Kyushu University Academic Staff Educational and Research Activities Database
List of Papers
Masanori Honsho Last modified date:2022.05.18

Associate Professor / Department of Neuroinflammation and Brain Fatigue Science / Faculty of Medical Sciences

1. Honsho M.*, Abe Y.*, Imoto Y., Chang Z.F., Mandel H., Falik-Zaccai T.C., and Fujiki Y.(*共筆頭著者), Mammalian homologue NME3 of DYNAMO1 regulates peroxisome division, Int J Mol Sci., 10.3390/ijms21218040, 21, 21, E8040, 2020.10, Peroxisomes proliferate by sequential processes comprising elongation, constriction,
and scission of peroxisomal membrane. It is known that the constriction step is mediated by a GTPase
named dynamin-like protein 1 (DLP1) upon e cient loading of GTP. However, mechanism of fuelling
GTP to DLP1 remains unknown in mammals. We earlier show that nucleoside diphosphate (NDP)
kinase-like protein, termed dynamin-based ring motive-force organizer 1 (DYNAMO1), generates
GTP for DLP1 in a red alga, Cyanidioschyzon merolae. In the present study, we identified that nucleoside
diphosphate kinase 3 (NME3), a mammalian homologue of DYNAMO1, localizes to peroxisomes.
Elongated peroxisomes were observed in cells with suppressed expression of NME3 and fibroblasts
from a patient lacking NME3 due to the homozygous mutation at the initiation codon of NME3.
Peroxisomes proliferated by elevation of NME3 upon silencing the expression of ATPase family
AAA domain containing 1, ATAD1. In the wild-type cells expressing catalytically-inactive NME3,
peroxisomes were elongated. These results suggest that NME3 plays an important role in peroxisome
division in a manner dependent on its NDP kinase activity. Moreover, the impairment of peroxisome
division reduces the level of ether-linked glycerophospholipids, ethanolamine plasmalogens, implying
the physiological importance of regulation of peroxisome morphology..
2. Abe Y., Nishimura Y., Nakamura K., Tamura S., Honsho M., Udo H., Yamashita T., and Fujiki Y., Peroxisome Deficiency Impairs BDNF Signaling and Memory, Front. Cell Dev. Biol., 10.3389/fcell.2020.567017, 8, 567017, 2020.10, Peroxisome is an intracellular organelle that functions in essential metabolic pathways including β-oxidation of very-long-chain fatty acids and biosynthesis of plasmalogens. Peroxisome biogenesis disorders (PBDs) manifest severe dysfunction in multiple organs including central nervous system (CNS), whilst the pathogenic mechanisms are largely unknown. We recently reported that peroxisome-deficient neural cells secrete an increased level of brain-derived neurotrophic factor (BDNF), resulting in the cerebellar malformation. Peroxisomal functions in adulthood brain have been little investigated. To induce the peroxisome deficiency in adulthood brain, we here established tamoxifen-inducible conditional Pex2-knockout mouse. Peroxisome deficiency in the conditional Pex2-knockout adult mouse brain induces the upregulated expression of BDNF and its inactive receptor TrkB-T1 in hippocampus, which notably results in memory disturbance. Our results suggest that peroxisome deficiency gives rise to the dysfunction of hippocampal circuit via the impaired BDNF signaling..
3. Yamashita K., Tamura S., Honsho M., Yada H., Yagita Y., Kosako H., and Fujiki Y., Mitotic phosphorylation of Pex14p regulates peroxisomal import machinery, J. Cell Biol., 10.1083/jcb.202001003, 219, 10, e202001003, 2020.10, [URL], 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..
4. Honsho M., Tanaka M., Zoeller RA, and Fujiki Y., Distinct functions of acyl/alkyl dihydroxyacetonephosphate reductase in peroxisomes and endoplasmic reticulum, Front. Cell Dev. Biol., 10.3389/fcell.2020.00855, 8, 855, 2020.09.
