Peroxisomes In Mammalian Cells Research Articles

The subset of peroxisomal tail-anchored proteins do not reach peroxisomes via ER, instead mitochondria can be involved.

Peroxisomes are membrane-enclosed organelles with important roles in fatty acid breakdown, bile acid synthesis and biosynthesis of sterols and ether lipids. Defects in peroxisomes result in severe genetic diseases, such as Zellweger syndrome and neonatal adrenoleukodystrophy. However, many aspects of peroxisomal biogenesis are not well understood. Here we investigated delivery of tail-anchored (TA) proteins to peroxisomes in mammalian cells. Using glycosylation assays we showed that peroxisomal TA proteins do not enter the endoplasmic reticulum (ER) in both wild type (WT) and peroxisome-lacking cells. We observed that in cells lacking the essential peroxisome biogenesis factor, PEX19, peroxisomal TA proteins localize mainly to mitochondria. Finally, to investigate peroxisomal TA protein targeting in cells with fully functional peroxisomes we used a proximity biotinylation approach. We showed that while ER-targeted TA construct was exclusively inserted into the ER, peroxisome-targeted TA construct was inserted to both peroxisomes and mitochondria. Thus, in contrast to previous studies, our data suggest that some peroxisomal TA proteins do not insert to the ER prior to their delivery to peroxisomes, instead, mitochondria can be involved.

Acyl CoA oxidase: from its expression, structure, folding, and import to its role in human health and disease.

β-oxidation of fatty acids is an important metabolic pathway and is a shared function between mitochondria and peroxisomes in mammalian cells. On the other hand, peroxisomes are the sole site for the degradation of fatty acids in yeast. The first reaction of this pathway is catalyzed by the enzyme acyl CoA oxidase housed in the matrix of peroxisomes. Studies in various model organisms have reported the conserved function of the protein in fatty acid oxidation. The importance of this enzyme is highlighted by the lethal conditions caused in humans due to its altered function. In this review, we discuss various aspects ranging from gene expression, structure, folding, and import of the protein in both yeast and human cells. Further, we highlight recent findings on the role of the protein in human health and aging, and discuss the identified mutations in the protein associated with debilitating conditions in patients.

Competitive Microtubule Binding of PEX14 Coordinates Peroxisomal Protein Import and Motility

Human PEX14 plays a dual role as docking protein in peroxisomal protein import and as peroxisomal anchor for microtubules (MT), which relates to peroxisome motility. For docking, the conserved N-terminal domain of PEX14 (PEX14-NTD) binds amphipathic alpha-helical ligands, typically comprising one or two aromatic residues, of which human PEX5 possesses eight. Here, we show that the PEX14-NTD also binds to microtubular filaments in vitro with a dissociation constant in nanomolar range. PEX14 interacts with two motifs in the C-terminal region of human ß-tubulin. At least one of the binding motifs is in spatial proximity to the binding site of microtubules (MT) for kinesin. Both PEX14 and kinesin can bind to MT simultaneously. Notably, binding of PEX14 to tubulin can be prevented by its association with PEX5. The data suggest that PEX5 competes peroxisome anchoring to MT by occupying the ß-tubulin-binding site of PEX14. The competitive correlation of matrix protein import and motility may facilitate the homogeneous dispersion of peroxisomes in mammalian cells.

A bacteria-derived tail anchor localizes to peroxisomes in yeast and mammalian cells

Prokaryotes can provide new genetic information to eukaryotes by horizontal gene transfer (HGT), and such transfers are likely to have been particularly consequential in the era of eukaryogenesis. Since eukaryotes are highly compartmentalized, it is worthwhile to consider the mechanisms by which newly transferred proteins might reach diverse organellar destinations. Toward this goal, we have focused our attention upon the behavior of bacteria-derived tail anchors (TAs) expressed in the eukaryote Saccharomyces cerevisiae. In this study, we report that a predicted membrane-associated domain of the Escherichia coli YgiM protein is specifically trafficked to peroxisomes in budding yeast, can be found at a pre-peroxisomal compartment (PPC) upon disruption of peroxisomal biogenesis, and can functionally replace an endogenous, peroxisome-directed TA. Furthermore, the YgiM(TA) can localize to peroxisomes in mammalian cells. Since the YgiM(TA) plays no endogenous role in peroxisomal function or assembly, this domain is likely to serve as an excellent tool allowing further illumination of the mechanisms by which TAs can travel to peroxisomes. Moreover, our findings emphasize the ease with which bacteria-derived sequences might target to organelles in eukaryotic cells following HGT, and we discuss the importance of flexible recognition of organelle targeting information during and after eukaryogenesis.

