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• W2092504384 abstract "High affinity, retinoid-specific binding proteins chaperone retinoids to manage their transport and metabolism. Proposing mechanisms of retinoid transfer between these binding proteins and membrane-associated retinoid-metabolizing enzymes requires insight into enzyme topology. We therefore determined the topology of mouse retinol dehydrogenase type 1 (Rdh1) and cis-retinoid androgen dehydrogenase type 1 (Crad1) in the endoplasmic reticulum of intact mammalian cells. The properties of Rdh1 were compared with a chimera with a luminal signaling sequence (11β-hydroxysteroid dehydrogenase (11β-HSD1)(1-41)/Rdh1(23-317); the green fluorescent protein (GFP) fusion proteins Rdh1(1-22)/GFP, Crad1(1-22)/GFP, and 11β-HSD1(1-41)/GFP; and signaling sequence charge difference mutants using confocal immunofluorescence, antibody access, proteinase K sensitivity, and deglycosylation assays. An N-terminal signaling sequence of 22 residues, consisting of a hydrophobic helix ending in a net positive charge, anchors Rdh1 and Crad1 in the endoplasmic reticulum facing the cytoplasm. Mutating arginine to glutamine in the signaling sequence did not affect topology. Inserting one or two arginine residues near the N terminus of the signaling sequence caused 28-95% inversion from cytoplasmic to luminal, depending on the net positive charge remaining at the C terminus of the signaling sequence; e.g. the mutant L3R,L5R,R16Q,R19Q,R21Q faced the lumen. Experiments with N- and C-terminal epitope-tagged Rdh1 and molecular modeling indicated that a hydrophobic helix-turn-helix near the C terminus of Rdh1 (residues 289-311) projects into the cytoplasm. These data provide insight into the features necessary to orient type III (reverse signal-anchor) proteins and demonstrate that Rdh1, Crad1, and other short-chain dehydrogenases/reductases, which share similar N-terminal signaling sequences such as human Rdh5 and mouse Rdh4, orient with their catalytic domains facing the cytoplasm. High affinity, retinoid-specific binding proteins chaperone retinoids to manage their transport and metabolism. Proposing mechanisms of retinoid transfer between these binding proteins and membrane-associated retinoid-metabolizing enzymes requires insight into enzyme topology. We therefore determined the topology of mouse retinol dehydrogenase type 1 (Rdh1) and cis-retinoid androgen dehydrogenase type 1 (Crad1) in the endoplasmic reticulum of intact mammalian cells. The properties of Rdh1 were compared with a chimera with a luminal signaling sequence (11β-hydroxysteroid dehydrogenase (11β-HSD1)(1-41)/Rdh1(23-317); the green fluorescent protein (GFP) fusion proteins Rdh1(1-22)/GFP, Crad1(1-22)/GFP, and 11β-HSD1(1-41)/GFP; and signaling sequence charge difference mutants using confocal immunofluorescence, antibody access, proteinase K sensitivity, and deglycosylation assays. An N-terminal signaling sequence of 22 residues, consisting of a hydrophobic helix ending in a net positive charge, anchors Rdh1 and Crad1 in the endoplasmic reticulum facing the cytoplasm. Mutating arginine to glutamine in the signaling sequence did not affect topology. Inserting one or two arginine residues near the N terminus of the signaling sequence caused 28-95% inversion from cytoplasmic to luminal, depending on the net positive charge remaining at the C terminus of the signaling sequence; e.g. the mutant L3R,L5R,R16Q,R19Q,R21Q faced the lumen. Experiments with N- and C-terminal