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Ethanolamine-containing alkenyl ether glycerophospholipids, plasmalogens, are major cell membrane components of mammalian cells that activate membrane protein receptors such as ion transporters and G-protein coupled receptors. However, the mechanism by which plasmalogens modulate receptor function is unknown. Here, we found that exogenously added plasmalogens activate transient receptor potential cation channel subfamily C member 4 (TRPC4) to increase Ca2+ influx, followed by calcium/calmodulin-dependent protein kinase 2-mediated phosphorylation of AMP-activated protein kinase (AMPK). Upon topical application of plasmalogens to the skin of mice, AMPK activation was observed in TRPC4-expressing hair bulbs and hair follicles. Here, TRPC4 was co-localized with the leucine-rich repeat containing G protein-coupled receptor 5, a marker of hair-follicle stem cells, leading to hair growth. Collectively, this study indicates that plasmalogens could function as gate openers for TRPC4, followed by activating AMPK, which likely accelerates hair growth in mice.

Plasmalogens are a subclass of glycerophospholipid containing vinyl-ether bond at the sn-1 position of glycerol backbone. Ethanolamine-containing plasmalogens (plasmalogens) are major constituents of cellular membranes in mammalian cells and de novo synthesis of plasmalogens largely contributes to the homeostasis of plasmalogens. Plasmalogen biosynthesis is regulated by a feedback mechanism that senses the plasmalogen level in the inner leaflet of the plasma membrane and regulates the stability of fatty acyl-CoA reductase 1 (Far1), a rate-limiting enzyme for plasmalogen biosynthesis. However, the molecular mechanism underlying the localization of plasmalogens in cytoplasmic leaflet of plasma membrane remains unknown. To address this issue, we attempted to identify a potential transporter of plasmalogens from the outer to the inner leaflet of plasma membrane by focusing on phospholipid flippases, type-IV P-type adenosine triphosphatases (P4-ATPase), localized in the plasma membranes. We herein show that knockdown of ATP8B2 belonging to the class-1 P4-ATPase enhances localization of plasmalogens but not phosphatidylethanolamine in the extracellular leaflet and impairs plasmalogen-dependent degradation of Far1. Furthermore, phosphorylation of protein kinase B (AKT) is downregulated by lowering the expression of ATP8B2, which leads to suppression of cell growth. Taken together, these results suggest that enrichment of plasmalogens in the cytoplasmic leaflet of plasma membranes is mediated by ATP8B2 and this asymmetric distribution of plasmalogens is required for sensing plasmalogens as well as phosphorylation of AKT.

Plasmalogens are a subclass of ether glycerophospholipids characterized by a vinyl-ether bond at the sn-1 position of the glycerol backbone. Plasmalogen biosynthesis is initiated in peroxisomes. At the third step of plasmalogen synthesis, alkyl-dihydroxyacetonephosphate (DHAP) is enzymatically reduced to 1-alkyl-sn-glycero-3-phospate by acyl/alkyl DHAP reductase (ADHAPR), whose activity is found in both peroxisomes and microsomes. We herein show that knockdown of ADHAPR in HeLa cells reduced the synthesis of ethanolamine plasmalogen (PlsEtn), similar to the Chinese hamster ovary cell mutant FAA.K1B deficient in ADHAPR activity. Endogenous ADHAPR and ectopically expressed FLAG-tagged ADHAPR were localized to peroxisomes and endoplasmic reticulum (ER) as a type I integral membrane protein in HeLa cells. ADHAPR targets to peroxisomes via a Pex19p-dependent class I pathway. In addition, it is also inserted into the ER via the SRP-dependent mechanism. The ADHAPR mutant lacking the N-terminal domain preferentially targets to the ER, restoring the reduced level of PlsEtn synthesis in FAA.K1B cell. In contrast, the expression of full-length ADHAPR in the mutant cells elevates the synthesis of phosphatidylethanolamine, but not PlsEtn. Taken together, these results suggest that the third step of plasmalogen synthesis is mediated by ER-localized ADHAPR.

The peroxisome is a subcellular organelle that functions in essential metabolic pathways, including biosynthesis of plasmalogens, fatty acid β-oxidation of very-long-chain fatty acids, and degradation of hydrogen peroxide. Peroxisome biogenesis disorders (PBDs) manifest as severe dysfunction in multiple organs, including the central nervous system (CNS), but the pathogenic mechanisms in PBDs are largely unknown. Because CNS integrity is coordinately established and maintained by neural cell interactions, we here investigated whether cell-cell communication is impaired and responsible for the neurological defects associated with PBDs. Results from a noncontact co-culture system consisting of primary hippocampal neurons with glial cells revealed that a peroxisome-deficient astrocytic cell line secretes increased levels of brain-derived neurotrophic factor (BDNF), resulting in axonal branching of the neurons. Of note, the BDNF expression in astrocytes was not affected by defects in plasmalogen biosynthesis and peroxisomal fatty acid β-oxidation in the astrocytes. Instead, we found that cytosolic reductive states caused by a mislocalized catalase in the peroxisome-deficient cells induce the elevation in BDNF secretion. Our results suggest that peroxisome deficiency dysregulates neuronal axogenesis by causing a cytosolic reductive state in astrocytes. We conclude that astrocytic peroxisomes regulate BDNF expression and thereby support neuronal integrity and function.