The cytosolic peroxisome-targeting signal (PTS)-receptors, Pex7p and Pex5pL, are sufficient to transport PTS2 proteins to peroxisomes

Proteins harboring peroxisome-targeting signal type-2 (PTS2) are recognized in the cytosol by mobile PTS2 receptor Pex7p and associate with a longer isoform Pex5pL of the PTS1 receptor. Trimeric PTS2 protein-Pex7p-Pex5pL complexes are translocated to peroxisomes in mammalian cells. However, it remains unclear whether Pex5pL and Pex7p are sufficient cytosolic components in transporting of PTS2 proteins to peroxisomes. Here, we construct a semi-intact cell import system to define the cytosolic components required for the peroxisomal PTS2 protein import and show that the PTS2 pre-import complexes comprising Pex7p, Pex5p, and Hsc70 isolated from the cytosol of pex14 Chinese hamster ovary cell mutant ZP161 is import-competent. PTS2 reporter proteins are transported to peroxisomes by recombinant Pex7p and Pex5pL in semi-intact cells devoid of the cytosol. Furthermore, PTS2 proteins are translocated to peroxisomes in the presence of a non-hydrolyzable ATP analogue, adenylyl imidodiphosphate, and N-ethylmaleimide, suggesting that ATP-dependent chaperones including Hsc70 are dispensable for PTS2 protein import. Taken together, we suggest that Pex7p and Pex5pL are the minimal cytosolic factors in the transport of PTS2 proteins to peroxisomes.

New splicing variants of mitochondrial Rho GTPase-1 (Miro1) transport peroxisomes.

Microtubule-dependent long-distance movement of peroxisomes occurs in mammalian cells. However, its molecular mechanisms remain undefined. In this study, we identified three distinct splicing variants of human mitochondrial Rho GTPase-1 (Miro1), each containing amino acid sequence insertions 1 (named Miro1-var2), 2 (Miro1-var3), and both 1 and 2 (Miro1-var4), respectively, at upstream of the transmembrane domain. Miro1-var4 and Miro1-var2 are localized to peroxisomes in a manner dependent on the insertion 1 that is recognized by the cytosolic receptor Pex19p. Exogenous expression of Miro1-var4 induces accumulation of peroxisomes at the cell periphery and augments long-range movement of peroxisomes along microtubules. Depletion of all Miro1 variants by knocking down MIRO1 suppresses the long-distance movement of peroxisomes. Such abrogated movement is restored by reexpression of peroxisomal Miro1 variants. Collectively, our findings identify for the first time peroxisome-localized Miro1 variants as adapter proteins that link peroxisomes to the microtubule-dependent transport complexes including TRAK2 in the intracellular translocation of peroxisomes in mammalian cells.

The making of a mammalian peroxisome, version 2.0: mitochondria get into the mix.

A recent report from the Laboratory of Heidi McBride (McGill University) presents a role for mitochondria in the de novo biogenesis of peroxisomes in mammalian cells. Peroxisomes are essential organelles responsible for a wide variety of biochemical functions, from the generation of bile to plasmalogen synthesis, reduction of peroxides, and the oxidation of very-long-chain fatty acids. Like mitochondria, peroxisomes proliferate primarily through growth and division of pre-existing peroxisomes. However, unlike mitochondria, peroxisomes do not fuse; further, and perhaps most importantly, they can also be born de novo, a process thought to occur through the generation of pre-peroxisomal vesicles that originate from the endoplasmic reticulum. De novo peroxisome biogenesis has been extensively studied in yeast, with a major focus on the role of the ER in this process; however, in the mammalian system this field is much less explored. By exploiting patient cells lacking mature peroxisomes, the McBride laboratory now assigns a role to ER and mitochondria in de novo mammalian peroxisome biogenesis by showing that the formation of immature pre-peroxisomes occurs through the fusion of Pex3-/Pex14-containing mitochondria-derived vesicles with Pex16-containing ER-derived vesicles.

