Mammalian alanine/glyoxylate aminotransferase 1 is imported into peroxisomes via the PTS1 translocation pathway. Increased degeneracy and context specificity of the mammalian PTS1 motif and implications for the peroxisome-to-mitochondrion mistargeting of AGT in primary hyperoxaluria type 1

Alanine/glyoxylate aminotransferase 1 (AGT) is peroxisomal in most normal humans, but in some patients with the hereditary disease primary hyperoxaluria type 1 (PH1), AGT is mislocalized to the mitochondria. In an attempt to identify the sequences in AGT that mediate its targeting to peroxisomes, and to determine the mechanism by which AGT is mistargeted in PH1, we have studied the intracellular compartmentalization of various normal and mutant AGT polypeptides in normal human fibroblasts and cell lines with selective deficiencies of peroxisomal protein import, using immunofluorescence microscopy after intranuclear microinjection of AGT expression plasmids. The results show that AGT is imported into peroxisomes via the peroxisomal targeting sequence type 1 (PTS1) translocation pathway. Although the COOH-terminal KKL of human AGT was shown to be necessary for its peroxisomal import, this tripeptide was unable to direct the peroxisomal import of the bona fide peroxisomal protein firefly luciferase or the reporter protein bacterial chloramphenicol acetyltransferase. An ill-defined region immediately upstream of the COOH-terminal KKL was also found to be necessary for the peroxisomal import of AGT, but again this region was found to be insufficient to direct the peroxisomal import of chloramphenicol acetyltransferase. Substitution of the COOH-terminal KKL of human AGT by the COOH-terminal tripeptides found in the AGTs of other mammalian species (SQL, NKL), the prototypical PTS1 (SKL), or the glycosomal PTS1 (SSL) also allowed peroxisomal targeting, showing that the allowable PTS1 motif in AGT is considerably more degenerate than, or at least very different from, that acceptable in luciferase. AGT possessing the two amino acid substitutions responsible for its mistargeting in PH1 (i.e., Pro11-- >Leu and Gly170-->Arg) was targeted mainly to the mitochondria. However, AGTs possessing each amino acid substitution on its own were targeted normally to the peroxisomes. This suggests that Gly170-->Arg- mediated increased functional efficiency of the otherwise weak mitochondrial targeting sequence (generated by the Pro11-->Leu polymorphism) is not due to interference with the peroxisomal targeting or import of AGT.     

Enzymological and mutational analysis of a complex primary hyperoxaluria type 1 phenotype involving alanine:Glyoxylate aminotransferase peroxisome-to-mitochondrion mistargeting and intraperoxisomal aggregation

Primary hyperoxaluria type 1 (PH1) is a rare autosomal recessive disease caused by a deficiency of the liver-specific peroxisomal enzyme alanine:glyoxylate aminotransferase (AGT). Three unrelated PH1 patients, who possess a novel complex phenotype, are described. At the enzymological level, this phenotype is characterized by a complete, or nearly complete, absence of AGT catalytic activity and reduced AGT immunoreactivity. Unlike normal individuals in whom the AGT is confined to the peroxisomal matrix, the immunoreactive AGT in these three patients was distributed approximately equally between the peroxisomes and mitochondria. The peroxisomal AGT appeared to be aggregated into amorphous core-like structures in which no other peroxisomal enzymes could be identified. Mutational analysis of the AGT gene showed that two of the three patients were compound heterozygotes for two previously unrecognized point mutations which caused Gly41→Arg and Phe152→Iso amino acid substitutions. The third patient was shown to be a compound heterozygote for the Gly41→Arg mutation and a previously recognized Gly170→Arg mutation. All three patients were homozygous for the Pro11→Leu polymorphism that had been found previously with a high allelic frequency in normal populations. It is suggested that the Phe152→Iso and Gly170→Arg substitutions, which are only eighteen residues apart and located in the same highly conserved internal region of 58 amino acids, might be involved in the inhibition of peroxisomal targeting and/or import of AGT and, in combination with the Pro11→Leu polymorphism, be responsible for its aberrant mitochondrial compartmentalization. On the other hand, the Gly41→Arg substitution, either in combination with the Pro11→Leu polymorphism or by itself, is predicted to be responsible for the intraperoxisomal aggregation of the AGT protein.     