Plasmalogens, mostly ethanolamine-containing alkenyl ether phospholipids, are a major subclass of glycerophospholipids. Plasmalogen synthesis is initiated in peroxisomes and completed in the endoplasmic reticulum. The absence of plasmalogens in several organs of peroxisome biogenesis-defective patients suggests that the de novo synthesis of plasmalogens plays a pivotal role in its homeostasis in tissues. Plasmalogen synthesis is regulated by modulating the stability of fatty acyl-CoA reductase 1 on peroxisomal membranes, a rate-limiting enzyme in plasmalogen synthesis, by sensing plasmalogens in the inner leaflet of plasma membranes. Dysregulation of plasmalogen homeostasis impairs cholesterol biosynthesis by altering the stability of squalene monooxygenase, a key enzyme in cholesterol biosynthesis, implying physiological consequences of plasmalogen homeostasis with respect to cholesterol metabolism in cells, as well as in organs such as the liver.

Peroxisome number and quality are maintained by its biogenesis and turnover and are important for the homeostasis of peroxisomes. Peroxisomes are increased in number by division with dynamic morphological changes including elongation, constriction, and fission. In the course of peroxisomal division, peroxisomal morphogenesis is orchestrated by Pex11β, dynamin-like protein 1 (DLP1), and mitochondrial fission factor (Mff). Conversely, peroxisome number is reduced by its degradation. Peroxisomes are mainly degraded by pexophagy, a type of autophagy specific for peroxisomes. Upon pexophagy, an adaptor protein translocates on peroxisomal membrane and connects peroxisomes to autophagic machineries. Molecular mechanisms of pexophagy are well studied in yeast systems where several specific adaptor proteins are identified. Pexophagy in mammals also proceeds in a manner dependent on adaptor proteins. In this review, we address the recent progress in studies on peroxisome morphogenesis and pexophagy.

The peroxisomal membrane protein Pex14p is phosphorylated in vivo, whereas no function has been assigned to Pex14p phosphorylation in yeast and mammalian cells. Mitotic phosphorylation of Pex14p and consequent suppression of catalase import are a mechanism of protecting DNA upon nuclear envelope breakdown at mitosis., Peroxisomal matrix proteins are imported into peroxisomes via membrane-bound docking/translocation machinery. One central component of this machinery is Pex14p, a peroxisomal membrane protein involved in the docking of Pex5p, the receptor for peroxisome targeting signal type 1 (PTS1). Studies in several yeast species have shown that Pex14p is phosphorylated in vivo, whereas no function has been assigned to Pex14p phosphorylation in yeast and mammalian cells. Here, we investigated peroxisomal protein import and its dynamics in mitotic mammalian cells. In mitotically arrested cells, Pex14p is phosphorylated at Ser-232, resulting in a lower import efficiency of catalase, but not the majority of proteins including canonical PTS1 proteins. Conformational change induced by the mitotic phosphorylation of Pex14p more likely increases homomeric interacting affinity and suppresses topological change of its N-terminal part, thereby giving rise to the retardation of Pex5p export in mitotic cells. Taken together, these data show that mitotic phosphorylation of Pex14p and consequent suppression of catalase import are a mechanism of protecting DNA upon nuclear envelope breakdown at mitosis.

Synthesis of ethanolamine plasmalogen (PlsEtn) is regulated by modulating the stability of fatty acyl-CoA reductase 1 (Far1) on peroxisomal membrane, a rate-limiting enzyme in plasmalogen synthesis. Dysregulation of plasmalogen homeostasis impairs cholesterol biosynthesis in cultured cells by altering the stability of squalene epoxidase (SQLE). However, regulation of PlsEtn synthesis and physiological consequences of plasmalogen homeostasis in tissues remain unknown. In the present study, we found that the protein but not the transcription level of Far1 in the cerebellum of the Pex14 mutant mouse expressing Pex14p lacking its C-terminal region (Pex14ΔC/ΔC) is higher than that from wild-type mouse, suggesting that Far1 is stabilized by the lowered level of PlsEtn. The protein level of SQLE was increased, whereas the transcriptional activity of the liver X receptors (LXRs), ligand-activated transcription factors of the nuclear receptor superfamily, is lowered in the cerebellum of Pex14ΔC/ΔC and the mice deficient in dihydroxyacetonephosphate acyltransferase, the initial enzyme for the synthesis of PlsEtn. These results suggest that the reduction of plasmalogens in the cerebellum more likely compromises the cholesterol homeostasis, thereby reducing the transcriptional activities of LXRs, master regulators of cholesterol homeostasis.