epitope-tagged Rdh1 and molecular modeling indicated that a hydrophobic helix-turn-helix near the C terminus of Rdh1 (residues 289-311) projects into the cytoplasm. These data provide insight into the features necessary to orient type III (reverse signal-anchor) proteins and demonstrate that Rdh1, Crad1, and other short-chain dehydrogenases/reductases, which share similar N-terminal signaling sequences such as human Rdh5 and mouse Rdh4, orient with their catalytic domains facing the cytoplasm. Retinol requires conversion into all-trans-retinoic acid to fulfill the vitamin A function of regulating gene expression that begins shortly after conception and continues throughout vertebrate life (1Wolf G. Physiol. Rev. 1984; 64: 873-938Crossref PubMed Scopus (289) Google Scholar, 2De Luca L.M. Basic Life Sci. 1993; 61: 17-25PubMed Google Scholar). Retinoid-active SDRs 1The abbreviations used are: SDR, short-chain dehydrogenase/reductase; 11β-HSD1, 11β-hydroxysteroid dehydrogenase, type 1; Crad1, mouse cis-retinol/androgen dehydrogenase type 1; CRBP, cellular retinol-binding protein type I; ER, endoplasmic reticulum; Rdh1, mouse retinol dehydrogenase type 1; SLO, streptolysin O; SRP, signal recognition particle; GFP, green fluorescent protein; PNGase F, peptide-N-glycosidase F; PBS, phosphate-buffered saline; ConA, concanavalin A; DAPI, 4,6-diamidino-2-phenylindole.1The abbreviations used are: SDR, short-chain dehydrogenase/reductase; 11β-HSD1, 11β-hydroxysteroid dehydrogenase, type 1; Crad1, mouse cis-retinol/androgen dehydrogenase type 1; CRBP, cellular retinol-binding protein type I; ER, endoplasmic reticulum; Rdh1, mouse retinol dehydrogenase type 1; SLO, streptolysin O; SRP, signal recognition particle; GFP, green fluorescent protein; PNGase F, peptide-N-glycosidase F; PBS, phosphate-buffered saline; ConA, concanavalin A; DAPI, 4,6-diamidino-2-phenylindole. recognize the major physiological form of retinol, retinol bound with CRBP, to generate retinal for irreversible conversion into all-trans-retinoic acid (3Napoli J.L. FASEB J. 1996; 10: 993-1001Crossref PubMed Scopus (324) Google Scholar). The most active of the retinol dehydrogenases include mouse Rdh1 and its orthologs, rat Rodh1 and -2 and human RDH-E/RoDH4 (4Zhang M. Chen W. Smith S.M. Napoli J.L. J. Biol. Chem. 2001; 276: 44083-44090Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 5Chai X. Boerman M.H.E.M. Zhai Y. Napoli J.L. J. Biol. Chem. 1995; 270: 3900-3904Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar, 6Chai X. Zhai Y. Popescu G. Napoli J.L. J. Biol. Chem. 1995; 270: 28408-28412Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 7Gough W.H. Van Ooteghem S. Sint T. Kedishvili N.Y. J. Biol. Chem. 1998; 272: 19778-19785Abstract Full Text Full Text PDF Scopus (108) Google Scholar, 8Jurukovski V. Markova N.G. Karaman-Jurukovska N. Randolph R.K. Su J. Napoli J.L. Simon M. Mol. Genet. Metab. 1999; 67: 62-73Crossref PubMed Scopus (49) Google Scholar). rRodh2 e.g. has lower apparent Km values with holo-CRBP than with unbound retinol and catalyzes retinal synthesis in the presence of excess apoCRBP. Dehydrogenation of retinol in the presence of excess CRBP with concentrations of both ligand and binding protein in the micromolar range indicates that the reaction proceeds through the SDR accessing CRBP-bound retinol because the CRBP-retinol complex has a Kd value ∼0.1 nm, which would virtually eliminate unbound retinol. Close approach of the two has been confirmed by cross-linking of holo-CRBP with microsomal Rdh (9Boerman M.H.E.M. Napoli J.L. Biochemistry. 