Multiple targeting modules on peroxisomal proteins are not redundant: discrete functions of targeting signals within Pmp47 and Pex8p

Several peroxisomal proteins have two nonoverlapping targeting signals. These signals have been termed “redundant” because targeting can still occur with only one signal. We now report that separate targeting motifs within both Pmp47 and Pex8 provide complementary function. Pmp47 is an ATP translocator that contains six transmembrane domains (TMDs). We had previously shown that the TMD2 region (termed TMD2R, consisting of TMD2 and a short adjacent segment of cytosolic loop) was required for targeting to proliferated peroxisomes in Saccharomyces cerevisiae. We now report that the analogous TMD4R, which cannot target to proliferated peroxisomes, targets at least as well, or much better (depending on strain and growth conditions) in cells containing only basal (i.e., nonproliferated) peroxisomes. These data suggest differences in the targeting pathway among peroxisome populations. Pex8p, a peripheral protein facing the matrix, contains a typical carboxy terminal targeting sequence (PTS1) that has been shown to be nonessential for targeting, indicating the existence of a second targeting domain (not yet defined in S. cerevisiae); thus, its function was unknown. We show that targeting to basal peroxisomes, but not to proliferated peroxisomes, is more efficient with the PTS1 than without it. Our results indicate that multiple targeting signals within peroxisomal proteins extend coverage among heterogeneous populations of peroxisomes and increase efficiency of targeting in some metabolic states.     

The Arabidopsis tail-anchored protein PEROXISOMAL AND MITOCHONDRIAL DIVISION FACTOR1 is involved in the morphogenesis and proliferation of peroxisomes and mitochondria

Peroxisomes and mitochondria are multifunctional eukaryotic organelles that are not only interconnected metabolically but also share proteins in division. Two evolutionarily conserved division factors, dynamin-related protein (DRP) and its organelle anchor FISSION1 (FIS1), mediate the fission of both peroxisomes and mitochondria. Here, we identified and characterized a plant-specific protein shared by these two types of organelles. The Arabidopsis thaliana PEROXISOMAL and MITOCHONDRIAL DIVISION FACTOR1 (PMD1) is a coiled-coil protein tethered to the membranes of peroxisomes and mitochondria by its C terminus. Null mutants of PMD1 contain enlarged peroxisomes and elongated mitochondria, and plants overexpressing PMD1 have an increased number of these organelles that are smaller in size and often aggregated. PMD1 lacks physical interaction with the known division proteins DRP3 and FIS1; it is also not required for DRP3's organelle targeting. Affinity purifications pulled down PMD1's homolog, PMD2, which exclusively targets to mitochondria and plays a specific role in mitochondrial morphogenesis. PMD1 and PMD2 can form homo- and heterocomplexes. Organelle targeting signals reside in the C termini of these proteins. Our results suggest that PMD1 facilitates peroxisomal and mitochondrial proliferation in a FIS1/DRP3-independent manner and that the homologous proteins PMD1 and PMD2 perform nonredundant functions in organelle morphogenesis.     

Alternative topogenic signals in peroxisomal citrate synthase of Saccharomyces cerevisiae.

The tripeptide serine-lysine-leucine (SKL) occurs at the carboxyl terminus of many peroxisomal proteins and serves as a peroxisomal targeting signal. Saccharomyces cerevisiae has two isozymes of citrate synthase. The peroxisomal form, encoded by CIT2, terminates in SKL, while the mitochondrial form, encoded by CIT1, begins with an amino-terminal mitochondrial signal sequence and ends in SKN. We analyzed the importance of SKL as a topogenic signal for citrate synthase, using oleate to induce peroxisomes and density gradients to fractionate organelles. Our experiments revealed that SKL was necessary for directing citrate synthase to peroxisomes. C-terminal SKL was also sufficient to target a leaderless version of mitochondrial citrate synthase to peroxisomes. Deleting this tripeptide from the CIT2 protein caused peroxisomal citrate synthase to be missorted to mitochondria. These experiments suggest that the CIT2 protein contains a cryptic mitochondrial targeting signal.     