Ether glycerolipids, plasmalogens are found in various mammalian cells and tissues. However, physiological role of plasmalogens in epithelial cells remains unknown. We herein show that synthesis of ethanolamine-containing plasmalogens, plasmenylethanolamine (PlsEtn), is deficient in MCF7 cells, an epithelial cell line, with severely reduced expression of alkyl-dihydroxyacetonephosphate synthase (ADAPS), the second enzyme in the PlsEtn biosynthesis. Moreover, expression of ADAPS or supplementation of PlsEtn containing C18-alkenyl residue delays the migration of MCF7 cells as compared to that mock-treated MCF7 and C16-alkenyl-PlsEtn-supplemented MCF7 cells. Localization of E-cadherin to cell–cell junctions is highly augmented in cells containing C18-alkenyl-PlsEtn. Together, these results suggest that PlsEtn containing C18-alkenyl residue plays a distinct role in the integrity of E-cadherin-mediated adherens junction.

Plasmalogen biosynthesis is regulated by modulating fatty acyl-CoA reductase 1 stability in a manner dependent on cellular plasmalogen level. However, physiological significance of the regulation of plasmalogen biosynthesis remains unknown. Here we show that elevation of the cellular plasmalogen level reduces cholesterol biosynthesis without affecting the isoprenylation of proteins such as Rab and Pex19p. Analysis of intermediate metabolites in cholesterol biosynthesis suggests that the first oxidative step in cholesterol biosynthesis catalyzed by squalene monooxygenase (SQLE), an important regulator downstream HMG-CoA reductase in cholesterol synthesis, is reduced by degradation of SQLE upon elevation of cellular plasmalogen level. By contrast, the defect of plasmalogen synthesis causes elevation of SQLE expression, resulting in the reduction of 2,3-epoxysqualene required for cholesterol synthesis, hence implying a novel physiological consequence of the regulation of plasmalogen biosynthesis.

Peroxisomes are essential intracellular organelles that involve many metabolic processes, such as β-oxidation of very long-chain fatty acids and synthesis of plasmalogen and bile acids as well as generation and degradation of hydrogen peroxide. These peroxisomal functions are fulfilled by strictly and spatiotemporally regulated compartmentation of the proteins catalysing these reactions. Defects in peroxisomal protein import results in inherited peroxisome biogenesis disorders in humans. Peroxisomal matrix and membrane proteins are synthesised on free ribosomes but transported into peroxisomes by distinct pathways determined by specific targeting signals and their receptors. The mechanism by which this is achieved has been clarified by identification of many PEX genes and the products named peroxins, the essential factors for peroxisome biogenesis. This article introduces several basic methods to investigate protein import into peroxisomes. Key Concepts Peroxisome plays an essential role in various metabolic pathways. Deficiency of peroxisomes in human causes severe foetal genetic diseases, peroxisome-deficient disorders. Functions and the integrity of peroxisomes are archived by proper import of both matrix and membrane proteins into peroxisomes. Peroxisomal proteins are encoded by nuclear DNA, synthesised in cytosolic free ribosomes and post-translationally imported into pre-existing peroxisomes. Peroxisomal matrix and membrane proteins are translocated into peroxisomes via different pathways in a manner dependent on their distinct targeting signals. Many of peroxins encoded by PEX genes are responsible factors for peroxisome biogenesis and are involved in the import of peroxisomal proteins. Various experimental approaches have elucidated the molecular mechanisms underlying peroxisome biogenesis. Post-translational import of peroxisomal proteins into isolated peroxisomes can be reproduced in vitro. Semi-permeabilised cells, in which only the plasma membrane is selectively permeabilised, are used to investigate peroxisomal protein import, as with living cells. Keywords: peroxisomes; protein translocation; peroxisome targeting signals; import receptors; PEX genes; peroxins; peroxisome biogenesis disorders

Alkenyl ether phospholipids are a major sub-class of ethanolamine- and choline-phospholipids in which a long chain fatty alcohol is attached at the sn-1 position through a vinyl ether bond. Biosynthesis of ethanolamine-containing alkenyl ether phospholipids, plasmalogens, is regulated by modulating the stability of fatty acyl-CoA reductase 1 (Far1) in a manner dependent on the level of cellular plasmalogens. However, precise molecular mechanisms underlying the regulation of plasmalogen synthesis remain poorly understood. Here we show that degradation of Far1 is accelerated by inhibiting dynamin-, Src kinase-, or flotillin-1-mediated endocytosis without increasing the cellular level of plasmalogens. By contrast, Far1 is stabilized by sequestering cholesterol with nystatin. Moreover, abrogation of the asymmetric distribution of plasmalogens in the plasma membrane by reducing the expression of CDC50A encoding a β-subunit of flippase elevates the expression level of Far1 and plasmalogen synthesis without reducing the total cellular level of plasmalogens. Together, these results support a model that plasmalogens localised in the inner leaflet of the plasma membranes are sensed for plasmalogen homeostasis in cells, thereby suggesting that plasmalogen synthesis is spatiotemporally regulated by monitoring cellular level of plasmalogens.