5. Abe Y., Honsho M., Kawaguchi R., Matsuzaki T., Ichiki Y., Fujitani M., Fujiwara K., Hirokane M., Oku M., Sakai Y., Yamashita T., and Fujiki Y., A peroxisome deficiency-induced reductive cytosol state up-regulates the brain-derived neurotrophic factor pathway, J. Biol. Chem., 10.1074/jbc.RA119.011989, 295, 16, 5321-5334, 2020.04, [URL], 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..
6. Dubreuil M.M., Morgens D.W., Okumoto K., Honsho M., Contrepois K., Lee-McMullen B., Traber G., Sood R., Dixon S.J., Snyder M.P., Fujiki Y., and Bassik M.C., Systematic identification of regulators of oxidative stress reveals non-canonical roles for peroxisomal import and the pentose phosphate pathway, Cell Rep., 10.1016/j.celrep.2020.01.013, 30, 5, 1417-1433.e7, 2020.02, Reactive oxygen species (ROS) play critical roles in metabolism and disease, yet a comprehensive analysis of the cellular response to oxidative stress is lacking. To systematically identify regulators of oxidative stress, we conducted genome-wide Cas9/CRISPR and shRNA screens. This revealed a detailed picture of diverse pathways that control oxidative stress response, ranging from the TCA cycle and DNA repair machineries to iron transport, trafficking, and metabolism. Paradoxically, disrupting the pentose phosphate pathway (PPP) at the level of phosphogluconate dehydrogenase (PGD) protects cells against ROS. This dramatically alters metabolites in the PPP, consistent with rewiring of upper glycolysis to promote antioxidant production. In addition, disruption of peroxisomal import unexpectedly increases resistance to oxidative stress by altering the localization of catalase. Together, these studies provide insights into the roles of peroxisomal matrix import and the PPP in redox biology and represent a rich resource for understanding the cellular response to oxidative stress. Despite its importance in metabolism and disease, a comprehensive analysis of the cellular response to oxidative stress is lacking. Here, Dubreuil et al. use genome-wide screens to identify cellular regulators of oxidative stress. They investigate paradoxical mechanisms by which disruption of the pentose phosphate and peroxisomal import pathways protect cells..
7. Takahashi T*., Honsho M*., Abe Y., and Fujiki Y.(*共筆頭著者), Plasmalogen mediates integration of adherens junction, J. Biochem., 10.1093/jb/mvz049, 166, 5, 423-432, 2019.11, 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..
8. Exner T., Romero-Brey I., Yifrach E., Rivera-Monroy J., Schrul B., Zouboulis C.C., Stremmel W., Honsho M., Bartenschlager R., Zalckvar E., Poppelreuther M., and Fullekrug J., An alternative membrane topology permits lipid droplet localization of peroxisomal fatty acyl-CoA reductase, J. Cell Sci. , 10.1242/jcs.223016, 132, 6, 2019.03, Fatty acyl-CoA reductase 1 (Far1) is a ubiquitously expressed peroxisomal membrane protein that generates the fatty alcohols required for the biosynthesis of ether lipids. Lipid droplet localization of exogenously expressed and endogenous human Far1 was observed by fluorescence microscopy under conditions of increased triglyceride synthesis in tissue culture cells. This unexpected finding was supported further by correlative light electron microscopy and subcellular fractionation. Selective permeabilization, protease sensitivity and N-glycosylation tagging suggested that Far1 is able to assume two different membrane topologies, differing in the orientation of the short hydrophilic C-terminus towards the lumen or the cytosol, respectively. Two closely spaced hydrophobic domains are contained within the C-terminal region. When analyzed separately, the second domain was sufficient for the localization of a fluorescent reporter to lipid droplets. Targeting of Far1 to lipid droplets was not impaired in either Pex19 or ASNA1 (also known as TRC40) CRISPR/Cas9 knockout cells. In conclusion, our data suggest that Far1 is a novel member of the rather exclusive group of dual topology membrane proteins. At the same time, Far1 shows lipid metabolism-dependent differential subcellular localizations to peroxisomes and lipid droplets..