Conserved targeting information in mammalian and fungal peroxisomal tail-anchored proteins.

The targeting signals and mechanisms of soluble peroxisomal proteins are well understood, whereas less is known about the signals and targeting routes of peroxisomal membrane proteins (PMP). Pex15 and PEX26, tail-anchored proteins in yeast and mammals, respectively, exert a similar cellular function in the recruitment of AAA peroxins at the peroxisomal membrane. But despite their common role, Pex15 and PEX26 are neither homologs nor they are known to follow similar targeting principles. Here we show that Pex15 targets to peroxisomes in mammalian cells, and PEX26 reaches peroxisomes when expressed in yeast cells. In both proteins C-terminal targeting information is sufficient for correct sorting to the peroxisomal membrane. In yeast, PEX26 follows the pathway that also ensures correct targeting of Pex15: PEX26 enters the endoplasmic reticulum (ER) in a GET-dependent and Pex19-independent manner. Like in yeast, PEX26 enters the ER in mammalian cells, however, independently of GET/TRC40. These data show that conserved targeting information is employed in yeast and higher eukaryotes during the biogenesis of peroxisomal tail-anchored proteins.

Flavivirus Infection Impairs Peroxisome Biogenesis and Early Antiviral Signaling.

Flaviviruses are significant human pathogens that have an enormous impact on the global health burden. Currently, there are very few vaccines against or therapeutic treatments for flaviviruses, and our understanding of how these viruses cause disease is limited. Evidence suggests that the capsid proteins of flaviviruses play critical nonstructural roles during infection, and therefore, elucidating how these viral proteins affect cellular signaling pathways could lead to novel targets for antiviral therapy. We used affinity purification to identify host cell proteins that interact with the capsid proteins of West Nile and dengue viruses. One of the cellular proteins that formed a stable complex with flavivirus capsid proteins is the peroxisome biogenesis factor Pex19. Intriguingly, flavivirus infection resulted in a significant loss of peroxisomes, an effect that may be due in part to capsid expression. We posited that capsid protein-mediated sequestration and/or degradation of Pex19 results in loss of peroxisomes, a situation that could result in reduced early antiviral signaling. In support of this hypothesis, we observed that induction of the lambda interferon mRNA in response to a viral RNA mimic was reduced by more than 80%. Together, our findings indicate that inhibition of peroxisome biogenesis may be a novel mechanism by which flaviviruses evade the innate immune system during early stages of infection. RNA viruses infect hundreds of millions of people each year, causing significant morbidity and mortality. Chief among these pathogens are the flaviviruses, which include dengue virus and West Nile virus. Despite their medical importance, there are very few prophylactic or therapeutic treatments for these viruses. Moreover, the manner in which they subvert the innate immune response in order to establish infection in mammalian cells is not well understood. Recently, peroxisomes were reported to function in early antiviral signaling, but very little is known regarding if or how pathogenic viruses affect these organelles. We report for the first time that flavivirus infection results in significant loss of peroxisomes in mammalian cells, which may indicate that targeting of peroxisomes is a key strategy used by viruses to subvert early antiviral defenses.

Pex11mediates peroxisomal proliferation by promoting deformation of the lipid membrane.

Pex11p family proteins are key players in peroxisomal fission, but their molecular mechanisms remains mostly unknown. In the present study, overexpression of Pex11pβ caused substantial vesiculation of peroxisomes in mammalian cells. This vesicle formation was dependent on dynamin-like protein 1 (DLP1) and mitochondrial fission factor (Mff), as knockdown of these proteins diminished peroxisomal fission after Pex11pβ overexpression. The fission-deficient peroxisomes exhibited an elongated morphology, and peroxisomal marker proteins, such as Pex14p or matrix proteins harboring peroxisomal targeting signal 1, were discernible in a segmented staining pattern, like beads on a string. Endogenous Pex11pβ was also distributed a striped pattern, but which was not coincide with Pex14p and PTS1 matrix proteins. Altered morphology of the lipid membrane was observed when recombinant Pex11p proteins were introduced into proteo-liposomes. Constriction of proteo-liposomes was observed under confocal microscopy and electron microscopy, and the reconstituted Pex11pβ protein localized to the membrane constriction site. Introducing point mutations into the N-terminal amphiphathic helix of Pex11pβ strongly reduced peroxisomal fission, and decreased the oligomer formation. These results suggest that Pex11p contributes to the morphogenesis of the peroxisomal membrane, which is required for subsequent fission by DLP1.