1996; 34: 7027-7037Crossref Scopus (72) Google Scholar). Although Rdhs that recognize holo-CRBP also occur in cytoplasm, they are inhibited severely by a small excess of apoCRBP (10Boerman M.H.E.M. Napoli J.L. J. Biol. Chem. 1996; 271: 5610-5616Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). Even uninhibited cytosolic enzymes make a quantitatively minor contribution to the total retinal-generating units. In contrast to SDR, the cytosolic alcohol dehydrogenase isozymes do not recognize CRBP-retinol in vitro (10Boerman M.H.E.M. Napoli J.L. J. Biol. Chem. 1996; 271: 5610-5616Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar, 11Kedishvili N.Y. Gough W.H. Davis W.I. Parsons S. Li T.K. Bosron W.F. Biochem. Biophys. Res. Commun. 1998; 24: 191-196Crossref Scopus (48) Google Scholar). Moreover alcohol dehydrogenase access to physiological amounts of unbound retinol in intact cells may be limited because retinol has very low solubility in aqueous media when not sequestered tightly with CRBP. Notably gene knock-out experiments did not provide evidence for an alcohol dehydrogenase contribution to retinol metabolism under normal circumstances, because Adh1-/-, Adh3-/-, Adh4-/-, and dual Adh1/Adh4-/- null mice showed no retinoic acid deficiency phenotype nor any disturbance of retinoid metabolism or compensatory response (12Molotkov A. Deltour L. Foglio M.H. Cuenca A.E. Duester G. J. Biol. Chem. 2002; 277: 13804-13811Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 13Molotkov A. Fan X. Deltour L. Foglio M.H. Martras S. Farres J. Pares X. Duester G. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 5337-5342Crossref PubMed Scopus (126) Google Scholar). The importance of high affinity, specific binding proteins chaperoning retinoids in vivo has been illustrated through gene ablation experiments (14Noy N. Biochem. J. 2000; 348: 481-495Crossref PubMed Scopus (353) Google Scholar). CRBP null mice e.g. seem morphologically normal but eliminate retinyl esters 6-fold faster than wild-type mice via an unknown mechanism (15Ghyselinck N.B. Båvik C. Sapin V. Mark M. Bonnier D. Hindelang C. Dierich A. Nilsson C.B. Håkansson H. Sauvant P. Azaïs-Braesco V. Frasson M. Picaud S. Chambon P. EMBO J. 1999; 18: 4903-4914Crossref PubMed Scopus (257) Google Scholar). Mice null in the intestinal cellular retinol-binding protein type II die from vitamin A deficiency within 24 h after birth when delivered by dams fed a diet marginal in vitamin A (16E X. Zhang L. Lu J. Tso P. Blaner W.S. Levin M.S. Li E. J. Biol. Chem. 2002; 277: 36617-36623Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). Mice null in the retinal pigment epithelium retinyl ester-binding protein RPE65 cannot produce 11-cis-retinol for production of rhodopsin, and mutations in RPE65 cause blindness (Leber's congenital amaurosis) (17Gollapalli D.R. Maiti P. Rando R.R. Biochemistry. 2003; 42: 11824-11830Crossref PubMed Scopus (65) Google Scholar, 18Mata N.L. Moghrabi W.N. Lee J.S. Bui T.V. Radu R.A. Horwitz J. Travis G.H. J. Biol. Chem. 2004; 279: 635-643Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). Mutations in another retinal pigment epithelium-binding protein, cellular retinal-binding protein, cause night blindness and photoreceptor degeneration from inefficient metabolic processing of 11-cis-retinol (19Saari J.C. Nawrot M. Kennedy B.N. Garwin G.G. Hurley J.B. Huang J. Possin D.E. Crabb J.E. Neuron. 