Rhizomelic chondrodysplasia punctata (RCDP) is an autosomal recessive disorder due to the deficiency in ether lipid synthesis. RCDP type 1, the most prominent type, is caused by the dysfunction of the receptor of peroxisome targeting signal type 2, Pex7 (peroxisomal biogenesis factor 7), and the rest of the patients, RCDP types 2 and 3, have defects in peroxisomal enzymes catalyzing the initial two steps of alkyl-phospholipid synthesis, glyceronephosphate O-acyltransferase and alkylglycerone phosphate synthase (Agps). We herein investigated defects of two patients with RCDP type 3. Patient 1 had a novel missense mutation, T1533G, resulting in the I511M substitution in Agps. The plasmalogen level was mildly reduced, whereas the protein level and peroxisomal localization of Agps-I511M in fibroblasts were normal as in the control fibroblasts. Structure prediction analysis suggested that the mutated residue was located in the helix α15 on the surface of V-shaped active site tunnel in Agps, likely accounting for the mild defects of plasmalogen synthesis. These results strongly suggest that an individual with mildly affected level of plasmalogen synthesis develops RCDP. In fibroblasts from patient 2, the expression of AGPS mRNA and Agps protein was severely affected, thereby giving rise to the strong reduction of plasmalogen synthesis.

Peroxisomes are subcellular organelles that function in multiple anabolic and catabolic processes, including β-oxidation of very-long-chain fatty acids (VLCFA) and biosynthesis of ether phospholipids. Peroxisomal disorders caused by defects in peroxisome biogenesis or peroxisomal β-oxidation manifest as severe neural disorders of the central nervous system. Abnormal peroxisomal metabolism is thought to be responsible for the clinical symptoms of these diseases, but their molecular pathogenesis remains to be elucidated. We performed lipidomic analysis to identify aberrant metabolites in fibroblasts from patients with Zellweger syndrome (ZS), acyl-CoA oxidase1 (AOx) deficiency, D-bifunctional protein (D-BP) and X-linked adrenoleukodystrophy (X-ALD), as well as in peroxisome-deficient Chinese hamster ovary cell mutants. In cells deficient in peroxisomal biogenesis, plasmenylethanolamine was remarkably reduced and phosphatidylethanolamine was increased. Marked accumulation of very-long-chain saturated fatty acid and monounsaturated fatty acids in phosphatidylcholine was observed in all mutant cells. Very-long-chain polyunsaturated fatty acid (VLC-PUFA) levels were significantly elevated, whilst phospholipids containing docosahexaenoic acid (DHA, C22:6n-3) were reduced in fibroblasts from patients with ZS, AOx deficiency, and D-BP deficiency, but not in fibroblasts from an X-ALD patient. Because patients with AOx deficiency suffer from more severe symptoms than those with X-ALD, accumulation of VLC-PUFA and/or reduction of DHA may be associated with the severity of peroxisomal diseases.

Peroxisomal fatty acyl-CoA reductase 1 (Far1) is essential for supplying fatty alcohols required for ether bond formation in ether glycerophospholipid synthesis. The stability of Far1 is regulated by a mechanism that is dependent on cellular plasmalogen levels. However, the membrane topology of Far1 and how Far1 is targeted to membranes remain largely unknown. Here, Far1 is shown to be a peroxisomal tail-anchored protein. The hydrophobic C terminus of Far1 binds to Pex19p, a cytosolic receptor harboring a C-terminal CAAX motif, which is responsible for the targeting of Far1 to peroxisomes. Far1, but not Far2, was preferentially degraded in response to the cellular level of plasmalogens. Experiments in which regions of Far1 or Far2 were replaced with the corresponding region of the other protein showed that the region flanking the transmembrane domain of Far1 is required for plasmalogen-dependent modulation of Far1 stability. Expression of Far1 increased plasmalogen synthesis in wild-type Chinese hamster ovary cells, strongly suggesting that Far1 is a rate-limiting enzyme for plasmalogen synthesis.