9. Abe Y., Honsho M., Itoh R., Kawaguchi R., Fujitani M., Fujiwara K., Hirokane M., Matsuzaki T., Nakayama K., Ohgi R., Marutani T., Nakayama K.I., Yamashita T., and Fujiki Y., Peroxisome biogenesis deficiency attenuates the BDNF-TrkB pathway-mediated development of the cerebellum, Life Sci. Alliance, 10.26508/lsa.201800062, 1, 6, e201800062-e201800062, 2018.12, Peroxisome biogenesis disorders (PBDs) manifest as neurological deficits in the central nervous system, including neuronal migration defects and abnormal cerebellum development. However, the mechanisms underlying pathogenesis remain enigmatic. Here, to investigate how peroxisome deficiency causes neurological defects of PBDs, we established a new PBD model mouse defective in peroxisome assembly factor Pex14p, termed Pex14ΔC/ΔC mouse. Pex14ΔC/ΔC mouse manifests a severe symptom such as disorganization of cortical laminar structure and dies shortly after birth, although peroxisomal biogenesis and metabolism are partially defective. The Pex14ΔC/ΔC mouse also shows malformation of the cerebellum including the impaired dendritic development of Purkinje cells. Moreover, extracellular signal-regulated kinase and AKT signaling are attenuated in this mutant mouse by an elevated level of brain-derived neurotrophic factor (BDNF) together with the enhanced expression of TrkB-T1, a dominant-negative isoform of the BDNF receptor. Our results suggest that dysregulation of the BDNF-TrkB pathway, an essential signaling for cerebellar morphogenesis, gives rise to the pathogenesis of the cerebellum in PBDs..
10. Imoto Y., Abe Y., Honsho M., Okumoto K., Ohnuma M., Kuroiwa H., Kuroiwa T., and Fujiki Y., Onsite GTP fuelling via DYNAMO1 drives division of mitochondria and peroxisomes, Nat. Commun., 10.1038/s41467-018-07009-z, 9, 1, 4634, 2018.11, Mitochondria and peroxisomes proliferate by division. During division, a part of their membrane is pinched off by constriction of the ring-shaped mitochondrial division (MD) and peroxisome-dividing (POD) machinery. This constriction is mediated by a dynamin-like GTPase Dnm1 that requires a large amount of GTP as an energy source. Here, via proteomics of the isolated division machinery, we show that the 17-kDa nucleoside diphosphate kinase-like protein, dynamin-based ring motive-force organizer 1 (DYNAMO1), locally generates GTP in MD and POD machineries. DYNAMO1 is widely conserved among eukaryotes and colocalizes with Dnm1 on the division machineries. DYNAMO1 converts ATP to GTP, and disruption of its activity impairs mitochondrial and peroxisomal fissions. DYNAMO1 forms a ring-shaped complex with Dnm1 and increases the magnitude of the constricting force. Our results identify DYNAMO1 as an essential component of MD and POD machineries, suggesting that local GTP generation in Dnm1-based machinery regulates motive force for membrane severance..