2001; 29: 739-748Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar). Mutations in hRdh5, which serves as one of the retinal pigment epithelium 11-cis-retinol dehydrogenases, are linked with the rare, autosomal recessive disease fundus albipunctatus, i.e. night blindness from delayed photopigment regeneration (20Yamamoto H. Simon A. Eriksson U. Harris E. Berson E.L. Dryja T.P. Nat. Genet. 1999; 22: 188-191Crossref PubMed Scopus (238) Google Scholar, 21Jang G.F. Van Hooser J.P. Kuksa V. McBee J.K. He Y.G. Janssen J.J. Driessen C.A. Palczewski K. J. Biol. Chem. 2001; 276: 32456-32465Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). Serum retinol-binding protein null mice are unable to mobilize hepatic retinyl esters and suffer visual impairment (22Quadro L. Blaner W.S. Salchow D.J. Vogel S. Piantedosi R. Gouras P. Freeman S. Cosma M.P. Colantuoni V. Gottesman M.E. EMBO J. 1999; 18: 4633-4644Crossref PubMed Scopus (396) Google Scholar). Proposing precise models of retinoid transfer between these retinoid-binding proteins and retinoid-metabolizing enzymes requires understanding the topology of the enzymes. The retinoid-metabolizing, membrane-associated SDRs, including rRodh1, Rdh1, hRdh-E/Rodh4, and bovine 11-cis-retinol dehydrogenase/hRdh5/mRdh4, consist of N-terminal hydrophobic helices of ∼18 residues flanked on the C terminus by net positive charges, midprotein hydrophobic helix-loop-helix transmembrane regions, and hydrophobic helices of ∼23 residues close to their C termini. Several models have been proposed for their association with the ER. An early model suggested that all four of the hRoDH4 hydrophobic helices transected the ER membrane (7Gough W.H. Van Ooteghem S. Sint T. Kedishvili N.Y. J. Biol. Chem. 1998; 272: 19778-19785Abstract Full Text Full Text PDF Scopus (108) Google Scholar). A second model presented 11-cis-retinol dehydrogenase with its N- and C-terminal hydrophobic helices projecting through the ER membrane with the bulk of the protein facing the lumen (23Simon A. Romert A. Gustafson A.L. McCaffery J.M. Eriksson U. J. Cell Sci. 1999; 112: 549-558Crossref PubMed Google Scholar). The SDR Crad1 and the 11-cis-retinol dehydrogenase orthologs Rdh4/5 also have been proposed as luminal facing (24Tryggvason K. Romert A. Eriksson U. J. Biol. Chem. 2001; 276: 19253-19258Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). Another model presented rRodh1 as a type III (also known as reverse signal-anchor) protein with the N-terminal signaling sequence buried in the ER membrane but as a globular SDR projecting into the cytoplasm (25Wang J. Bongianni J.K. Napoli J.L. Biochemistry. 2001; 40: 12533-12540Crossref PubMed Scopus (20) Google Scholar). In this model, the C-terminal helix associated with the surface of the ER. All these models cannot be accurate in view of the amino acid similarity among these closely related SDRs especially in their putative N-terminal signaling sequences. The goals of this study were to determine whether the Rdh1 localizes exclusively to the ER membrane; the topology of Rdh1 by comparing its behavior in intact cells with luminal facing proteins for proteinase sensitivity, access to antibodies, and glycosylation; and the molecular features that establish ER retinoid dehydrogenase membrane expression locus and topology. cDNA Constructs—Constructs were made by PCR (primers are detailed in the Supplemental material) and were sequenced. pcDNA3/Rdh1 was used as the template for the Rdh1 mutants (4Zhang M. Chen W. Smith S.M. Napoli J.L. J. Biol. Chem. 2001; 276: 