Acyl-CoA Oxidase is Imported As a Heteropentameric, Cofactor-containing Complex into Peroxisomes of Yarrowia Lipolytica

The Journal of Cell Biology. Feb, 2002  |  Pubmed ID: 11815635

Five isoforms of acyl-CoA oxidase (Aox), designated Aox1p to Aox5p, constitute a 443-kD heteropentameric complex containing one polypeptide chain of each isoform within the peroxisomal matrix of the yeast Yarrowia lipolytica. Assembly of the Aox complex occurs in the cytosol and precedes its import into peroxisomes. Peroxisomal targeting of the Aox complex is abolished in a mutant lacking the peroxin Pex5p, a component of the matrix protein targeting machinery. Import of the Aox complex into peroxisomes does not involve the cytosolic chaperone Pex20p, which mediates the oligomerization and import of peroxisomal thiolase. Aox2p and Aox3p play a pivotal role in the formation of the Aox complex in the cytosol and can substitute for one another in promoting assembly of the complex. In vitro, these subunits retard disassembly of the Aox complex and increase the efficiency of its reassembly. Neither Aox2p nor Aox3p is required for acquisition of the cofactor FAD by other components of the complex. We provide evidence that the Aox2p- and Aox3p-assisted assembly of the Aox complex in the cytosol is mandatory for its import into peroxisomes and that no component of the complex can penetrate the peroxisomal matrix as a monomer.

RNA Interference of Peroxisome-related Genes in C. Elegans: a New Model for Human Peroxisomal Disorders

Physiological Genomics. Aug, 2002  |  Pubmed ID: 12181365

RNA-mediated interference (RNAi) for the posttranscriptional silencing of genes was used to evaluate the importance of various peroxisomal enzymes and peroxins for the development of Caenorhabditis elegans and to compare the roles of these proteins in the nematode to their roles in yeasts and humans. The nematode counterparts of the human ATP-binding cassette half-transporters, the enzymes alkyldihydroxyacetonephosphate synthase and Delta(3,5)-Delta (2,4)-dienoyl-CoA isomerase, the receptors for peroxisomal membrane and matrix proteins (Pex19p and Pex5p), and components of the docking and translocation machineries for matrix proteins (Pex13p and Pex12p) are essential for the development of C. elegans. Unexpectedly, RNAi silencing of the acyl-CoA synthetase-mediated activation of fatty acids, the alpha- and beta-oxidation of fatty acids, the intraperoxisomal decomposition of hydrogen peroxide, and the peroxins Pex1p, Pex2p, and Pex6p had no apparent effect on C. elegans development. The described analysis of functional gene knockouts through RNAi provides a basis for the use of C. elegans as a valuable model system with which to study the molecular and physiological defects underlying the human peroxisomal disorders.

Peroxisome Division in the Yeast Yarrowia Lipolytica is Regulated by a Signal from Inside the Peroxisome

The Journal of Cell Biology. Sep, 2003  |  Pubmed ID: 14504266

We describe an unusual mechanism for organelle division. In the yeast Yarrowia lipolytica, only mature peroxisomes contain the complete set of matrix proteins. These mature peroxisomes assemble from several immature peroxisomal vesicles in a multistep pathway. The stepwise import of distinct subsets of matrix proteins into different immature intermediates along the pathway causes the redistribution of a peroxisomal protein, acyl-CoA oxidase (Aox), from the matrix to the membrane. A significant redistribution of Aox occurs only in mature peroxisomes. Inside mature peroxisomes, the membrane-bound pool of Aox interacts with Pex16p, a membrane-associated protein that negatively regulates the division of early intermediates in the pathway. This interaction inhibits the negative action of Pex16p, thereby allowing mature peroxisomes to divide.

The Peroxisome: Orchestrating Important Developmental Decisions from Inside the Cell

The Journal of Cell Biology. Mar, 2004  |  Pubmed ID: 14981090

The peroxisome has long been known for its role in lipid metabolism and hydrogen peroxide detoxification. However, growing evidence supports the view that this organelle can also function both as an intracellular signaling compartment and as an organizing platform that orchestrates certain developmental decisions from inside the cell. This review highlights various strategies that peroxisomes employ to regulate the processes of development, differentiation, and morphogenesis and critically evaluates several molecular mechanisms by which peroxisomes promote these processes.