The membrane peroxin PEX3 induces peroxisome-ubiquitination-linked pexophagy

Peroxisomes are degraded by a selective type of autophagy known as pexophagy. Several different types of pexophagy have been reported in mammalian cells. However, the mechanisms underlying how peroxisomes are recognized by autophagy-related machinery remain elusive. PEX3 is a peroxisomal membrane protein (PMP) that functions in the import of PMPs into the peroxisomal membrane and has been shown to interact with pexophagic receptor proteins during pexophagy in yeast. Thus, PEX3 is important not only for peroxisome biogenesis, but also for peroxisome degradation. However, whether PEX3 is involved in the degradation of peroxisomes in mammalian cells is unclear. Here, we report that high levels of PEX3 expression induce pexophagy. In PEX3-loaded cells, peroxisomes are ubiquitinated, clustered, and degraded in lysosomes. Peroxisome targeting of PEX3 is essential for the initial step of this degradation pathway. The degradation of peroxisomes is inhibited by treatment with autophagy inhibitors or siRNA against NBR1, which encodes an autophagic receptor protein. These results indicate that ubiquitin- and NBR1-mediated pexophagy is induced by increased expression of PEX3 in mammalian cells. In addition, another autophagic receptor protein, SQSTM1/p62, is required only for the clustering of peroxisomes. Expression of a PEX3 mutant with substitution of all lysine and cysteine residues by arginine and alanine, respectively, also induces peroxisome ubiquitination and degradation, hence suggesting that ubiquitination of PEX3 is dispensable for pexophagy and an endogenous, unidentified peroxisomal protein is ubiquitinated on the peroxisomal membrane.

PEX16 contributes to peroxisome maintenance by constantly trafficking PEX3 via the ER

ABSTRACTThe endoplasmic reticulum (ER) is required for the de novo biogenesis of peroxisomes in mammalian cells. However, its role in peroxisome maintenance is unclear. To explore ER involvement in the maintenance of peroxisomes, we redirect a peroxisomal membrane protein (PMP), PEX3, to directly target to the ER using the N-terminal ER signal sequence from preprolactin. Using biochemical techniques and fluorescent imaging, we find that ER-targeting PEX3 (ssPEX3) is continuously imported into pre-existing peroxisomes. This suggests that the ER constitutively provides membrane proteins and associated lipids to pre-existing peroxisomes. Using quantitative time-lapse live-cell fluorescence microscopy applied to cells that were either depleted of or exogenously expressing PEX16, we find that PEX16 mediates the peroxisomal trafficking of two distinct peroxisomal membrane proteins, PEX3 and PMP34, via the ER. These results not only provide insight into peroxisome maintenance and PMP trafficking in mammalian cells but also highlight important similarities and differences in the mechanisms of PMP import between the mammalian and yeast systems.

Dual function of Sec16B

The origin of peroxisomes has long been disputed. However, recent evidence suggests that peroxisomes can be formed de novo from the endoplasmic reticulum (ER) in yeast and higher eukaryotes. Sec16A and Sec16B, mammalian orthologs of yeast Sec16, are scaffold proteins that organize ER exit sites by interacting with COPII components. We recently demonstrated that Sec16B, but not Sec16A, regulates the transport of peroxisomal biogenesis factors from the ER to peroxisomes in mammalian cells. The C-terminal region of Sec16B, which is not conserved in Sec16A, is required for this function. The data suggest that Sec16B in ER areas other than ER exit sites plays this role. Our findings provide an unexpected connection between at least part of the COPII machinery and the formation of preperoxisomal vesicles at the ER, and offer an explanation of how secretory and peroxisomal trafficking from the ER are distinguished.