44083-44090Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). Mutants and their primers (forward/reverse, respectively) included RD(1-18), F1/R1; RD(1-30), F2/R1; RD(289-317), F3/R2; and RD2, F1/R2. Point mutations were introduced into Rdh1 by overlapping PCR. Mutant glycosylation site at residue 213 was made using the primer pairs F3/R3 and F4/R1 to amplify the 5′- and 3′-sections of Rdh1. The gel-purified products were used as templates with primers F3/R1 to make the mutant V213N. Similarly mutant G251N was obtained with primer pairs F5/R4 and F3/R1 and was used to make the mutant L253T. Additional mutants and primer pairs included R19Q, F6/R5 and F3/R1; R21Q, F7/R6 and F3/R1; and R(19,21)2Q, F8/R7 and F3/R1. Mutant R(16,19,21)3Q was obtained with primer pairs F9/R8 and F3/R1 using the double mutant R(19,21)2Q as template. Mutants L(3,5)2R, L(3,5)2R/R(19,21)2Q, and L(3,5)2R/R(16,19,21)3Q were generated with primers F10/R1. Mutants with an arginine residue inserted after the second Rdh1 residue R3, R3/19Q, R3/21Q, R3/(19,21)2Q, and R3/(16,19,21)3Q, were made with the primer pair F11/R1. To obtain the chimeric 11β-HSD1(1-41)/Rdh1(23-317), a PCR product coding for the first 41 amino acid residues of human 11β-HSD1 was amplified with primers F12/R9 and digested with EcoRI/StuI. The DNA fragment encoding amino acids 23-317 of Rdh1 was obtained with primers F13/R1 and digested with PmlI/XhoI. A sequence coding for the FLAG epitope (DYKDDDDK) was attached to the N or C termini of Rdh1 to generate FLAG-Rdh and Rdh-FLAG with primer pairs F14/R1 and F3/R10, respectively. Rdh1 mutants were subcloned into the EcoRI/XhoI site of pcDNA3 (Invitrogen). GFP constructs were cloned into the EcoRI/NotI sites of pEGFP-N1 (BD Biosciences). To obtain Rdh(1-22)/GFP, a DNA fragment encoding the first 22 residues of Rdh1 was amplified with primers F3/R14 and digested with EcoRI/PvuII. A second DNA fragment encoding the coding region of GFP was amplified with primers F16/R11 and digested with PmlI/NotI. The two fragments were cloned. Rdh(1-18)/GFP was made with primers F3/R12, digested with EcoRI/DraI, and cloned with the second DNA fragment described above. Overlapping PCR was used to generate mutant GFP/Rdh(289-317) with primers F18/R13 and F19/R14 and cloned. Plasmid Crad1(1-22)/GFP was constructed with primers F3/R15 and F20/R11, and the chimeric DNA fragment was cloned. Subcellular Fractionation and Western Blotting—COS cells (ATCC) were grown at 37 °C with 5% CO2 in Dulbecco's modified essential medium supplemented with 10% fetal bovine serum, 100 units/ml penicillin, and 100 μg/ml streptomycin. Cells on 100-mm plates were transfected with 8 μg of plasmid DNA using 40 μl of SuperFect (Qiagen). Twenty-four hours post-transfection, 5 × 107 cells were resuspended in 2 ml of ice-cold HB (150 μm MgCl2,10mm KCl, 10 mm Tris-HCl, pH 6.7) plus protease inhibitors and left on ice for 5 min. The cells were disrupted by nitrogen cavitation at 4 °C (80 p.s.i./30 min). The suspension was then homogenized by five gentle strokes of a Dounce grinder with a loose pestle. An additional ⅓ volume ice-cold HB with 1 m sucrose was added to the cell suspension and mixed well. The nuclei, cell debris, and unbroken cells were removed by centrifugation at 700 × g for 10 min. The postnuclear supernatant was centrifuged at 5000 × g for 10 min, and the pellet was resuspended with 5 ml of buffer B (250 mm sucrose, 1 mm EDTA, 10 mm Tris-HCl, pH 7.4) plus protease inhibitors. This homogenate was centrifuged at 5000 × g for 10 min to