A New Definition for the Consensus Sequence of the Peroxisome Targeting Signal Type 2

Journal of Molecular Biology. Jul, 2004  |  Pubmed ID: 15312767

All organisms except the nematode Caenorhabditis elegans have been shown to possess an import system for peroxisomal proteins containing a peroxisome targeting signal type 2 (PTS2). The currently accepted consensus sequence for this amino-terminal nonapeptide is -(R/K)(L/V/I)X(5)(H/Q)(L/A)-. Some C.elegans proteins contain putative PTS2 motifs, including the ortholog (CeMeK) of human mevalonate kinase, an enzyme known to be targeted by PTS2 to mammalian peroxisomes. We cloned the gene for CeMeK (open reading frame Y42G9A.4) and examined the subcellular localization of CeMeK and of two other proteins with putative PTS2s at their amino termini encoded by the open reading frames D1053.2 and W10G11.11. All three proteins localized to the cytosol, confirming and extending the finding that C.elegans lacks PTS2-dependent peroxisomal protein import. The putative PTS2s of the proteins encoded by D1053.2 and W10G11.11 did not function in targeting to peroxisomes in yeast or mammalian cells, suggesting that the current PTS2 consensus sequence is too broad. Analysis of available experimental data on both functional and nonfunctional PTS2s led to two re-evaluated PTS2 consensus sequences: -R(L/V/I/Q)XX(L/V/I/H)(L/S/G/A)X(H/Q)(L/A)-, describes the most common variants of PTS2, while -(R/K)(L/V/I/Q)XX(L/V/I/H/Q)(L/S/G/A/K)X(H/Q)(L/A/F)-, describes essentially all variants of PTS2. These redefined PTS2 consensus sequences will facilitate the identification of proteins of unknown cellular localization as possible peroxisomal proteins.

Dynamic Ergosterol- and Ceramide-rich Domains in the Peroxisomal Membrane Serve As an Organizing Platform for Peroxisome Fusion

The Journal of Cell Biology. Feb, 2005  |  Pubmed ID: 15738267

We describe unusual ergosterol- and ceramide-rich (ECR) domains in the membrane of yeast peroxisomes. Several key features of these detergent-resistant domains, including the nature of their sphingolipid constituent and its unusual distribution across the membrane bilayer, clearly distinguish them from well characterized detergent-insoluble lipid rafts in the plasma membrane. A distinct set of peroxisomal proteins, including two ATPases, Pex1p and Pex6p, as well as phosphoinositide- and GTP-binding proteins, transiently associates with the cytosolic face of ECR domains. All of these proteins are essential for the fusion of the immature peroxisomal vesicles P1 and P2, the earliest intermediates in a multistep pathway leading to the formation of mature, metabolically active peroxisomes. Peroxisome fusion depends on the lateral movement of Pex1p, Pex6p, and phosphatidylinositol-4,5-bisphosphate-binding proteins from ECR domains to a detergent-soluble portion of the membrane, followed by their release to the cytosol. Our data suggest a model for the multistep reorganization of the multicomponent peroxisome fusion machinery that transiently associates with ECR domains.

Peroxisome Biogenesis: the Peroxisomal Endomembrane System and the Role of the ER

The Journal of Cell Biology. Jul, 2006  |  Pubmed ID: 16801391

Peroxisomes have long been viewed as semiautonomous, static, and homogenous organelles that exist outside the secretory and endocytic pathways of vesicular flow. However, growing evidence supports the view that peroxisomes actually constitute a dynamic endomembrane system that originates from the endoplasmic reticulum. This review highlights the various strategies used by evolutionarily diverse organisms for coordinating the flow of membrane-enclosed carriers through the peroxisomal endomembrane system and critically evaluates the dynamics and molecular mechanisms of this multistep process.