Identification of a cryptic N-terminal signal in Saccharomyces cerevisiae peroxisomal citrate synthase that functions in both peroxisomal and mitochondrial targeting

Saccharomyces cerevisiae has three distinct citrate synthases, two located in mitochondria (mature Cit1p and Cit3p) and one in peroxisomes (mature Cit2p). While the precursor of the major mitochondrial enzyme, Cit1p, has a signal for mitochondrial targeting at its N-terminus (MTS), Cit2p has one for peroxisomal targeting (PTS1) at its C-terminus. We have previously shown that the N-terminal segment of Cit2p is removed during import into peroxisomes [Lee, H.S. et al. (1994) Kor. J. Microbiol. 32, 558-564], which implied the presence of an additional N-terminal sorting signal. To analyze the function of the N-terminal region of Cit2p in protein trafficking, we constructed the N-terminal domain-swapped versions of Cit1p and Cit2p. Both fusions, Cit1::Cit2 and Cit2::Cit1, complemented the glutamate auxotrophy caused by the double-disruption of the CIT1 and CIT2 genes. In addition, part of the Cit2::Cit1 fusion protein, as well as Cit1::Cit2, was shown to be transported into both mitochondria and peroxisomes. The subcellular localization of the recombinant fusion proteins containing various N-terminal segments of Cit2p fused to a mutant version of green fluorescent protein (GFP2) was also examined. As a result, we found that the 20-amino acid N-terminal segment of Cit2p contains a cryptic cleavable targeting signal for both peroxisomes and mitochondria. In addition, we show that the peroxisomal import process mediated by the N-terminal segment of Cit2p was not affected by the disruption of either PEX5 (encoding PTS1 receptor) or PEX7 (encoding PTS2 receptor).     

Sterol Carrier Protein-2, a Nonspecific Lipid-Transfer Protein,in Intracellular Cholesterol Trafficking in Testicular Leydig Cells

Sterol carrier protein-2 (SCP2), also called nonspecific lipid-transfer protein, is thought to play a major role in intracellular lipid transport and metabolism, and it has been associated with diseases involving abnormalities in lipid trafficking, such as Zellweger syndrome. The Scp2 gene encodes the 58 kDa sterol carrier protein-x (SCPX) and 15 kDa pro-SCP2 proteins, both of which contain a 13 kDa SCP2 domain in their C-termini. We found that 22-NBD-cholesterol, a fluorescent analog of cholesterol and a preferred SCP2 ligands, was not localized in the peroxisomes. This raises questions about previous reports on the localization of the SCPX and SCP2 proteins and their relationship to peroxisomes and mitochondria in intracellular cholesterol transport. Immunofluorescent staining of cryosections of mouse testis and of MA-10 mouse tumor Leydig cells showed that SCPX and SCP2 are present in both mouse testicular interstitial tissue and in MA-10 cells. Fluorescent fusion proteins of SCPX and SCP2, as well as confocal live-cell imaging, were used to investigate the subcellular targeting of these proteins and the function of the putative mitochondrial targeting sequence. The results showed that SCPX and SCP2 are targeted to the peroxisomes by the C-terminal PTS1 domain, but the putative N-terminal mitochondrial targeting sequence alone is not potent enough to localize SCPX and SCP2 to the mitochondria. Homology modeling and molecular docking studies indicated that the SCP2 domain binds cholesterol, but lacks specificity of the binding and/or transport. These findings further our understanding of the role of SCPX and SCP2 in intracellular cholesterol transport, and present a new point of view on the role of these proteins in cholesterol trafficking.     

Immunological detection of the mitochondrial F1-ATPase α subunit in the matrix of rat liver peroxisomes: A protein involved in organelle biogenesis?

Rat liver peroxisomes contain in their matrix the α-subunit of the mitochondrial F₁-ATPase complex. The identification of this protein in liver peroxisomes has been achieved by immunoelectron microscopy and subcellular fractionation. No β-subunit of the mitochondrial F₁-ATPase complex was detected in the peroxisomal fractions obtained in sucrose gradients or in Nycodenz pelletted peroxisomes. The consensus peroxisomal targeting sequence (Ala-Lys-Leu) is found at the carboxy terminus of the mature α-subunit from bovine heart and rat liver mitochondria. Due to the dual subcellular localization of the α-subunit and to the structural homologies that exist between this protein and molecular chaperones [(1990) Biol. Chem. 265, 7713-7716] it is suggested that the protein should perform another functional role(s) in both organelles, plus to its characteristic involvement in the regulation of mitochondrial ATPase activity.     