obtain a crude mitochondrial pellet. The crude mitochondria pellet was resuspended by five gentle strokes with a Dounce grinder, layered on a discontinuous sucrose gradient (30, 40, and 55%), and centrifuged at 150,000 × g with an SW41 rotor for 20 min at 4 °C. The mitochondria fraction was collected from the 40/55% interface, diluted with 5 volumes of buffer B, and centrifuged at 10,000 × g for 20 min. The resulting pellet was solubilized in buffer B. The microsomal fraction was obtained by centrifugation of the postmitochondrial supernatant at 100,000 × g for 1 h and was rehomogenized in buffer B. For Western blotting, 10 μg of protein were analyzed by 12.5% SDS-PAGE. Results were imaged with an ECL-Plus Western blotting detection system (Amersham Biosciences). Deglycosylation—Deglycosylation was done with PNGase F (New England Biolabs). A mixture of denatured proteins (1× Glycoprotein Denature Buffer, 100 °C, 10 min) with 0.1 volume each of 10× G7 buffer and 10% Nonidet P-40 and 2 μl of PNGase F (500,000 units/ml) was incubated at 37 °C for 1 h. Mixtures were resolved by 12.5% SDS-PAGE and analyzed by Western blot. Protease Protection—cDNA constructs were transfected into COS cells cultured in 6-well plates with SuperFect reagent (Qiagen). Twenty-four hours post-transfection, cells were washed three times in PBS and trypsinized with 300 μl of trypsin-EDTA (0.25%) for 10 min at 37 °C. Trypsinized cells were resuspended in PBS, washed twice with PBS, and permeabilized (5 min, 37 °C) by addition of 200 μl of SLO in PBS (1000 units/ml). Alternatively cells were treated with 200 μl of PBS or 200 μl of 0.5% Triton X-100 in PBS under the same conditions. Cells were then treated with proteinase K (50 μg/ml) at 37 °C for 15 min. Digestion was stopped by addition of 1 mm phenylmethylsulfonyl fluoride (Roche Applied Science) and 100 μl of 60 mm HEPES, pH 7.2, 1.5 m sucrose, 6 mm EDTA containing protease inhibitor mixture (BD Biosciences). Cells were lysed by sonication. Immunoblotting was done as described above using anti-Rdh1 or anti-GFP monoclonal antibody. Antibodies and Fluorescence Markers—Subconfluent COS cells grown on coverslips were transfected with SuperFect reagent (Qiagen). Twenty-four hours post-transfection, cells were incubated in serum-free medium for 1 h. Cells were washed three times in SLO buffer (75 mm potassium acetate, 25 mm HEPES buffer, pH 7.2) and fixed with paraformaldehyde (4% in PBS) at room temperature for 15 min. After washing, cells were incubated for 15 min at room temperature with 500 units/ml activated SLO (Sigma). SLO was removed, and the cells were washed once and incubated in SLO buffer for 15 min at 37 °C to allow pore formation. Alternatively fixed cells were incubated in PBS or 0.2% Triton X-100 (containing 1% bovine serum albumin in PBS) for 15 min at room temperature. Some fixed cells were permeabilized with digitonin (5 μg/ml) for 15 min at 4 °C in 20 mm HEPES, pH 6.9, 0.3 m sucrose, 0.1 m KCl, 2.5 mm MgCl2, 1 mm EDTA. Permeabilized cells were incubated at room temperature for 2 h with anti-Rdh1 (1:200, raised in rabbits against the peptide DRLSSNTKMIWDKASSEVK), anti-GFP (1:200, monoclonal antibody JL-8, BD Biosciences), or anti-calnexin (1:200, Sigma) followed by Alexa Fluor 488-conjugated goat anti-rabbit (5 μg/ml) or Alexa Fluor 594-conjugated goat anti-mouse immunoglobulins (5 μg/ml) for another 1 h. Fluorescein isothiocyanate-conjugated anti-FLAG M2 monoclonal antibody (10 μg/ml) was used for FLAG epitope detection (Sigma). Alexa