Lipids and Lipid Domains in the Peroxisomal Membrane of the Yeast Yarrowia Lipolytica

Biochimica Et Biophysica Acta. Dec, 2006  |  Pubmed ID: 17023063

Biological membranes have unique and highly diverse compositions of their lipid constituents. At present, we have only partial understanding of how membrane lipids and lipid domains regulate the structural integrity and functionality of cellular organelles, maintain the unique molecular composition of each organellar membrane by orchestrating the intracellular trafficking of membrane-bound proteins and lipids, and control the steady-state levels of numerous signaling molecules generated in biological membranes. Similar to other organellar membranes, a single lipid bilayer enclosing the peroxisome, an organelle known for its essential role in lipid metabolism, has a unique lipid composition and organizes some of its lipid and protein components into distinctive assemblies. This review highlights recent advances in our knowledge of how lipids and lipid domains of the peroxisomal membrane regulate the processes of peroxisome assembly and maintenance in the yeast Yarrowia lipolytica. We critically evaluate the molecular mechanisms through which lipid constituents of the peroxisomal membrane control these multistep processes and outline directions for future research in this field.

Overproduction of Translation Elongation Factor 1-alpha (eEF1A) Suppresses the Peroxisome Biogenesis Defect in a Hansenula Polymorpha Pex3 Mutant Via Translational Read-through

FEMS Yeast Research. Oct, 2007  |  Pubmed ID: 17425673

In eukaryotes, elongation factor 1-alpha (eEF1A) is required during the elongation phase of translation. We observed that a portion of the cellular eEF1A colocalizes with purified peroxisomes from the methylotrophic yeast Hansenula polymorpha. We have isolated two genes (TEF1 and TEF2) that encode eEF1A, and which are constitutively expressed. We observed that overproduction of eEF1A suppressed the peroxisome deficient phenotype of an H. polymorpha pex3-1 mutant, which was not observed in a strain deleted for PEX3. The pex3-1 allele contains a UGG to UGA mutation, thereby truncating Pex3p after amino acid 242, suggesting that the suppression effect might be the result of translational read-through. Consistent with this hypothesis, overexpression of the pex3-1 gene itself (including its now untranslated part) partly restored peroxisome biogenesis in a PEX3 null mutant. Subsequent co-overexpression of TEF2 in this strain fully restored its peroxisome biogenesis defect and resulted in the formation of major amounts of full-length Pex3p, presumably via translational read-through.

A Signal from Inside the Peroxisome Initiates Its Division by Promoting the Remodeling of the Peroxisomal Membrane

The Journal of Cell Biology. Apr, 2007  |  Pubmed ID: 17438077

We define the dynamics of spatial and temporal reorganization of the team of proteins and lipids serving peroxisome division. The peroxisome becomes competent for division only after it acquires the complete set of matrix proteins involved in lipid metabolism. Overloading the peroxisome with matrix proteins promotes the relocation of acyl-CoA oxidase (Aox), an enzyme of fatty acid beta-oxidation, from the matrix to the membrane. The binding of Aox to Pex16p, a membrane-associated peroxin required for peroxisome biogenesis, initiates the biosynthesis of phosphatidic acid and diacylglycerol (DAG) in the membrane. The formation of these two lipids and the subsequent transbilayer movement of DAG initiate the assembly of a complex between the peroxins Pex10p and Pex19p, the dynamin-like GTPase Vps1p, and several actin cytoskeletal proteins on the peroxisomal surface. This protein team promotes membrane fission, thereby executing the terminal step of peroxisome division.

Spatiotemporal Dynamics of the ER-derived Peroxisomal Endomembrane System

International Review of Cell and Molecular Biology. 2009  |  Pubmed ID: 19121819

Recent studies have provided evidence that peroxisomes constitute a multicompartmental endomembrane system. The system begins to form with the targeting of certain peroxisomal membrane proteins to the ER and their exit from the ER via preperoxisomal carriers. These carriers undergo a multistep maturation into metabolically active peroxisomes containing the entire complement of peroxisomal membrane and matrix proteins. At each step, the import of a subset of proteins and the uptake of certain membrane lipids result in the formation of a distinct, more mature compartment of the peroxisomal endomembrane system. Individual peroxisomal compartments proliferate by undergoing one or several rounds of division. Herein, we discuss various strategies that evolutionarily diverse organisms use to coordinate compartment formation, maturation, and division in the peroxisomal endomembrane system. We also critically evaluate the molecular and cellular mechanisms governing these processes, outline the most important unanswered questions, and suggest directions for future research.