Molecular chaperones and the biogenesis of mitochondria and peroxisomes

Summary— A review of the proteinaceous machinery involved in protein sorting pathways and protein folding and assembly in mitochondria and peroxisomes is presented. After considering the various sorting pathways and targeting signals of mitochondrial and peroxisomal proteins, we make a comparative dissection of the protein factors involved in: i) the stabilization of cytosolic precursor proteins in a translocation competent conformation; ii) the membrane import apparatus of mitochondria and peroxisomes; iii) the processing of mitochondrial precursor proteins, and the eventual processing of certain peroxisomal precursor, in the interior of the organelles; and iv) the requirement of molecular chaperones for appropriate folding and assembly of imported proteins in the matrix of both organelles. Those aspects of mitochondrial biogenesis that have developed rapidly during the last few years, such as the requirement of molecular chaperones, are stressed in order to stimulate further parallel investigations aimed to understand the origin, biochemistry, molecular biology and pathology of peroxisomes. In this regard, a brief review of findings from our group and others is presented in which the role of the F₁-ATPase α-subunit is pointed out as a molecular chaperone of mitochondria and chloroplasts. In addition, data are presented that could question our previous indication that the immunoreactive protein found in the rat liver peroxisomes is due to the presence of the F₁-ATPase α-subunit.     

A role for Fis1 in both mitochondrial and peroxisomal fission in mammalian cells

The mammalian dynamin-like protein DLP1/Drp1 has been shown to mediate both mitochondrial and peroxisomal fission. In this study, we have examined whether hFis1, a mammalian homologue of yeast Fis1, which has been shown to participate in mitochondrial fission by an interaction with DLP1/Drp1, is also involved in peroxisomal growth and division. We show that hFis1 localizes to peroxisomes in addition to mitochondria. Through differential tagging and deletion experiments, we demonstrate that the transmembrane domain and the short C-terminal tail of hFis1 is both necessary and sufficient for its targeting to peroxisomes and mitochondria, whereas the N-terminal region is required for organelle fission. hFis1 promotes peroxisome division upon ectopic expression, whereas silencing of Fis1 by small interfering RNA inhibited fission and caused tubulation of peroxisomes. These findings provide the first evidence for a role of Fis1 in peroxisomal fission and suggest that the fission machinery of mitochondria and peroxisomes shares common components.     

Targeting of hFis1 to peroxisomes is mediated by Pex19p

The processes of peroxisome formation and proliferation are still a matter of debate. We have previously shown that peroxisomes share some components of their division machinery with mitochondria. hFis1, a tail-anchored membrane protein, regulates the membrane fission of both organelles by DLP1/Drp1 recruitment, but nothing is known about the mechanisms of the dual targeting of hFis1. Here we demonstrate for the first time that peroxisomal targeting of hFis1 depends on Pex19p, a peroxisomal membrane protein import factor. hFis1/Pex19p binding was demonstrated by expression and co-immunoprecipitation studies. Using mutated versions of hFis1 an essential binding region for Pex19p was located within the last 26 C-terminal amino acids of hFis1, which are required for proper targeting to both mitochondria and peroxisomes. The basic amino acids in the very C terminus are not essential for Pex19p binding and peroxisomal targeting, but are instead required for mitochondrial targeting. Silencing of Pex19p by small interference RNA reduced the targeting of hFis1 to peroxisomes, but not to mitochondria. In contrast, overexpression of Pex19p alone was not sufficient to shift the targeting of hFis1 to peroxisomes. Our findings indicate that targeting of hFis1 to peroxisomes and mitochondria are independent events and support a direct, Pex19p-dependent targeting of peroxisomal tail-anchored proteins.     