Inhibition of alanine:glyoxylate aminotransferase 1 dimerization is a prerequisite for its peroxisome-to-mitochondrion mistargeting in primary hyperoxaluria type 1

Peroxisome-to-mitochondrion mistargeting of the homodimeric enzyme alanine:glyoxylate aminotransferase 1 (AGT) in the autosomal recessive disease primary hyperoxaluria type 1 (PH1) is associated with the combined presence of a normally occurring Pro(11)Leu polymorphism and a PH1-specific Gly170Arg mutation. The former leads to the formation of a novel NH2-terminal mitochondrial targeting sequence (MTS), which although sufficient to direct the import of in vitro-translated AGT into isolated mitochondria, requires the additional presence of the Gly170Arg mutation to function efficiently in whole cells. The role of this mutation in the mistargeting phenomenon has remained elusive. It does not interfere with the peroxisomal targeting or import of AGT. In the present study, we have investigated the role of the Gly170Arg mutation in AGT mistargeting. In addition, our studies have led us to examine the relationship between the oligomeric status of AGT and the peroxisomal and mitochondrial import processes. The results obtained show that in vitro-translated AGT rapidly forms dimers that do not readily exchange subunits. Although the presence of the Pro(11)Leu or Gly170Arg substitutions alone had no effect on dimerization, their combined presence abolished homodimerization in vitro. However, AGT containing both substitutions was still able to form heterodimers in vitro with either normal AGT or AGT containing either substitution alone. Expression of various combinations of normal and mutant, as well as epitope-tagged and untagged forms of AGT in whole cells showed that normal AGT rapidly dimerizes in the cytosol and is imported into peroxisomes as a dimer. This dimerization prevents mitochondrial import, even when the AGT possesses an MTS generated by the Pro(11)Leu substitution. The additional presence of the Gly170Arg substitution impairs dimerization sufficiently to allow mitochondrial import. Pharmacological inhibition of mitochondrial import allows AGT containing both substitutions to be imported into peroxisomes efficiently, showing that AGT dimerization is not a prerequisite for peroxisomal import.     

Primary hyperoxaluria type 1: AGT mistargeting highlights the fundamental differences between the peroxisomal and mitochondrial protein import pathways

Primary hyperoxaluria type 1 (PH1) is an atypical peroxisomal disorder, as befits a deficiency of alanine:glyoxylate aminotransferase (AGT), which is itself an atypical peroxisomal enzyme. PH1 is characterized by excessive synthesis and excretion of the metabolic end-product oxalate and the progressive accumulation of insoluble calcium oxalate in the kidney and urinary tract. Disease in many patients is caused by a unique protein trafficking defect in which AGT is mistargeted from peroxisomes to mitochondria, where it is metabolically ineffectual, despite remaining catalytically active. Although the peroxisomal import of human AGT is dependent upon the PTS1 import receptor PEX5p, its PTS1 is exquisitely specific for mammalian AGT, suggesting the presence of additional peroxisomal targeting information elsewhere in the AGT molecule. This and many other functional peculiarities of AGT are probably a consequence of its rather chequered evolutionary history, during which much of its time has been spent being a mitochondrial, rather than a peroxisomal, enzyme. Analysis of the molecular basis of AGT mistargeting in PH1 has thrown into sharp relief some of the fundamental differences between the requirements of the peroxisomal and mitochondrial protein import pathways, particularly the properties of peroxisomal and mitochondrial matrix targeting sequences and the different conformational limitations placed upon importable cargos.     

Peroxisomal and mitochondrial targeting of serine: Pyruvate/alanine: Glyoxylate aminotransferase in rat liver

Serine: pyruvate/alanine:glyoxylate aminotransferase (SPT or SPT/AGT) of rat liver is a unique enzyme of dual subcellular localization, and exists in both mitochondria and peroxisomes. To characterize a peroxisomal targeting signal of rat liver SPT, a number of C-terminal mutants were constructed and their subcellular localization in transfected COS-1 cells was examined. Deletion of C-terminal NKL, and point mutation of K2 (the second Lys from the C-terminus), K4 and E15 caused accumulation of translated products in the cytoplasm. This suggests that the PTS of SPT is not identical to PTS1 (the C-terminal SKL motif) in that it is not restricted to the C-terminal tripeptide. In vitro synthesized precursor for mitochondrial SPT was highly sensitive to the proteinase K digestion, whereas peroxisomal SPT (SPTp) was fairly resistant to the protease. In in vitro import experiment with purified peroxisomes, however, STPp recovered in the peroxisomal fraction was very sensitive to the protease. These results suggest that the mitochondrial precursor is synthesized as an unfolded form and is translocated into the mitochondrial matrix, whereas SPTp is synthesized as a folded form and its conformation changes to an unfolded form just before translocation into peroxisomes.     