Pex11pβ-mediated maturation of peroxisomes

Peroxisomes are highly dynamic, multifunctional organelles that display remarkable changes in morphology, number and enzyme content. Peroxisomes multiply by growth and division of pre-existing organelles, but they can also form de novo from the ER. Growth and division of peroxisomes in mammalian cells involves elongation, membrane constriction, and final fission and requires the peroxisome biogenesis Pex11 proteins as well as the recruitment of Dynamin-like protein DLP1/Drp1. We recently exploited the division-inhibiting properties of a unique Pex11pβ-YFP fusion protein to further dissect the process of peroxisomal growth and division. By applying life cell imaging and the HaloTag technology, our study revealed that Pex11pβ-mediated growth (elongation) and division of peroxisomes follows a multistep maturation pathway, which is initiated by the formation of an early peroxisomal membrane compartment from a pre-existing peroxisome and its stepwise conversion into a mature, metabolically active peroxisome compartment. Our observations support the view that peroxisomes formed by growth and division of pre-existing ones contain new membrane and matrix components. Peroxisome division is an asymmetric process, which is more complex than simple (symmetric) division of a pre-existing organelle and equal distribution of the protein content. Our findings are in favor of Pex11pβ acting as a peroxisomal membrane shaping protein.

Pex11pβ-mediated growth and division of mammalian peroxisomes follows a maturation pathway

Peroxisomes are ubiquitous subcellular organelles, which multiply by growth and division but can also form de novo via the endoplasmic reticulum. Growth and division of peroxisomes in mammalian cells involves elongation, membrane constriction and final fission. Dynamin-like protein (DLP1/Drp1) and its membrane adaptor Fis1 function in the later stages of peroxisome division, whereas the membrane peroxin Pex11pbeta appears to act early in the process. We have discovered that a Pex11pbeta-YFP(m) fusion protein can be used as a specific tool to further dissect peroxisomal growth and division. Pex11pbeta-YFP(m) inhibited peroxisomal segmentation and division, but resulted in the formation of pre-peroxisomal membrane structures composed of globular domains and tubular extensions. Peroxisomal matrix and membrane proteins were targeted to distinct regions of the peroxisomal structures. Pex11pbeta-mediated membrane formation was initiated at pre-existing peroxisomes, indicating that growth and division follows a multistep maturation pathway and that formation of mammalian peroxisomes is more complex than simple division of a pre-existing organelle. The implications of these findings on the mechanisms of peroxisome formation and membrane deformation are discussed.

The peroxin Pex14p is involved in LC3-dependent degradation of mammalian peroxisomes

As a step toward understanding the homeostasis of peroxisomes in mammalian cells, we investigated a degradation system of peroxisomes in Chinese hamster ovary (CHO)-K1 cells in response to the nutrient-starvation. Peroxisomal proteins were degraded apparently in a preferential manner as compared to cytosolic proteins, when CHO-K1 cells were starved in Hank's solution and then re-cultured in a normal medium. We verified whether microtubule-associated protein I light chain 3 (LC3), an essential factor for autophagy, was involved in the degradation of peroxisomal proteins. In the LC3-knocked-down CHO-K1 cells, the specific degradation of peroxisomal proteins was no longer observed and proteins including peroxisomal and cytosolic proteins were rather non-selectively degraded under the starvation condition. The starvation-dependent non-selective protein degradation was inhibited with proteasome inhibitors, MG132 and Epoxomicin. The integral membrane peroxin, Pex14p interacted with membrane-bound LC3-II, the modified form of LC3, via microtubules under the starvation condition. Taken together, these results suggest that peroxisomal proteins are degraded by two degradation systems involving autophagy and proteasomes depending on various cell-culture conditions, and that Pex14p plays a pivotal role as a prerequisite factor for the degradation of peroxisomal proteins by autophagy with the aid of microtubules.

Special delivery from mitochondria to peroxisomes

Inter-organellar communication and interactions are necessary and accepted consequences of the segregation of biochemical functions into subcellular organelles. Recently, Heidi McBride and her collaborators found a novel link between mitochondria and peroxisomes in their discovery of mitochondria-derived vesicles (MDVs), which appear to fuse with a fraction of pre-existing peroxisomes in mammalian cells. We discuss the potential role of this vesicle population in the context of pathways for the exchange of metabolites and/or macromolecules between these compartments.