Betanodavirus B2 Causes ATP Depletion-induced Cell Death via Mitochondrial Targeting and Complex II Inhibition in Vitro and in Vivo

The betanodavirus non-structural protein B2 is a newly discovered necrotic death factor with a still unknown role in regulation of mitochondrial function. In the present study, we examined protein B2-mediated inhibition of mitochondrial complex II activity, which results in ATP depletion and thereby in a bioenergetic crisis in vitro and in vivo. Expression of protein B2 was detected early at 24 h postinfection with red-spotted grouper nervous necrosis virus in the cytoplasm. Later B2 was found in mitochondria using enhanced yellow fluorescent protein (EYFP) and immuno-EM analysis. Furthermore, the B2 mitochondrial targeting signal peptide was analyzed by serial deletion and specific point mutation. The sequence of the B2 targeting signal peptide (⁴¹RTFVISAHAA⁵⁰) was identified and its presence correlated with loss of mitochondrial membrane potential in fish cells. Protein B2 also was found to dramatically inhibit complex II (succinate dehydrogenase) activity, which impairs ATP synthesis in fish GF-1 cells as well as human embryonic kidney 293T cells. Furthermore, when B2 was injected into zebrafish embryos at the one-cell stage to determine its cytotoxicity and ability to inhibit ATP synthesis, we found that B2 caused massive embryonic cell death and depleted ATP resulting in further embryonic death at 10 and 24 h post-fertilization. Taken together, our results indicate that betanodavirus protein B2-induced cell death is due to direct targeting of the mitochondrial matrix by a specific signal peptide that targets mitochondria and inhibits mitochondrial complex II activity thereby reducing ATP synthesis.     

Role of the Ascorbate-Glutathione Cycle of Mitochondria and Peroxisomes in the Senescence of Pea Leaves

We investigated the relationship between H₂O₂ metabolism and the senescence process using soluble fractions, mitochondria, and peroxisomes from senescent pea (Pisum sativum L.) leaves. After 11 d of senescence the activities of Mn-superoxide dismutase, dehydroascorbate reductase (DHAR), and glutathione reductase (GR) present in the matrix, and ascorbate peroxidase (APX) and monodehydroascorbate reductase (MDHAR) activities localized in the mitochondrial membrane, were all substantially decreased in mitochondria. The mitochondrial ascorbate and dehydroascorbate pools were reduced, whereas the oxidized glutathione levels were maintained. In senescent leaves the H₂O₂ content in isolated mitochondria and the NADH- and succinate-dependent production of superoxide (O₂^·−) radicals by submitochondrial particles increased significantly. However, in peroxisomes from senescent leaves both membrane-bound APX and MDHAR activities were reduced. In the matrix the DHAR activity was enhanced and the GR activity remained unchanged. As a result of senescence, the reduced and the oxidized glutathione pools were considerably increased in peroxisomes. A large increase in the glutathione pool and DHAR activity were also found in soluble fractions of senescent pea leaves, together with a decrease in GR, APX, and MDHAR activities. The differential response to senescence of the mitochondrial and peroxisomal ascorbate-glutathione cycle suggests that mitochondria could be affected by oxidative damage earlier than peroxisomes, which may participate in the cellular oxidative mechanism of leaf senescence longer than mitochondria.     

Participation of Mitochondrial Metabolism in Photorespiration : Reconstituted System of Peroxisomes and Mitochondria from Spinach Leaves

In this study the interplay of mitochondria and peroxisomes in photorespiration was simulated in a reconstituted system of isolated mitochondria and peroxisomes from spinach (Spinacia oleracea L.) leaves. The mitochondria oxidizing glycine produced serine, which was reduced in the peroxisomes to glycerate. The required reducing equivalents were provided by the mitochondria via the malate-oxaloacetate (OAA) shuttle, in which OAA was reduced in the mitochondrial matrix by NADH generated during glycine oxidation. The rate of peroxisomal glycerate formation, as compared with peroxisomal protein, resembled the corresponding rate required during leaf photosynthesis under ambient conditions. When the reconstituted system produced glycerate at this rate, the malate-to-OAA ratio was in equilibrium with a ratio of NADH/NAD of 8.8 × 10⁻³. This low ratio is in the same range as the ratio of NADH/NAD in the cytosol of mesophyll cells of intact illuminated spinach leaves, as we had estimated earlier. This result demonstrates that in the photorespiratory cycle a transfer of redox equivalents from the mitochondria to peroxisomes, as postulated from separate experiments with isolated mitochondria and peroxisomes, can indeed operate under conditions of the very low reductive state of the NADH/NAD system prevailing in the cytosol of mesophyll cells in a leaf during photosynthesis.     