11. Hossain M. S., Abe Y., Ali F., Youssef M., Honsho M., Fujiki Y., and Katafuchi T., Reduction of Ether-Type Glycerophospholipids, Plasmalogens, by NF-B Signal Leading to Microglial Activation, J. Neurosci., 10.1523/JNEUROSCI.3941-15.2017, 37, 15, 4074-4092, 2017.04, Neuroinflammation characterized by activation of glial cells is observed in various neurodegenerative diseases including Alzheimer’s disease (AD). Although the reduction of ether-type glycerophospholipids, plasmalogens (Pls), in the brain is reported in AD patients, the mechanism of the reduction and its impact on neuroinflammation remained elusive. In the present study, we found for the first time that various inflammatory stimuli reduced Pls levels in murine glial cells via NF-κB activation, which then downregulated a Pls-synthesizing enzyme, glycerone phosphate O-acyltransferase (Gnpat) through increased c-Myc recruitment onto the Gnpat promoter. We also found that systemic injection of lipopolysaccharide, aging, and chronic restraint stress reduced brain Pls contents that were associated with glial NF-κB activation, an increase in c-Myc expression, and downregulation of Gnpat in the mouse cortex and hippocampus. More interestingly, the reduction of Pls contents in the murine cortex itself could increase the activated phenotype of microglial cells and the expression of proinflammatory cytokines, suggesting further acceleration of neuroinflammation by reduction of brain Pls. A similar mechanism of Gnpat reduction was also found in human cell lines, triple-transgenic AD mouse brain, and postmortem human AD brain tissues. These findings suggest a novel mechanism of neuroinflammation that may explain prolonged progression of AD and help us to explore preventive and therapeutic strategies to treat neurodegenerative diseases..
12. Honsho M., Abe Y., and Fujiki Y., Plasmalogen biosynthesis is spatiotemporally regulated by sensing plasmalogens in the inner leaflet of plasma membranes, Sci. Rep., 10.1038/srep43936, 7, 43936, 2017.03, 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..
13. Imoto Y., Abe Y., Okumoto K., Honsho M., Kuroiwa H., Kuroiwa T., and Fujiki Y., Defining the dynamin-based ring organizing center on the peroxisome-dividing machinery isolated from Cyanidioschyzon merolae, J. Cell Sci., 10.1242/jcs.199182, 130, 5, 853-867, 2017.01, Organelle division is executed through contraction of a ring-shaped supramolecular dividing machinery. A core component of the machinery is the dynamin-based ring conserved during the division of mitochondrion, plastid and peroxisome. Here, using isolated peroxisome-dividing (POD) machinery from a unicellular red algae, Cyanidioschyzon merolae, we identified a dynamin-based ring organizing center (DOC) that acts as an initiation point for formation of the dynamin-based ring. C. merolae contains a single peroxisome, the division of which can be highly synchronized by light-dark stimulation; thus, intact POD machinery can be isolated in bulk. Dynamin-based ring homeostasis is maintained by the turnover of the GTP-bound form of the dynamin-related protein Dnm1 between the cytosol and division machinery via the DOC. A single DOC is formed on the POD machinery with a diameter of 500-700 nm, and the dynamin-based ring is unidirectionally elongated from the DOC in a manner that is dependent on GTP concentration. During the later step of membrane fission, the second DOC is formed and constructs the double dynamin-based ring to make the machinery thicker. These findings provide new insights to define fundamental mechanisms underlying the dynamin-based membrane fission in eukaryotic cells..
14. Honsho M., Abe Y., and Fujiki Y., Dysregulation of Plasmalogen Homeostasis Impairs Cholesterol Biosynthesis, J. Biol. Chem., 10.1074/jbc.M115.656983, 290, 48, 28822-28833, 2015.11, 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..
15. Yoshida Y., Niwa H., Honsho M., Itoyama A., Fujiki Y., Pex11mediates peroxisomal proliferation by promoting deformation of the lipid membrane, Biol. Open., 10.1242/bio.201410801, 4, 6, 710-721, 2015.06, Pex11p family proteins are key players in peroxisomal fission, but their molecular mechanisms remains mostly unknown. In the present study, overexpression of Pex11pb 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 Pex11pb 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 Pex11pb 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 Pex11pb protein localized to the membrane constriction site. Introducing point mutations into the N-terminal amphiphathic helix of Pex11pb 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..
16. Noguchi M., Honsho M., Abe Y., Toyama R., Niwa H., Sato Y., Ghaedi K., Rahmanifar A., Shafeghati Y., and Fujiki Y., Mild reduction of plasmalogens causes rhizomelic chondrodysplasia punctata: functional characterization of a novel mutation, J. Human Genet., 10.1038/jhg.2014.39, 59, 7, 387-392, 2014.07, 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..