Fluor 594-conjugated ConA or MitoTracker Red CMXRos (both from Molecular Probes) were used as ER or mitochondria markers, respectively. DAPI was used for nuclear counterstaining. Cells were mounted in ProLong Antifade reagents (Molecular Probes), viewed under a Zeiss 510 laser scanning confocal microscope, and processed with Zeiss Bitplane Imaris 3.3 software. Molecular Modeling—The molecular model was generated with Swiss-Model Version 36.0002, based on three-dimensional structures of soluble SDR, using an optimal amino acid sequence alignment as described previously (26Song M.-S. Chen W. Zhang M. Napoli J.L. J. Biol. Chem. 2003; 278: 40079-40087Abstract Full Text Full Text PDF PubMed Scopus (7) Google Scholar). The model relied on x-ray structures of human 17β-hydroxysteroid dehydrogenase type I complexed with cofactor and/or substrate (39.8% identity; Protein Data Bank codes 1FDS, 1A27, and 1EQU) (27Ghosh D. Pletnev V.Z. Zhu D.W. Wawrzak Z. Duax W.L. Pangborn W. Labrie F. Lin S.X. Structure. 1995; 3: 503-513Abstract Full Text Full Text PDF PubMed Scopus (252) Google Scholar, 28Azzi A. Rehse P.H. Zhu D.-W. Campbell R.I. Labrie F. Lin S.-X. Nat. Struct. Biol. 1996; 3: 665-668Crossref PubMed Scopus (132) Google Scholar, 29Breton R. Housset D. Mazza C. Fontecilla-Camps J.C. Structure (Lond.). 1996; 4: 905-915Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar), 20β-hydroxysteroid dehydrogenase (51.5%; Protein Data Bank code 1HU4) (30Ghosh D. Weeks C.M. Grochulski P. Duax W.L. Erman M. Rimsay R.L. Orr J.C. Proc. Natl. Acad. Sci. U. S. A. 1996; 88: 10064-10068Crossref Scopus (231) Google Scholar), and β-ketoacyl-[acyl-carrier protein]-reductase (41% identity; Protein Data Bank codes 1I0E and 1I0B) (31Fisher M. Kroon J.T. Martindale W. Stuitje A.R. Slabas A.R. Rafferty J.B. Struct. Fold. Des. 2000; 15: 339-347Abstract Full Text Full Text PDF Scopus (87) Google Scholar). Only the ER Expresses Rdh1—The N terminus of Rdh1 has characteristics of a dual mitochondria/microsomal signaling sequence. The first 18 amino acid residues form a hydrophobic helix sufficiently long to span a lipid bilayer membrane and are flanked by the positively charged residues Arg19, Arg21, and Lys30 (Fig. 1). This resembles N-terminal signaling sequences in enzymes expressed dually by ER and mitochondria, such as CYP1A1, CYP2E1, and P450MT2 (32Addya S. Anandatheerthavarada H.K. Biswas G. Bhagwat S.V. Mullick J. Avadhani N.G. J. Cell Biol. 1997; 139: 589-599Crossref PubMed Scopus (133) Google Scholar, 33Bhagwat S.V. Biswas G. Anandatheerthavarada H.K. Addya S. Pandak W. Avadhani N.G. J. Biol. Chem. 1999; 274: 24014-24022Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 34Robin M.A. Anandatheerthavarada H.K. Biswas G. Sepuri N.B.V. Gordon D.M. Pain D. Avadhani N.G. J. Biol. Chem. 2002; 277: 40583-40593Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar). We expressed Rdh1 in COS cells and determined its locus in subcellular fractions by immunoblotting and in intact cells by immunofluorescence. Immunoblotting (Fig. 1) revealed Rdh1 in the microsomal and mitochondria fractions, distributing with calnexin, an ER membrane protein (35Bergeron J.J. Brenner M.B. Thomas D.Y. Williams D.B. Trends Biochem. Sci. 1994; 19: 124-128Abstract Full Text PDF PubMed Scopus (455) Google Scholar). Cytochrome c, a mitochondria intermembrane space protein, appeared only in the mitochondria fraction. These results are consistent with reports of ER protein contamination of mitochondria even after their isolation by