and sorting functions. It is recognized by all three known mitochondrial import receptors, demonstrating their capacity to decode various targeting signals. We suggest that the BCS1 precursor crosses the TOM complex as a loop structure and that once the precursor emerges from the TOM complex, all three structural elements are essential for the intramitochondrial sorting to the inner membrane.     

Mitochondrial disulfide relay: redox-regulated protein import into the intermembrane space

99% of all mitochondrial proteins are synthesized in the cytosol, from where they are imported into mitochondria. In contrast to matrix proteins, many proteins of the intermembrane space (IMS) lack presequences and are imported in an oxidation-driven reaction by the mitochondrial disulfide relay. Incoming polypeptides are recognized and oxidized by the IMS-located receptor Mia40. Reoxidation of Mia40 is facilitated by the sulfhydryl oxidase Erv1 and the respiratory chain. Although structurally unrelated, the mitochondrial disulfide relay functionally resembles the Dsb (disufide bond) system of the bacterial periplasm, the compartment from which the IMS was derived 2 billion years ago.     

Sequential processing of a mitochondrial tandem protein: insights into protein import in Schizosaccharomyces pombe

The sequencing of the genome of Schizosaccharomyces pombe revealed the presence of a number of genes encoding tandem proteins, some of which are mitochondrial components. One of these proteins (pre-Rsm22-Cox11) consists of a fusion of Rsm22, a component of the mitochondrial ribosome, and Cox11, a factor required for copper insertion into cytochrome oxidase. Since in Saccharomyces cerevisiae, Cox11 is physically attached to the mitochondrial ribosome, it was suggested that the tandem organization of Rsm22-Cox11 is used to covalently tie the mitochondrial ribosome to Cox11 in S. pombe. We report here that pre-Rsm22-Cox11 is matured in two subsequent processing events. First, the mitochondrial presequence is removed. At a later stage of the import process, the Rsm22 and Cox11 domains are separated by cleavage of the mitochondrial processing peptidase at an internal processing site. In vivo data obtained using a tagged version of pre-Rsm22-Cox11 confirmed the proteolytic separation of Cox11 from the Rsm22 domain. Hence, the tandem organization of pre-Rsm22-Cox11 does not give rise to a persistent fusion protein but rather might be used to increase the import efficiency of Cox11 and/or to coordinate expression levels of Rsm22 and Cox11 in S. pombe.     

Mitochondrial protein import: from transport pathways to an integrated network

Mitochondria, the powerhouses of the cell, import most of their proteins from the cytosol. It was originally assumed that mitochondria imported precursor proteins via a general pathway but recent studies have revealed a remarkable variety of import pathways and mechanisms. Currently, five different protein import pathways can be distinguished. However, the import machineries cooperate with each other and are connected to other systems that function in the respiratory chain, mitochondrial membrane organization, protein quality control and endoplasmic reticulum-mitochondria junctions. In this Opinion, we propose that mitochondrial protein import should not be seen as an independent task of the organelle and that a network of cooperating machineries is responsible for major mitochondrial functions.     

Mitochondrial protein import in plants – Signals,Sorting, Targeting,Processing and Regulation

Plant Molecular Biology - Mitochondrial biogenesis requires a coordinated expression of both the nuclear and the organellar genomes and specific intracellular protein trafficking, processing and...