17. Abe Y., Honsho M., Nakanishi H., Taguchi R., and Fujiki Y., Very-long-chain polyunsaturated fatty acids accumulate in phosphatidylcholine of fibroblasts from patients with Zellweger syndrome and acyl-CoA oxidase1 deficiency, Biochim. Biophys. Acta- Mol. Cell Biol. Lipids , 10.1016/j.bbalip.2014.01.001, 1841, 4, 610-619, 2014.04, 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..
18. Honsho M., Asaoku S., Fukumoto K., and Fujiki Y., Topogenesis and Homeostasis of Fatty Acyl-CoA Reductase 1, J. Biol. Chem., 10.1074/jbc.M113.498345, 288, 48, 34588-34598, 2013.11, 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..
19. Itoyama A., Michiyuki S., Honsho M., Yamamoto T., Moser A., Yoshida Y., and Fujiki Y., Mff functions with Pex11pβ and DLP1 in peroxisomal fission, Biol. Open., 10.1242/bio.20135298, 2, 10, 998-1006, 2013.10, 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..
20. Itoyama A., Honsho M., Abe Y., Moser A., Yoshida Y., and Fujiki Y., Docosahexaenoic acid mediates peroxisomal elongation, a prerequisite for peroxisome division, J. Cell Sci., 10.1242/​jcs.087452, 125, 589-602, 2012.02, [URL], 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 b-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 hyperoligomerization
of Pex11pb and the formation of Pex11pb-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..
21. Honsho M., Hashigushi Y., Ghaedi K., and Fujiki Y., Interaction defect of the medium isoform of PTS1-receptor Pex5p with PTS2-receptor Pex7p abrogates the PTS2 protein import into peroxisomes in mammals, J. Biochem., 10.1093/jb/mvq130, 149, 2, 203-210, 2011.02, 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..
22. Honsho M., Asaoku S., and Fujiki Y., Posttranslational Regulation of Fatty Acyl-CoA Reductase 1, Far1, Controls Ether Glycerophospholipid Synthesis, J. Biol. Chem., 10.1074/jbc.M109.083311, 285, 12, 8537-8542, 2010.03, 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..
23. Honsho M*., Yagita Y*., Kinoshita N. and Fujiki Y.(*共筆頭著者), Isolation and characterization of mutant animal cell line defective in alkyl-dihydroxyacetonephosphate synthase: Localization and transport of plasmalogens to post-Golgi compartments, Biochim Biophys Acta.-Mol. Cell Res., 10.1016/j.bbamcr.2008.05.018, 1783, 10, 1857-1865, 2008.10, 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..
24. Schneider A., Rajendran L., Honsho M., Gralle M., Donnert G., Wouters F., Hell SW., and Simons M., Flotillin-Dependent Clustering of the Amyloid Precursor Protein Regulates Its Endocytosis and Amyloidogenic
Processing in Neurons, J. Neurosci. , 10.1523/JNEUROSCI.5345-07.2008, 28, 11, 2874-2882, 2008.03, The flotillins/reggie proteins are associated with noncaveolar membrane microdomains and have been implicated in the regulation of a clathrin- and caveolin-independent endocytosis pathway. Endocytosis is required for the amyloidogenic processing of the amyloid precursor protein (APP) and thus to initiate the release of the neurotoxic β-amyloid peptide (Aβ), the major component of extracellular plaques found in the brains of Alzheimer's disease patients. Here, we report that small interference RNA-mediated downregulation of flotillin-2 impairs the endocytosis of APP, in both neuroblastoma cells and primary cultures of hippocampal neurons, and reduces the production of Aβ. Similar to tetanus neurotoxin endocytosis, but unlike the internalization of transferrin, clathrin-dependent endocytosis of APP requires cholesterol and adaptor protein-2 but is independent of epsin1 function. Moreover, on a nanoscale resolution using stimulated emission depletion microscopy and by Förster resonance energy transfer with fluorescence lifetime imaging microscopy, we provide evidence that flotillin-2 promotes the clustering of APP at the cell surface. We show that the interaction of flotillin-2 with APP is dependent on cholesterol and that clustering of APP enhances its endocytosis rate. Together, our data suggest that cholesterol/flotillin-dependent clustering of APP may stimulate the internalization into a specialized clathrin-dependent endocytosis pathway to promote amyloidogenic processing..