Summary Peroxisomal division comprises three steps: elongation, constriction, and fission. Translocation of dynamin-like protein 1 (DLP1), a member of the large GTPase family, from the cytosol to peroxisomes is a prerequisite for membrane fission; however, the molecular machinery for peroxisomal targeting of DLP1 remains unclear. This study investigated whether mitochondrial fission factor (Mff), which targets DLP1 to mitochondria, may also recruit DLP1 to peroxisomes. Results show that endogenous Mff is localized to peroxisomes, especially at the membrane-constricted regions of elongated peroxisomes, in addition to mitochondria. Knockdown of MFF abrogates the fission stage of peroxisomal division and is associated with failure to recruit DLP1 to peroxisomes, while ectopic expression of MFF increases the peroxisomal targeting of DLP1. Co-expression of MFF and PEX11β, the latter being a key player in peroxisomal elongation, increases peroxisome abundance. Overexpression of MFF also increases the interaction between DLP1 and Pex11pβ, which knockdown of MFF, but not Fis1, abolishes. Moreover, results show that Pex11pβ interacts with Mff in a DLP1-dependent manner. In conclusion, Mff contributes to the peroxisomal targeting of DLP1 and plays a key role in the fission of the peroxisomal membrane by acting in concert with Pex11pβ and DLP1.

Peroxisome division is regulated by several factors, termed fission factors, as well as the conditions of the cellular environment. Over the past decade, the idea of metabolic control of peroxisomal morphogenesis has been postulated, but remains largely undefined to date. In the current study, docosahexaenoic acid (DHA, C22:6n-3) was identified as an inducer of peroxisome division. In fibroblasts isolated from patients that carry defects in peroxisomal fatty acid β-oxidation, peroxisomes are much less abundant than normal cells. Treatment of these patient fibroblasts with DHA induced the proliferation of peroxisomes to the level seen in normal fibroblasts. DHA-induced peroxisomal proliferation was abrogated by treatment with a small inhibitory RNA (siRNA) targeting dynamin-like protein 1 and with dynasore, an inhibitor of dynamin-like protein 1, which suggested that DHA stimulates peroxisome division. DHA augmented the hyper-oligomerization of Pex11pβ and the formation of Pex11pβ-enriched regions on elongated peroxisomes. Time-lapse imaging analysis of peroxisomal morphogenesis revealed a sequence of steps involved in peroxisome division, including elongation in one direction followed by peroxisomal fission. DHA enhanced peroxisomal division in a microtubule-independent manner. These results suggest that DHA is a crucial signal for peroxisomal elongation, a prerequisite for subsequent fission and peroxisome division.

Plasmalogens are a major subclass of ethanolamine and choline glycerophospholipids in which a long chain fatty alcohol is attached at the sn-1 position through a vinyl ether bond. This ether-linked alkyl bond is formed in peroxisomes by replacement of a fatty acyl chain in the intermediate 1-acyl-dihydroxyacetone phosphate with a fatty alcohol in a reaction catalyzed by alkyl dihydroxyacetone phosphate synthase. Here, we demonstrate that the enzyme fatty acyl-CoA reductase 1 (Far1) supplies the fatty alcohols used in the formation of ether-linked alkyl bonds. Far1 activity is elevated in plasmalogen-deficient cells, and conversely, the levels of this enzyme are restored to normal upon plasmalogen supplementation. Down-regulation of Far1 activity in response to plasmalogens is achieved by increasing the rate of degradation of peroxisomal Far1 protein. Supplementation of normal cells with ethanolamine and 1-O-hexadecylglycerol, which are intermediates in plasmalogen biosynthesis, accelerates degradation of Far1. Taken together, our results indicate that ether lipid biosynthesis in mammalian cells is regulated by a negative feedback mechanism that senses cellular plasmalogen levels and appropriately increases or decreases Far1.

We herein isolated plasmalogen-deficient Chinese hamster ovary (CHO) mutant, ZPEG251, with a phenotype of normal import of peroxisomal matrix and membrane proteins. In ZPEG251, plasmenylethanolamine (PlsEtn) was severely reduced. Complementation analysis by expression of genes responsible for the plasmalogen biogenesis suggested that alkyl-dihydroxyacetonephosphate synthase (ADAPS), catalyzing the second step of plasmalogen biogenesis, was deficient in ZPEG251. ADAPS mRNA was barely detectable as verified by Northern blot and reverse transcription-PCR analyses. Defect of ADAPS expression was also assessed by immunoblot. As a step toward delineating functional roles of PlsEtn, we investigated its subcellular localization. PlsEtn was localized to post-Golgi compartments and enriched in detergent-resistant membranes. Transport of PlsEtn to post-Golgi compartments was apparently affected by lowering cellular ATP, but not by inhibitors of microtubule assembly and vesicular transport. Partitioning of cholesterol and sphingomyelin, a typical feature of lipid rafts, was not impaired in plasmalogen-deficient cells, including peroxisome assembly-defective mutants, hence suggesting that PlsEtn was not essential for lipid-raft architecture in CHO cells.