Antioxidant cytoprotection by peroxisomal peroxiredoxin-5

Peroxiredoxin-5 (PRDX5) is a thioredoxin peroxidase that reduces hydrogen peroxide, alkyl hydroperoxides, and peroxynitrite. This enzyme is present in the cytosol, mitochondria, peroxisomes, and nucleus in human cells. Antioxidant cytoprotective functions have been previously documented for cytosolic, mitochondrial, and nuclear mammalian PRDX5. However, the exact function of PRDX5 in peroxisomes is still not clear. The aim of this work was to determine the function of peroxisomal PRDX5 in mammalian cells and, more specifically, in glial cells. To study the role of PRDX5 in peroxisomes, the endogenous expression of PRDX5 in murine oligodendrocyte 158 N cells was silenced by RNA interference. In addition, human PRDX5 was also overexpressed in peroxisomes using a vector coding for human PRDX5, whose unconventional peroxisomal targeting sequence 1 (PTS1; SQL) was replaced by the prototypical PTS1 SKL. Stable 158 N clones were obtained. The antioxidant cytoprotective function of peroxisomal PRDX5 against peroxisomal and mitochondrial KillerRed-mediated reactive oxygen species production as well as H₂O₂ was examined using MTT viability assays, roGFP2, and C11-BOBIPY probes. Altogether our results show that peroxisomal PRDX5 protects 158 N oligodendrocytes against peroxisomal and mitochondrial KillerRed- and H₂O₂-induced oxidative stress.     

Charcot‐Marie‐Tooth disease‐associated mutants of GDAP1 dissociate its roles in peroxisomal and mitochondrial fission

Mitochondria and peroxisomes can be fragmented by the process of fission. The fission machineries of both organelles share a set of proteins. GDAP1 is a tail‐anchored protein of mitochondria and induces mitochondrial fragmentation. Mutations in GDAP1 lead to Charcot‐Marie‐Tooth disease (CMT), an inherited peripheral neuropathy, and affect mitochondrial dynamics. Here, we show that GDAP1 is also targeted to peroxisomes mediated by the import receptor Pex19. Knockdown of GDAP1 leads to peroxisomal elongation that can be rescued by re‐expressing GDAP1 and by missense mutated forms found in CMT patients. GDAP1‐induced peroxisomal fission is dependent on the integrity of its hydrophobic domain 1, and on Drp1 and Mff, as is mitochondrial fission. Thus, GDAP1 regulates mitochondrial and peroxisomal fission by a similar mechanism. However, our results reveal also a more critical role of the amino‐terminal GDAP1 domains, carrying most CMT‐causing mutations, in the regulation of mitochondrial compared to peroxisomal fission.     

Polyglutamine expansion inhibits respiration by increasing reactive oxygen species in isolated mitochondria

Huntington’s disease results from expansion of the polyglutamine (PolyQ) domain in the huntingtin protein. Although the cellular mechanism by which pathologic-length PolyQ protein causes neurodegeneration is unclear, mitochondria appear central in pathogenesis. We demonstrate in isolated mitochondria that pathologic-length PolyQ protein directly inhibits ADP-dependent (state 3) mitochondrial respiration. Inhibition of mitochondrial respiration by PolyQ protein is not due to reduction in the activities of electron transport chain complexes, mitochondrial ATP synthase, or the adenine nucleotide translocase. We show that pathologic-length PolyQ protein increases the production of reactive oxygen species in isolated mitochondria. Impairment of state 3 mitochondrial respiration by PolyQ protein is reversed by addition of the antioxidants N-acetyl-l-cysteine or cytochrome c. We propose a model in which pathologic-length PolyQ protein directly inhibits mitochondrial function by inducing oxidative stress.     