25. Rajendran L., Honsho M., Zahn TR, Keller P., Geiger KD., Verkade P. and Simons K., Alzheimer's disease β-amyloid peptides are released in association with exosomes, Proc. Natl. Acad. Sci. USA. , 10.1073/pnas.0603838103, 103, 30, 11172-11177, 2006.07, Although the exact etiology of Alzheimer's disease (AD) is a topic of debate, the consensus is that the accumulation of β-amyloid (Aβ) peptides in the senile plaques is one of the hallmarks of the progression of the disease. The Aβ peptide is formed by the amyloidogenic cleavage of the amyloid precursor protein (APP) by β- and γ-secretases. The endocytic system has been implicated, in the cleavages leading to the formation of Aβ. However, the identity of the intracellular compartment where the amyloidogenic secretases cleave and the mechanism by which the intracellularly generated Aβ is released into the extracellular milieu are not clear. Here, we show that β-cleavage occurs in early endosomes followed by routing of Aβ to multivesicular bodies (MVBs) in HeLa and N2a cells. Subsequently, a minute fraction of Aβ 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..
26. Schuck S., Manninen A., Honsho M., Fullekrug J. and Simons K., Generation of single and double knockdowns in polarized epithelial cells by retrovirus-mediated RNA interference, Proc. Natl. Acad. Sci. USA. , 10.1073/pnas.0401285101, 101, 14, 4912-4917, 2004.04, RNA interference (RNAi) is a ubiquitous mechanism of eukaryotic gene regulation that can be exploited for specific gene silencing. Retroviruses have been successfully used for stable expression of short hairpin RNAs in mammalian cells, leading to persistent inhibition of gene expression by RNAi. Here, we apply retrovirus-mediated RNAi to epithelial Madin-Darby canine kidney cells, whose properties limit the applicability of other RNAi methods. We demonstrate efficient suppression of a set of 13 target genes by retroviral coexpression of short hairpin RNAs and a selectable marker. We characterize the resulting knockdown cell populations with regard to composition and stability, and examine the usefulness of proposed guidelines for choosing RNAi target sequences. Finally, we show that this system can be used to simultaneously target two genes, giving rise to double knockdowns. Thus, retrovirus-mediated RNAi is a convenient method for gene silencing in Madin-Darby canine kidney cells, and is likely to be applicable to virtually any mammalian cell..
27. Schuck S*., Honsho M*., Ekroos K., Shevchenko A. and Simons K.(*共筆頭著者), Resistance of cell membranes to different detergents, Proc. Natl. Acad. Sci. USA. , 10.1073/pnas.0631579100, 100, 10, 5795-5800, 2003.05, 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..
28. Lahtinen U., Honsho M., Parton RG., Simons K. and Verkade P., Involvement of caveolin-2 in caveolar biogenesis in MDCK cells, FEBS Lett. , 10.1016/S0014-5793(03)00135-2, 538, 1-3, 85-88, 2003.03, 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..
29. Zhao J., Onduka T., Kinoshita JY., Honsho M., Kinoshita T., Shimazaki K., and Ito A., Dual Subcellular Distribution of Cytochrome b5 in Plant, Cauliflower, Cells, J. Biochem., 10.1093/jb/mvg009, 133, 1, 115-121, 2003.01, Subfractionation studies showed that cytochrome b5 (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..