Although the exact etiology of Alzheimer's disease (AD) is a topic of debate, the consensus is that the accumulation of beta-amyloid (Abeta) peptides in the senile plaques is one of the hallmarks of the progression of the disease. The Abeta peptide is formed by the amyloidogenic cleavage of the amyloid precursor protein (APP) by beta- and gamma-secretases. The endocytic system has been implicated in the cleavages leading to the formation of Abeta. However, the identity of the intracellular compartment where the amyloidogenic secretases cleave and the mechanism by which the intracellularly generated Abeta is released into the extracellular milieu are not clear. Here, we show that beta-cleavage occurs in early endosomes followed by routing of Abeta to multivesicular bodies (MVBs) in HeLa and N2a cells. Subsequently, a minute fraction of Abeta peptides can be secreted from the cells in association with exosomes, intraluminal vesicles of MVBs that are released into the extracellular space as a result of fusion of MVBs with the plasma membrane. Exosomal proteins were found to accumulate in the plaques of AD patient brains, suggesting a role in the pathogenesis of AD.

Previously we isolated human PEX16 encoding 336-amino acid-long peroxin Pex16p and showed that its dysfunction was responsible for Zellweger syndrome of complementation group D (group 9). Here we have determined the membrane topology of Pex16p by differential permeabilization method: both N- and C-terminal parts are exposed to the cytosol. In the search for Pex16p topogenic sequence, basic amino acids clustered sequence, RKELRKKLPVSLSQQK, at positions 66-81 and the first transmembrane segment locating far downstream, nearly by 40 amino acids, of this basic region were defined to be essential for integration into peroxisome membranes. Localization to peroxisomes of membrane proteins such as Pex14p, Pex13p, and PMP70 was interfered with in CHO-K1 cells by a higher level expression of the pex16 patient-derived dysfunctional but topogenically active Pex16pR176ter comprising resides 1-176 or of the C-terminal cytoplasmic part starting from residues at 244 to the C terminus. Furthermore, Pex16p C-terminal cytoplasmic part severely abrogated peroxisome restoration in pex mutants such as matrix protein import-defective pex12 and membrane assembly impaired pex3 by respective PEX12 and PEX3 expression, whereas the N-terminal cytosolic region did not affect restoration. These results imply that Pex16p functions in peroxisome membrane assembly, more likely upstream of Pex3p.

Peroxisomal division comprises three stages: elongation, constriction, and fission. Potential candidates thus far studied for the factors involved in these stages include Pex11pβ, dynamin-like protein 1 (DLP1), mitochondrial fission factor (Mff), and Fission 1 (Fis1). A poly-unsaturated fatty acid of peroxisomal β-oxidation metabolites, docosahexaenoic acid (C22:6n-3), augments hyper-oligomerization of Pex11pβ that gives rise to peroxisomal elongation, a prerequisite for subsequent fission and peroxisome division. Translocation of DLP1, a member of the large GTPase family, from the cytosol to peroxisomes is a prerequisite for membrane fission. However, the molecular machinery for peroxisomal targeting of DLP1 remains elusive. Mff is also localized to peroxisomes, especially at the membrane-constricted regions of elongated peroxisomes. Knockdown of Mff abrogates the fission stage of peroxisomal division and fails to recruit DLP1 to peroxisomes, while ectopic expression of Mff increases the peroxisomal targeting of DLP1. Co-expression of Mff and Pex11pβ increases peroxisome abundance. Overexpression of Mff also increases the interaction between DLP1 and Pex11pβ, which knockdown of Mff, but not Fis1, abolishes. Moreover, Pex11pβ interacts with Mff in a DLP1-dependent manner. Mff contributes to the peroxisomal targeting of DLP1 and plays a key role in the fission of the peroxisomal membrane by acting in concert with Pex11pβ and DLP1. The investigations performed to date suggest that a functional complex comprising Pex11pβ, Mff, and DLP1 promotes Mff-mediated fission during peroxisomal division. With regard to peroxisome morphogenesis, we address recent issues and findings and propose a model for peroxisome division.

Human 34-kDa peroxisomal membrane protein (PMP34) consisting of 307 amino acids was previously identified as an ortholog of, or a similar protein (with 27% identity) to the, 423-amino acid-long PMP47 of the yeast Candida boidinii. We investigated membrane topogenesis of PMP34 with six putative transmembrane segments, as a model peroxisomal membrane protein. PMP34 was characterized as an integral membrane protein of peroxisomes. Transmembrane topology of PMP34 was determined by differential permeabilization and immunofluorescent staining of HeLa cells ectopically expressing PMP34 as well as of Chinese hamster ovary-K1 expressing epitope-tagged PMP34. As opposed to PMP47, PMP34 was found to expose its N- and C-terminal parts to the cytosol. Various deletion variants of PMP34 and their fusion proteins with green fluorescent protein were expressed in Chinese hamster ovary-K1 and were verified with respect to intracellular localization. The loop region between transmembrane segments 4 and 5 was required for the peroxisome-targeting activity, in which Ala substitution for basic residues abrogated the activity. Three hydrophobic transmembrane segments linked in a flanking region of the basic loop were essential for integration of PMP34 to peroxisome membranes. Therefore, it is evident that the intervening basic loop plus three transmembrane segments of PMP34 function as a peroxisomal targeting and topogenic signal.