Effect of Calorie Restriction on the Metabolic History of Chronologically Aging Yeast

Experimental Gerontology. Sep, 2009  |  Pubmed ID: 19539741

Aging is a highly complex, multifactorial process. We use the yeast Saccharomyces cerevisiae as a model to study the mechanisms of cellular aging in multicellular eukaryotes. To address the inherent complexity of aging from a systems perspective and to build an integrative model of aging process, we investigated the effect of calorie restriction (CR), a low-calorie dietary regimen, on the metabolic history of chronologically aging yeast. We examined how CR influences the age-related dynamics of changes in the intracellular levels of numerous proteins and metabolites, carbohydrate and lipid metabolism, interorganellar metabolic flow, concentration of reactive oxygen species, mitochondrial morphology, essential oxidation-reduction processes in mitochondria, mitochondrial proteome, cardiolipin in the inner mitochondrial membrane, frequency of mitochondrial DNA mutations, dynamics of mitochondrial nucleoid, susceptibility to mitochondria-controlled apoptosis, and stress resistance. Based on the comparison of the metabolic histories of long-lived CR yeast and short-lived non-CR yeast, we propose that yeast define their long-term viability by designing a diet-specific pattern of metabolism and organelle dynamics prior to reproductive maturation. Thus, our data suggest that longevity in chronologically aging yeast is programmed by the level of metabolic capacity and organelle organization they developed, in a diet-specific fashion, prior to entry into a non-proliferative state.

A Novel Function of Lipid Droplets in Regulating Longevity

Biochemical Society Transactions. Oct, 2009  |  Pubmed ID: 19754450

Growing evidence supports the view that LDs (lipid droplets) are dynamic organelles that can serve both as an intracellular signalling compartment and as an organizing platform orchestrating many vital processes in eukaryotic cells. It has become clear that the LDs-confined deposition and lipolytic degradation of neutral lipids define longevity in multicellular eukaryotic organisms and yeast. We summarize the evidence in support of the essential role that LDs play in longevity regulation and propose several molecular mechanisms by which these dynamic organellar compartments control the aging process in multicellular eukaryotes and yeast.

Chemical Genetic Screen Identifies Lithocholic Acid As an Anti-aging Compound That Extends Yeast Chronological Life Span in a TOR-independent Manner, by Modulating Housekeeping Longevity Assurance Processes

Aging. Jul, 2010  |  Pubmed ID: 20622262

In chronologically aging yeast, longevity can be extended by administering a caloric restriction (CR) diet or some small molecules. These life-extending interventions target the adaptable target of rapamycin (TOR) and cAMP/protein kinase A (cAMP/PKA) signaling pathways that are under the stringent control of calorie availability. We designed a chemical genetic screen for small molecules that increase the chronological life span of yeast under CR by targeting lipid metabolism and modulating housekeeping longevity pathways that regulate longevity irrespective of the number of available calories. Our screen identifies lithocholic acid (LCA) as one of such molecules. We reveal two mechanisms underlying the life-extending effect of LCA in chronologically aging yeast. One mechanism operates in a calorie availability-independent fashion and involves the LCA-governed modulation of housekeeping longevity assurance pathways that do not overlap with the adaptable TOR and cAMP/PKA pathways. The other mechanism extends yeast longevity under non-CR conditions and consists in LCA-driven unmasking of the previously unknown anti-aging potential of PKA. We provide evidence that LCA modulates housekeeping longevity assurance pathways by suppressing lipid-induced necrosis, attenuating mitochondrial fragmentation, altering oxidation-reduction processes in mitochondria, enhancing resistance to oxidative and thermal stresses, suppressing mitochondria-controlled apoptosis, and enhancing stability of nuclear and mitochondrial DNA.

Xenohormetic, Hormetic and Cytostatic Selective Forces Driving Longevity at the Ecosystemic Level

Aging. Aug, 2010  |  Pubmed ID: 20693605

We recently found that lithocholic acid (LCA), a bile acid, extends yeast longevity. Unlike mammals, yeast do not synthesize bile acids. We therefore propose that bile acids released into the environment by mammals may act as interspecies chemical signals providing longevity benefits to yeast and, perhaps, other species within an ecosystem.