30. Honsho M., Hiroshige T., and Fujiki Y., The Membrane Biogenesis Peroxin Pex16p
TOPOGENESIS AND FUNCTIONAL ROLES IN PEROXISOMAL MEMBRANE ASSEMBLY, J. Biol. Chem. , 10.1074/jbc.M206139200, 277, 46, 44513-44524, 2002.11, 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..
31. Honsho M. and Fujiki Y., Topogenesis of Peroxisomal Membrane Protein Requires A Short, Positively Charged Intervening-loop Sequence and Flanking Hydrophobic Segments. STUDY USING HUMAN MEMBRANE PROTEIN PMP34, J. Biol. Chem., 10.1074/jbc.M003304200, 276, 12, 9375-9382, 2001.03, 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..
32. Ghaedi K., Honsho M., Shimozawa N., Suzuki Y., Kondo N. and Fujiki Y., PEX3 Is the Causal Gene Responsible for Peroxisome Membrane Assembly–Defective Zellweger Syndrome of Complementation Group G, Am. J. Hum. Genet. , 10.1086/303086, 67, 4, 976-981, 2000.10, Peroxisome biogenesis disorders (PBDs) such as Zellweger syndrome (ZS) and neonatal adrenoleukodystrophy are autosomal recessive diseases caused by defects in peroxisome assembly, for which 13 genotypes have been identified. Expression of the human peroxin Pex3p cDNA encoding a 373-amino-acid peroxisomal membrane protein morphologically and biochemically restored peroxisome biogenesis, including peroxisomal membrane assembly, in fibroblasts from PBDG-02, a patient with complementation group G (CG-G) ZS. Patient PBDG-02 carried a homozygous, inactivating mutation - a 97-bp deletion of nucleotide residues at positions 942-1038 - resulting in a 32-amino-acid truncation and in a frameshift inducing both a 3-amino-acid substitution and a termination codon. Genomic PCR analysis revealed mutation of T→G at eight bases upstream of the splicing site at the boundary of intron 10 and exon 11 of PEX3 gene, giving rise to a deletion of all of exon 11. When assessed by expression in a pex3 mutant of Chinese hamster ovary cells and the patient's fibroblasts, PBDG-02-derived PEX3 cDNA was found to be defective in peroxisome-restoring activity. These results provide evidence that PEX3 is a novel, pathogenic gene responsible for CG-G PBDs..
33. Otera H., Harano T., Honsho M., Ghaedi K., Mukai S., Tanaka A., Kawai A., Shimizu N. and Fujiki Y., The Mammalian Peroxin Pex5pL, the Longer Isoform of the Mobile Peroxisome Targeting Signal (PTS) Type 1 Transporter, Translocates the Pex7p-PTS2 Protein Complex into Peroxisomes via Its Initial Docking Site, Pex14p, J. Biol. Chem. , 10.1074/jbc.M000720200, 275, 28, 21703-21714, 2000.07, In mammals, two isoforms of the peroxisome targeting signal (PTS) type 1 receptor Pex5p, i.e. Pex5pS and Pex5pL with an internal 37-amino acid insertion, have previously been identified. Expression of either type of Pex5p complements the impaired PTS1 import in Chinese hamster ovary pex5 mutants, but only Pex5pL can rescue the PTS2 import defect noted in a subgroup of pex5 mutants such as ZP105. In this work, we found that Pex5pL directly interacts with the PTS2 receptor Pex7p, carrying its cargo PTS2 protein in the cytosol. Pex5pL, but not Pex5pS, mediated the binding of PTS2 protein to Pex14p by translocating Pex7p, demonstrating that Pex5pL plays a pivotal role in peroxisomal PTS2 import. Pex5p was localized mostly in the cytosol in wild-type CHO-K1 and Pex14p-deficient mutant cells, whereas it accumulated in the peroxisomal remnants in cell mutants defective in Pex13p or the RING family peroxins such as Pex2p and Pex12p. Furthermore, overexpression of Pex14p, but not Pex10p, Pex12p, or Pex13p, caused accumulation of Pex5p in peroxisomal membranes, with concomitant interference with PTS1 and PTS2 import. Therefore, Pex5p carrying the cargoes most likely docks with the initial site (Pex14p) in a putative import machinery, subsequently translocating to other components such as Pex13p, Pex2p, Pex10p, and Pex12p..