Outer mitochondrial membrane cytochromeb 5 (OMb), which is an isoform of cytochromeb 5 (cyt b 5) in the endoplasmic reticulum, is a typical tail-anchored protein of the outer mitochondrial membrane. We cloned cDNA containing the complete amino acid sequence of OMb and found that the protein has no typical structural feature common to the mitochondrial targeting signal at the amino terminus. To identify the region responsible for the mitochondrial targeting of OMb, various mutated proteins were expressed in cultured mammalian cells, and the subcellular localization of the expressed proteins was analyzed. The deletion of more than 11 amino acid residues from the carboxyl-terminal end of OMb abolished the targeting of the protein to the mitochondria. When the carboxyl-terminal 10 amino acids of OMb were fused to the cytb 5 that was previously deleted in the corresponding 10 residues, the fused protein localized in the mitochondria, thereby indicating that the carboxyl-terminal 10 amino acid residues of OMb have sufficient information to transport OMb to the mitochondria. The replacement of either of the two positively charged residues within the carboxyl-terminal 10 amino acids by alanine resulted in the transport of the mutant proteins to the endoplasmic reticulum. The mutant cyt b 5, in which the acidic amino acid in its carboxyl-terminal end was replaced by basic amino acid, could be transported to the mitochondria. It would thus seem that charged amino acids in the carboxyl-terminal portion of these proteins determine their locations in the cell.

Cytochrome b5 (b5), a typical tail-anchored protein of the endoplasmic reticulum (ER) membrane, is composed of three functionally different domains: amino-terminal heme-containing catalytic, central hydrophobic membrane-anchoring, and carboxyl-terminal ER-targeting domains (Mitoma, J., and Ito, A. (1992) EMBO J. 11, 4197-4203). To analyze the potential retention signal of b5, mutant proteins were prepared to replace each domain with natural or artificial sequences, and subcellular localizations were examined using immunofluorescence microscopy and cell fractionation. The transmembrane domain functioned to retain the cytochrome in the ER, and the mutation of all or part of the transmembrane domain with an artificial hydrophobic sequence had practically no effect on intracellular distribution of the cytochrome. However, when the transmembrane domain was extended systematically, a substantial portion of the protein with the domain of over 22 amino acid residues leaked from the organelle. Thus, the transmembrane length functions as the retention signal. When cytochromes with mutations at the carboxyl-terminal end were overexpressed in cells, a substantial portion of the protein was transported to the plasma membrane, indicating that the carboxyl-terminal luminal domain also has a role in retention of b5 in the ER. Carbohydrate moiety of the glycosylatably-mutated b5 was sensitive to endoglycosidase H but resistant to endoglycosidase D. Therefore, both transmembrane and carboxyl-terminal portions seems to function as the static retention signal.

Cytochrome b5 is tail-anchored in the ER membrane and is composed of three functionally different portions; amino-terminal heme-containing catalytic, central hydrophobic membrane-anchoring, and carboxy-terminal ER-targeting portions [Mitoma, J. and Ito, A. (1992) EMBO J. 11, 4197-4203]. In situ topology of cytochrome b5 in the ER-membrane was studied using immunofluorescence microscopy. Antibodies were raised against the hydrophilic portion (anti-b5) and the carboxy-terminal seven amino acid residues (anti-peptide) of cytochrome b5 and used for detection of the cytochrome in COS cells which expressed the rat cytochrome. Anti-b5 antibody detected the cytochrome in a reticular staining pattern characteristic of the ER, even when the cell plasma membrane was permeabilized with Streptolysin O. The anti-peptide displayed a fluorescence signal only with Triton-permeabilized cells in which the antibody was able to penetrate into the ER lumen. In a double immuno-staining of the cell using the antipeptide antibody and the antibody against protein disulfide isomerase, both antibodies showed the same staining pattern in the presence of either Triton X-100 or Streptolysin O. The results indicate that the carboxy-terminal hydrophilic stretch is exposed to the luminal side. Cytochrome b5 was tagged with c-myc peptide at the carboxy-terminal end and the topology of the c-myc peptide was analyzed by the same method. Anti c-myc monoclonal IgG detected the tagged cytochrome b5 only after Triton treatment of the fixed cells, suggesting that the addition of c-myc peptide to the carboxy-terminal end does not affect insertion or orientation of the cytochrome in the ER membrane.