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.     

Differential enzyme targeting as an evolutionary adaptation to herbivory in carnivora

Not all members of the order Carnivora are carnivorous. Some are omnivorous, and a few, such as the giant panda, Ailuropoda melanoleuca, are almost exclusively herbivorous. Although a number of adaptations to increased plant-eating are recognized within Carnivora, few have been studied at the molecular level. One molecular adaptation to diet that is spread widely across Mammalia is the differential intracellular targeting of the intermediary metabolic enzyme alanine:glyoxylate aminotransferase (AGT), which tends to be mitochondrial in carnivores, peroxisomal in herbivores, and both mitochondrial and peroxisomal in omnivores. In the present study, we have analyzed the targeting of AGT in Carnivora in relation to species' natural diets. We show not only that there has been an adaptive shift in AGT targeting from the mitochondrion toward the peroxisome as diets have shifted from being mainly carnivorous to ones that are more omnivorous and herbivorous but also that in one lineage, namely that of the giant panda, there is evidence for positive selection pressure at the molecular level on the AGT mitochondrial targeting sequence to decrease its efficiency, thereby allowing more AGT to be targeted to the peroxisomes.     

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.     

Alanine:glyoxylate aminotransferase peroxisome-to-mitochondrion mistargeting in human hereditary kidney stone disease

The pyridoxal-phosphate (PLP)-dependent enzyme alanine:glyoxylate aminotransferase (AGT) is mistargeted from peroxisomes to mitochondria in patients with the hereditary kidney stone disease primary hyperoxaluria type 1 (PH1) due to the synergistic interaction between a common Pro(11)Leu polymorphism and a PH1-specific Gly(170)Arg mutation. The kinetic partitioning of newly synthesised AGT between peroxisomes and mitochondria is determined by the combined effects of (1) the generation of cryptic mitochondrial targeting information, and (2) the inhibition of AGT dimerization. The crystal structure of AGT has recently been solved, allowing the effects of the various polymorphisms and mutations to be rationalised in terms of AGT's three-dimensional conformation. Procedures that increase dimer stability and/or increase the rate of dimer formation have potential in the formulation of novel strategies to treat this otherwise intractable life-threatening disease.     

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.     

Human peroxisomal L-alanine: glyoxylate aminotransferase. Evolutionary loss of a mitochondrial targeting signal by point mutation of the initiation codon.

The amino acid sequence of human hepatic peroxisomal L-alanine: glyoxylate aminotransferase 1 (AGTI) deduced from cDNA shows 78% sequence identity with that of rat mitochondrial AGTI, but lacks the N-terminal 22 amino acids (the putative mitochondrial targeting signal). In humans this signal appears to have been deleted during evolution by a point mutation of the initiation codon ATG to ATA. These data suggest that the targeting defect in primary hyperoxaluria type 1, in which AGT1 is diverted from the peroxisomes to the mitochondria, could be due to a point mutation that reintroduces all or part of the mitochondrial signal sequence.     

Metabolic interactions between peroxisomes and mitochondria with a special focus on acylcarnitine metabolism

Carnitine plays an essential role in mitochondrial fatty acid β-oxidation as a part of a cycle that transfers long-chain fatty acids across the mitochondrial membrane and involves two carnitine palmitoyltransferases (CPT1 and CPT2). Two distinct carnitine acyltransferases, carnitine octanoyltransferase (COT) and carnitine acetyltransferase (CAT), are peroxisomal enzymes, which indicates that carnitine is not only important for mitochondrial, but also for peroxisomal metabolism. It has been demonstrated that after peroxisomal metabolism, specific intermediates can be exported as acylcarnitines for subsequent and final mitochondrial metabolism. There is also evidence that peroxisomes are able to degrade fatty acids that are typically handled by mitochondria possibly after transport as acylcarnitines. Here we review the biochemistry and physiological functions of metabolite exchange between peroxisomes and mitochondria with a special focus on acylcarnitines.     

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.     