Trypanosome Alternative Oxidase Possesses both an N-Terminal and Internal Mitochondrial Targeting Signal

Recognition of mitochondrial targeting signals (MTS) by receptor translocases of outer and inner membranes of mitochondria is one of the prerequisites for import of nucleus-encoded proteins into this organelle. The MTS for a majority of trypanosomatid mitochondrial proteins have not been well defined. Here we analyzed the targeting signal for trypanosome alternative oxidase (TAO), which functions as the sole terminal oxidase in the infective form of Trypanosoma brucei. Deleting the first 10 of 24 amino acids predicted to be the classical N-terminal MTS of TAO did not affect its import into mitochondria in vitro. Furthermore, ectopically expressed TAO was targeted to mitochondria in both forms of the parasite even after deletion of first 40 amino acid residues. However, deletion of more than 20 amino acid residues from the N terminus reduced the efficiency of import. These data suggest that besides an N-terminal MTS, TAO possesses an internal mitochondrial targeting signal. In addition, both the N-terminal MTS and the mature TAO protein were able to target a cytosolic protein, dihydrofolate reductase (DHFR), to a T. brucei mitochondrion. Further analysis identified a cryptic internal MTS of TAO, located within amino acid residues 115 to 146, which was fully capable of targeting DHFR to mitochondria. The internal signal was more efficient than the N-terminal MTS for import of this heterologous protein. Together, these results show that TAO possesses a cleavable N-terminal MTS as well as an internal MTS and that these signals act together for efficient import of TAO into mitochondria.     

A Fox2-Dependent Fatty Acid ?-Oxidation Pathway Coexists Both in Peroxisomes and Mitochondria of the Ascomycete Yeast Candida lusitaniae

It is generally admitted that the ascomycete yeasts of the subphylum Saccharomycotina possess a single fatty acid ß-oxidation pathway located exclusively in peroxisomes, and that they lost mitochondrial ß-oxidation early during evolution. In this work, we showed that mutants of the opportunistic pathogenic yeast Candida lusitaniae which lack the multifunctional enzyme Fox2p, a key enzyme of the ß-oxidation pathway, were still able to grow on fatty acids as the sole carbon source, suggesting that C. lusitaniae harbored an alternative pathway for fatty acid catabolism. By assaying ¹⁴C_α-palmitoyl-CoA consumption, we demonstrated that fatty acid catabolism takes place in both peroxisomal and mitochondrial subcellular fractions. We then observed that a fox2Δ null mutant was unable to catabolize fatty acids in the mitochondrial fraction, thus indicating that the mitochondrial pathway was Fox2p-dependent. This finding was confirmed by the immunodetection of Fox2p in protein extracts obtained from purified peroxisomal and mitochondrial fractions. Finally, immunoelectron microscopy provided evidence that Fox2p was localized in both peroxisomes and mitochondria. This work constitutes the first demonstration of the existence of a Fox2p-dependent mitochondrial β-oxidation pathway in an ascomycetous yeast, C. lusitaniae. It also points to the existence of an alternative fatty acid catabolism pathway, probably located in peroxisomes, and functioning in a Fox2p-independent manner.     

Immunological detection of the mitochondrial F1-ATPase alpha subunit in the matrix of rat liver peroxisomes. A protein involved in organelle biogenesis?

Rat liver peroxisomes contain in their matrix the alpha-subunit of the mitochondrial F1-ATPase complex. The identification of this protein in liver peroxisomes has been achieved by immunoelectron microscopy and subcellular fractionation. No beta-subunit of the mitochondrial F1-ATPase complex was detected in the peroxisomal fractions obtained in sucrose gradients or in Nycodenz pelletted peroxisomes. The consensus peroxisomal targeting sequence (Ala-Lys-Leu) is found at the carboxy terminus of the mature alpha-subunit from bovine heart and rat liver mitochondria. Due to the dual subcellular localization of the alpha-subunit and to the structural homologies that exist between this protein and molecular chaperones [(1990) Biol. Chem. 265, 7713-7716] it is suggested that the protein should perform another functional role(s) in both organelles, plus to its characteristic involvement in the regulation of mitochondrial ATPase activity.