34. Honsho M., Tamura S., Shimozawa N., Suzuki Y., Kondo N. and Fujiki Y., Mutation in PEX16 Is Causal in the Peroxisome-Deficient Zellweger Syndrome of Complementation Group D, Am. J. Hum. Genet. , 10.1086/302161, 63, 6, 1622-1630, 1998.12, Peroxisome-biogenesis disorders (PBDs), including Zellweger syndrome (ZS), are autosomal recessive diseases caused by a deficiency in peroxisome assembly as well as by a malfunction of peroxisomes, among which >10 genotypes have been identified. We have isolated a human PEX16 cDNA (HsPEX16) by performing an expressed-sequence-tag homology search on a human DNA database, by using yeast PEX16 from Yarrowia lipolytica and then screening the human liver cDNA library. This cDNA encodes a peroxisomal protein (a peroxin Pex16p) made up of 336 amino acids. Among 13 peroxisome-deficiency complementation groups (CGs), HsPEX16 expression morphologically and biochemically restored peroxisome biogenesis only in fibroblasts from a CG-D patient with ZS in Japan (the same group as CG-IX in the United States). Pex16p was localized to peroxisomes through expression study of epitope- tagged Pex16p. One patient (PBDD-01) possessed a homozygous, inactivating nonsense mutation, C→T at position 526 in a codon (CGA) for 176 Arg, that resulted in a termination codon (TGA). This implies that the C-terminal half is required for the biological function of Pex16p. PBDD-01-derived PEX16 cDNA was defective in peroxisome-restoring activity when expressed in the patient's fibroblasts. These results demonstrate that mutation in PEX16 is the genetic cause of CG-D PBDs..
35. Kuroda R., Ikenoue T., Honsho M., Tsujimoto S., Mitoma JY. and Ito A., Charged Amino Acids at the Carboxy-Terminal Portions Determine the Intracellular Locations of Two Isoforms of Cytochrome b5, J. Biol. Chem., 10.1074/jbc.273.47.31097, 273, 47, 31097-31102, 1998.11, Outer mitochondrial membrane cytochrome b5 (OMb), which is an isoform of cytochrome b5 (cyt b5) 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 cyt b5 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 amine acids by alanine resulted in the transport of the mutant proteins to the endoplasmic reticulum. The mutant cyt b5, in which the acidic amine acid in its carboxyl-terminal end was replaced by basic amine acid, could be transported to the mitochondria. It would thus seem that charged amine acids in the carboxyl-terminal portion of these proteins determine their locations in the cell..
36. Honsho M., Mitoma JY. and Ito A., Retention of Cytochrome b5 in the Endoplasmic Reticulum Is Transmembrane and Luminal Domain-dependent, J. Biol. Chem. , 10.1074/jbc.273.33.20860, 273, 33, 20860-20866, 1998.08, Cytochrome b5 (b5), a typical tail-anchored protein of the endoplasmic reticulum (ER) membrane, is composed of three functionally different domains: amino-terminal heine-containing catalytic, central hydrophobic membrane- anchoring, and carboxyl-terminal ER-targeting domains (Mitoma, J., and Ito, A. (1992) EMBO J. 11, 41974203). 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 carboxylterminal portions seems to function as the static retention signal..
37. Kuroda R., Kinoshita J., Honsho M., Mitoma JY. and Ito A., In situ Topology of Cytochrome b5 in the Endoplasmic Reticulum Membrane, J. Biochem. , 10.1093/oxfordjournals.jbchem.a021486, 120, 4, 828-833, 1996.10, 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. 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 immune-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..