Excess Peroxisomes Are Degraded by Autophagic Machinery in Mammals

Peroxisomes are degraded by autophagic machinery termed "pexophagy" in yeast; however, whether this is essential for peroxisome degradation in mammals remains unknown. Here we have shown that Atg7, an essential gene for autophagy, plays a pivotal role in the degradation of excess peroxisomes in mammals. Following induction of peroxisomes by a 2-week treatment with phthalate esters in control and Atg7-deficient livers, peroxisomal degradation was monitored within 1 week after discontinuation of phthalate esters. Although most of the excess peroxisomes in the control liver were selectively degraded within 1 week, this rapid removal was exclusively impaired in the mutant liver. Furthermore, morphological analysis revealed that surplus peroxisomes, but not mutant hepatocytes, were surrounded by autophagosomes in the control. Our results indicated that the autophagic machinery is essential for the selective clearance of excess peroxisomes in mammals. This is the first direct evidence for the contribution of autophagic machinery in peroxisomal degradation in mammals.

The C-terminal tripeptide of glycosomal phosphoglycerate kinase is both necessary and sufficient for import into the glycosomes of Trypanosoma brucei

Glycosomal phosphoglycerate kinase (gPGK) of Trypanosoma brucei differs from the cytoplasmic isozyme (cPGK) in its higher isoelectric point characterized by clusters of positive charges along the polypeptide chain, and a 20 amino acid C-terminal extension ending in serine-serine-leucine (SSL). While a C-terminal SSL tripeptide is apparently not capable of directing luciferase to the peroxisomes in mammalian cells [J. Cell Biol. 108 (1989), 1657-1664], we show here that it is sufficient for the import of luciferase as well as an unrelated protein, β-glucuronidase, into the glycosomes of T. brucei, as determined by immunoelectron microscopy. The analysis of luciferase-gPGK fusion proteins indicates that the only targeting signal for import of gPGK into the glycosome resides in this C-terminal SSL sequence.

Peroxisomes and β-Oxidation of Long-Chain Unsaturated Carboxylic Acids

Degradation of unsaturated long-chain carboxylic acids by beta-oxidation, which is a compartmentalized process occurring in both mitochondria and peroxisomes in mammalian cells, was studied using rat liver as a model tissue. Inclusion of (poly)unsaturated fatty acids in the perfusion medium resulted in an increased concentration of catalase-H2O2 complex indicating on-going peroxisomal beta-oxidation. For this to occur, an active peroxisomal delta 3, delta 2-enoyl-CoA isomerase was required for metabolism of the double bonds at odd-numbered positions in acyl-CoA. Experiments with isolated subcellular organelles from rat liver confirmed the presence of isomerase in the peroxisomes, and the enzyme was subsequently isolated with apparent homogeneity. Comparison of amino acid sequences from the enzyme with a published sequence for a bifunctional protein from rat liver identified them representing the same molecule. The peroxisomal bifunctional protein can thus act as a multifunctional hydratase-dehydrogenase-isomerase enzyme. Examination of the mitochondrial isomerase revealed that rat liver mitochondria possess two isoenzymes: a long-chain isomerase not induced by clofibrate-treatment and showing a preference for C10-C12 substrates, and a clofibrate-inducible short-chain isomerase which gave the highest catalytic activity with C6 substrates. Experiments with isolated peroxisomes and unsaturated acyl-CoAs demonstrated that the beta-oxidation of fatty acids having double bonds at even-numbered positions was dependent on 2,4-dienoyl-CoA reductase in peroxisomes, as in mitochondria. Immunocytochemical experiments using the protein A-gold labelling technique, and comparison of their physicochemical properties indicated that all the mammalian reductases purified so far are mitochondrial isoenzymes. It turned out during the isolation of 3-hydroxyacyl-CoA epimerase that there was no monofunctional epimerase at all in the rat liver. Instead, the epimerization reaction occurred via dehydration-hydration catalyzed by two distinct stereospecific hydratases, 2-enoyl-CoA hydratase 1 (the classic hydratase) and 2-enoyl-CoA hydratase 2 (a novel hydratase). The present data demonstrate that peroxisomes contain all the enzymes required for the beta-oxidation of unsaturated fatty acids and support the notion that one of the physiological functions of peroxisomal beta-oxidation is to metabolize long-chain hydrophobic carboxylic acids to shorter, more polar metabolites which are then either metabolized further in the body or excreted.