We earlier isolated peroxisome biogenesis-defective Chinese hamster ovary (CHO) cell mutants, ZPEG241, by the 9-(1'-pyrene)nonanol/ultraviolet selection method, from TKaEG2, the wild-type CHO-K1 cells transformed with two cDNAs encoding rat Pex2p and peroxisome targeting signal type 2 (PTS2)-tagged enhanced green fluorescent protein (EGFP). Peroxisomal localization of PTS2-EGFP was specifically impaired in ZPEG241 due to the failure of Pex5pL expression. Analysis of partial genomic sequence of PEX5 revealed one-point nucleotide-mutation from G to A in the 3'-acceptor splice site located at 1 nt upstream of exon 7 encoding Pex5pL specific 37-amino acid insertion, thereby generating 21-nt deleted mRNA of PEX5L in ZPEG241. When ZPEG241-derived Pex5pL was ectopically expressed in ZPEG241, PTS2 import was not restored because of no interaction with Pex7p. Together, we confirm the pivotal role of Pex5pL in PTS2 import, showing that the N-terminal 7-amino acid residues in the 37-amino acid insertion of Pex5pL are essential for the binding to Pex7p.

Publisher Summary Isolation of detergent-resistant membranes (DRMs) is a simple and useful biochemical method for analyzing the possible raft association of proteins and lipids. Raft proteins do not become solubilized, but remain associated with lipids. As a result, they have a lower apparent density than detergent-soluble membrane proteins and can be isolated by equilibrium density. Detergent solubility of an otherwise detergent-insoluble protein after cholesterol depletion with methyl-β-cyclodextrin can therefore serve as an additional criterion for raft association. However, the composition of DRMs may only imperfectly reflect the association of membrane components with lipid rafts in cell membranes. Cyclodextrin might be more effective on cell homogenate than on living cells, but the reasons for this are not completely clear. Various density gradient media can be used, and the choice between sucrose and iodixanol is largely a question of personal preference. However, sucrose might be superior if lipids are to be analyzed by a very sensitive method such as mass spectrometry, as iodixanol seems to have a weak tendency to follow lipids during extraction from DRMs.

Subfractionation studies showed that cytochrome b(5) (cyt b5), which has been considered to be a typical ER protein, was localized in both the endoplasmic reticulum membrane (ER) and the outer membrane of mitochondria in cauliflower (Brassica olracea) cells and was a component of antimycin A-insensitive NADH-cytochrome c reductase system in both membranes. When cDNA for cauliflower cyt b5 was introduced into mammalian (COS-7) and yeast cells as well as into onion cells, the expressed cytochrome was localized both in the ER and mitochondria in those cells. On the other hand, rat and yeast cyt b5s were specifically localized in the ER membranes even in the onion cells. Mutation experiments showed that cauliflower cyt b5 carries information that targets it to the ER and mitochondria within the carboxy-terminal 10 amino acids, as in the case of rat and yeast cyt b5s, and that replacement of basic amino acids in this region of cauliflower cyt b5 with neutral or acidic ones resulted in its distribution only in the ER. Together with the established findings of the importance of basic amino acids in mitochondrial targeting signals, these results suggest that charged amino acids in the carboxy-terminal portion of cyt b5 determine its location in the cell, and that the same mechanism of signal recognition and of protein transport to organelles works in mammalian, plant, and yeast cells.

Partial resistance of cell membranes to solubilization with mild detergents and the analysis of isolated detergent-resistant membranes (DRMs) have been used operationally to define membrane domains. Given the multitude of detergents used for this purpose, we sought to investigate whether extraction with different detergents might reflect the same underlying principle of domain formation. We therefore compared the protein and lipid content of DRMs prepared with a variety of detergents from two cell lines. We found that the detergents differ considerably in their ability to selectively solubilize membrane proteins and to enrich sphingolipids and cholesterol over glycerophospholipids as well as saturated over unsaturated phosphatidylcholine. In addition, we observed cell type-dependent variations of the molecular characteristics of DRMs and the effectiveness of particular detergents. These results make it unlikely that different detergents reflect the same aspects of membrane organization and underscore both the structural complexity of cell membranes and the need for more sophisticated analytical tools to understand their architecture.

Caveolins have been identified as key components of caveolae, specialized cholesterol-enriched raft domains visible as small flask-shaped invaginations of the plasma membrane. In polarized MDCK cells caveolin-1 and -2 are found together on basolateral caveolae whereas the apical membrane, where only caveolin-1 is present, lacks caveolae. Expression of a caveolin mutant prevented the formation of the large caveolin-1/-2 hetero-oligomeric complexes, and led to intracellular retention of caveolin-2 and disappearance of caveolae from the basolateral membrane. Correspondingly, in MDCK cells over-expressing caveolin-2 the basolateral membrane exhibited an increased number of caveolae. These results indicate the involvement of caveolin-2 in caveolar biogenesis.