Peroxisome Metabolism and Cellular Aging

Traffic (Copenhagen, Denmark). Mar, 2011  |  Pubmed ID: 21083858

The essential role of peroxisomes in fatty acid oxidation, anaplerotic metabolism, and hydrogen peroxide turnover is well established. Recent findings suggest that these and other related biochemical processes governed by the organelle may also play a critical role in regulating cellular aging. The goal of this review is to summarize and integrate into a model the evidence that peroxisome metabolism actually helps define the replicative and chronological age of a eukaryotic cell. In this model, peroxisomal reactive oxygen species (ROS) are seen as altering organelle biogenesis and function, and eliciting changes in the dynamic communication networks that exist between peroxisomes and other cellular compartments. At low levels, peroxisomal ROS activate an anti-aging program in the cell; at concentrations beyond a specific threshold, a pro-aging course is triggered.

In Search of Housekeeping Pathways That Regulate Longevity

Cell Cycle (Georgetown, Tex.). Sep, 2011  |  Pubmed ID: 21862878

Lithocholic Bile Acid Selectively Kills Neuroblastoma Cells, While Sparing Normal Neuronal Cells

Oncotarget. Oct, 2011  |  Pubmed ID: 21992775

Aging is one of the major risk factors of cancer. The onset of cancer can be postponed by pharmacological and dietary anti-aging interventions. We recently found in yeast cellular models of aging that lithocholic acid (LCA) extends longevity. Here we show that, at concentrations that are not cytotoxic to primary cultures of human neurons, LCA kills the neuroblastoma (NB) cell lines BE(2)-m17, SK-n-SH, SK-n-MCIXC and Lan-1. In BE(2)-m17, SK-n-SH and SK-n-MCIXC cells, the LCA anti-tumor effect is due to apoptotic cell death. In contrast, the LCA-triggered death of Lan-1 cells is not caused by apoptosis. While low concentrations of LCA sensitize BE(2)-m17 and SK-n-MCIXC cells to hydrogen peroxide-induced apoptotic cell death controlled by mitochondria, these LCA concentrations make primary cultures of human neurons resistant to such a form of cell death. LCA kills BE(2)-m17 and SK-n-MCIXC cell lines by triggering not only the intrinsic (mitochondrial) apoptotic cell death pathway driven by mitochondrial outer membrane permeabilization and initiator caspase-9 activation, but also the extrinsic (death receptor) pathway of apoptosis involving activation of the initiator caspase-8. Based on these data, we propose a mechanism underlying a potent and selective anti-tumor effect of LCA in cultured human NB cells. Moreover, our finding that LCA kills cultured human breast cancer and rat glioma cells implies that it has a broad anti-tumor effect on cancer cells derived from different tissues and organisms.

Dynamics and Regulation of Lipid Droplet Formation in Lipopolysaccharide (LPS)-stimulated Microglia

Biochimica Et Biophysica Acta. Jan, 2012  |  Pubmed ID: 22289388

Lipid droplets (LDs) are neutral lipid-rich organelles involved in many cellular processes. A well-known example is their accumulation in leukocytes upon activation by pro-inflammatory stimuli such as lipopolysaccharides (LPS) derived from gram-negative bacteria. A role of LDs and LD-associated proteins during inflammation in the brain is unknown, however. We have now studied their dynamics and regulation in microglia, the resident immune cells in the brain. We find that LPS treatment of microglia leads to the accumulation in them of LDs, and enhancement of the size of LDs. This induction of LDs was abolished by triacsin C, an inhibitor of triglyceride biosynthesis. LPS strongly activated c-Jun N-terminal kinase (JNK) and p38 MAPK stress signaling pathways and increased the expression of LD-associated protein perilipin-2 (ADRP) in a time-dependent manner. Immunostaining showed that perilipin-2 in LPS-treated microglia predominantly colocalized with LDs. Inhibitors of p38 α/β (SB203580) and PI3K/Akt pathway (LY294002), but not that of JNK (SP600125), reduced LPS-induced LD accumulation and eliminated the activating effect of LPS on perilipin-2. In addition, cytosolic phospholipase A(2) (cPLA(2)-α), a key enzyme for arachidonic acid release, colocalized with LPS-induced LDs. These observations suggest that LDs may play an important role in eicosanoid synthesis in activated microglia; they provide a novel insight into the regulation of LDs in inflammatory cells of the brain and point to a potential role of p38 α/β in LPS-induced LD accumulation. Collectively, our findings imply that LD formation and perilipin-2 induction could be microglial biomarkers of inflammation in the central nervous system.