Role of carnitine acetyltransferases in acetyl coenzyme A metabolism in Aspergillus nidulans

The flow of carbon metabolites between cellular compartments is an essential feature of fungal metabolism. During growth on ethanol, acetate, or fatty acids, acetyl units must enter the mitochondrion for metabolism via the tricarboxylic acid cycle, and acetyl coenzyme A (acetyl-CoA) in the cytoplasm is essential for the biosynthetic reactions and for protein acetylation. Acetyl-CoA is produced in the cytoplasm by acetyl-CoA synthetase during growth on acetate and ethanol while β-oxidation of fatty acids generates acetyl-CoA in peroxisomes. The acetyl-carnitine shuttle in which acetyl-CoA is reversibly converted to acetyl-carnitine by carnitine acetyltransferase (CAT) enzymes is important for intracellular transport of acetyl units. In the filamentous ascomycete Aspergillus nidulans, a cytoplasmic CAT, encoded by facC, is essential for growth on sources of cytoplasmic acetyl-CoA while a second CAT, encoded by the acuJ gene, is essential for growth on fatty acids as well as acetate. We have shown that AcuJ contains an N-terminal mitochondrial targeting sequence and a C-terminal peroxisomal targeting sequence (PTS) and is localized to both peroxisomes and mitochondria, independent of the carbon source. Mislocalization of AcuJ to the cytoplasm does not result in loss of growth on acetate but prevents growth on fatty acids. Therefore, while mitochondrial AcuJ is essential for the transfer of acetyl units to mitochondria, peroxisomal localization is required only for transfer from peroxisomes to mitochondria. Peroxisomal AcuJ was not required for the import of acetyl-CoA into peroxisomes for conversion to malate by malate synthase (MLS), and export of acetyl-CoA from peroxisomes to the cytoplasm was found to be independent of FacC when MLS was mislocalized to the cytoplasm.     

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.     

Novel proteins, putative membrane transporters, and an integrated metabolic network are revealed by quantitative proteomic analysis of Arabidopsis cell culture peroxisomes

Peroxisomes play key roles in energy metabolism, cell signaling, and plant development. A better understanding of these important functions will be achieved with a more complete definition of the peroxisome proteome. The isolation of peroxisomes and their separation from mitochondria and other major membrane systems have been significant challenges in the Arabidopsis (Arabidopsis thaliana) model system. In this study, we present new data on the Arabidopsis peroxisome proteome obtained using two new technical advances that have not previously been applied to studies of plant peroxisomes. First, we followed density gradient centrifugation with free-flow electrophoresis to improve the separation of peroxisomes from mitochondria. Second, we used quantitative proteomics to identify proteins enriched in the peroxisome fractions relative to mitochondrial fractions. We provide evidence for peroxisomal localization of 89 proteins, 36 of which have not previously been identified in other analyses of Arabidopsis peroxisomes. Chimeric green fluorescent protein constructs of 35 proteins have been used to confirm their localization in peroxisomes or to identify endoplasmic reticulum contaminants. The distribution of many of these peroxisomal proteins between soluble, membrane-associated, and integral membrane locations has also been determined. This core peroxisomal proteome from nonphotosynthetic cultured cells contains a proportion of proteins that cannot be predicted to be peroxisomal due to the lack of recognizable peroxisomal targeting sequence 1 (PTS1) or PTS2 signals. Proteins identified are likely to be components in peroxisome biogenesis, beta-oxidation for fatty acid degradation and hormone biosynthesis, photorespiration, and metabolite transport. A considerable number of the proteins found in peroxisomes have no known function, and potential roles of these proteins in peroxisomal metabolism are discussed. This is aided by a metabolic network analysis that reveals a tight integration of functions and highlights specific metabolite nodes that most probably represent entry and exit metabolites that could require transport across the peroxisomal membrane.     

Immunocytochemical localization of human hepatic alanine: glyoxylate aminotransferase in control subjects and patients with primary hyperoxaluria type 1

Primary hyperoxaluria type 1 (PH1) is an inherited disorder of glyoxylate metabolism caused by a deficiency of the hepatic peroxisomal enzyme alanine: glyoxylate aminotransferase (AGT; EC 2.6.1.44) [FEBS Lett (1986) 201:20]. The aim of the present study was to investigate the intracellular distribution of immunoreactive AGT protein, using protein A-gold immunocytochemistry, in normal human liver and in livers of PH1 patients with (CRM+) or without (CRM-) immunologically crossreacting enzyme protein. In all CRM+ individuals, which included three controls, a PH1 heterozygote and a PH1 homozygote immunoreactive AGT protein was confined to peroxisomes, where it was randomly dispersed throughout the peroxisomal matrix with no obvious association with the peroxisomal membrane. No AGT protein could be detected in the peroxisomes or other cytoplasmic compartments in the livers of CRM- PH1 patients (homozygotes). The peroxisomal labeling density in the CRM+ PH1 patient, who was completely deficient in AGT enzyme activity, was similar to that of the controls. In addition, in the PH1 heterozygote, who had one third normal AGT enzyme activity, peroxisomal labeling density was reduced to 50% of normal.