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Masanori Honsho Last modified date:2021.07.16

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




Homepage
https://kyushu-u.pure.elsevier.com/en/persons/masanori-honsho
 Reseacher Profiling Tool Kyushu University Pure
Phone
092-642-6117
Academic Degree
Ph. D
Country of degree conferring institution (Overseas)
No
Field of Specialization
Cell biology,Biochemistry
Total Priod of education and research career in the foreign country
02years06months
Research
Research Interests
  • Regulation of plasmalogen synthesis
    Analysis of the peroxisome membane biogenesis
    keyword : plasmalogens peroxisome
    2011.01~2021.12.
Academic Activities
Books
1. Honsho M. and Fujiki Y., Homeostasis of Plasmalogens in Mammals, Elsevier Inc., 10.1016/B978-0-08-100596-5.21664-8, Encyclopedia of Food Chemistry, Elsevier Inc., Waltham, USA. Vol. 2, 218-223, 2019.01, Ethanolamine-containing alkenyl ether glycerophospholipid, plasmalogen, is characterized by the presence of vinyl-ether linkage at the sn-1 position. Plasmalogens are found in nearly all mammalian tissues, and important components of cellular membranes. De novo synthesis of plasmalogens initiated in peroxisomes plays a pivotal role in the homeostasis of cellular plasmalogens. Synthesis of plasmalogens is regulated by feedback mechanism via sensing the plasmalogens located in the inner leaflet of plasma membranes, followed by modulating the level of fatty acyl-CoA reductase 1, the enzyme responsible for the synthesis of long-chain alcohols which are essential for the ether-bond formation in peroxisomes. Plasmalogens can be synthesized from alkylglycerol in cells and peripheral tissues such as liver and heart in rodent, whereas plasmalogens essential for the function of brain are more likely supplied by de novo synthesis in brain rather than the transport of peripherally synthesized plasmalogens by crossing blood brain barrier, implying that regulation of plasmalogen synthesis plays an important role in the homeostasis of brain plasmalogens..
2. Honsho M., and Fujiki Y., Analysis of Plasmalogen Synthesis in Cultured Cells, Humana, 10.1007/978-1-4939-6937-1_6, Schrader, M. (ed.) Peroxisomes: Methods and Protocols, Methods in Molecular Biology 55-61, 2017.01, Plasmalogen synthesis can be analyzed by metabolic labeling, followed by the separation of ethanolamine plasmalogens from glycerophospholipids on one-dimensional thin-layer chromatography. The vinyl-ether bond of plasmalogens is acid-labile, which allows separating plasmalogens as 2-acyl-glycerophospholipids from diacyl-glycerophospholipids..
Reports
1. Honsho M. and Fujiki Y., ”Focus on Plasmalogens” issue: Plasmalogen homeostasis: regulation of plasmalogen biosynthesis and its physiological consequence in mammals, FEBS Lett., 10.1002/1873-3468.12743, 2017.07, 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..
2. Honsho M*., Yamashita S*., and Fujiki Y.(*共筆頭著者), Peroxisome homeostasis: Mechanisms of division and selective degradation of peroxisomes in mammals, Biochim Biophys Acta.-Mol. Cell Res., 10.1016/j.bbamcr.2015.09.032, 2016.05, 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. This article is part of a Special Issue entitled: Peroxisomes edited by Ralf Erdmann..
Papers
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. 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.
3. 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..
4. 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..
5. 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..
6. 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..
7. 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..
8. 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..
9. 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..
10. 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..
11. 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..
12. 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..
13. 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..
14. 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..
15. 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..
Membership in Academic Society
  • The Japanese Conference on the Biochemistry of Lipids
  • The Molecular Biology Society of Japan
  • The Japanese Biochemical Society