sucrose density gradient centrifugation (36Kuwana T. Mackey M.R. Perkins G. Ellisman M.H. Lattérich M. Schneider R. Green D.R. Niemeyer D.D. Cell. 2002; 111: 331-342Abstract Full Text Full Text PDF PubMed Scopus (1203) Google Scholar, 37Zong W.X. Li C. Hatzivassiliou G. Lindsten T. Yu Q.C. Yuan J. Thompson C.B. J. Cell Biol. 2003; 162: 59-69Crossref PubMed Scopus (496) Google Scholar). The density of the Rdh1 signal relative to the calnexin signal remained the same in both the microsomal and mitochondrial fractions, indicating that only the ER expresses Rdh1. Immunofluorescence of intact cells confirmed these observations (Fig. 2, a and b). Rdh1 colocalized with ConA, an ER marker, but did not colocalize with MitoTracker.Fig. 2Expression of Rdh1 and deletion mutants in intact cells. Rdh1 and deletion mutants were immunostained in fixed COS cells permeabilized with 0.2% Triton X-100. The left column shows immunostaining using anti-Rdh1 as the primary antibody and Alexa Fluor 488-conjugated goat anti-rabbit IgG as the secondary antibody (green). The middle column alternates showing Alexa Fluor 594-conjugated ConA (a, c, f, and h) or MitoTracker (Mito) Red CMXRos (b, d, e, g, and i) (both red) as microsomal and mitochondria markers, respectively, with the exception of RD(1-30), which shows only MitoTracker. The right column shows a merged image of the left and middle columns. Nuclei were stained with DAPI (blue): a and b, Rdh1; c and d, RD(1-18); e, RD(1-30); f and g, RD(289-317); h and i, RD2.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The N-terminal Signaling Sequence Targets Rdh1 to the ER—Rdh1 has hydrophobic helices at its N terminus (residues 1-18) and near its C terminus (residues 289-311). We made deletion mutants to determine whether one or both contribute to ER targeting (Fig. 1). Mutants RD(1-18) or RD(1-30), which lack the first 18 and 30 amino acid residues of Rdh1, respectively, did not localize to the ER. Surprisingly both distributed in the mitochondria fraction with cytochrome c and localized in mitochondria in intact cells (Figs. 1 and 2, c, d, and e). These data show that residues 1-18 and residues 1-30 are necessary for ER localization but do not demonstrate whether they are sufficient. The deletion mutant RD(289-317) produced immunoblot signals in both the microsomal and mitochondria fractions but predominantly in the mitochondria. Immunofluorescence confirmed this: only ∼20% of transfected cells showed ER localization, whereas the rest exhibited the punctate signal of mitochondria expression (Figs. 1 and 2, f and g). Deletion of both the 18 N-terminal and the 28 C-terminal residues, mutant RD2, generated a protein that localized overwhelmingly in the mitochondria (Figs. 1 and 2, h and i). Thus, the N-terminal signaling sequence directs Rdh1 to the ER, the C-region hydrophobic helix may promote ER retention, and removing both does not generate a soluble protein. To further define the function of the N- and C-terminal hydrophobic helices, we determined the loci of Rdh1/GFP fusion proteins by immunofluorescence. Native GFP had a diffuse pattern, distributing throughout the cell (not shown). Fusing the first 22 residues of Rdh1 to GFP (Rdh1(1-22)/GFP) produced a protein with an ER expression pattern, demonstrating that these residues are sufficient to cause ER targeting (Fig. 3). In contrast, the fusion protein Rdh1(1-18)/GFP, with only the first 18 N-terminal residues fused to GFP, showed a diffuse signal, indicating that the first 18 residues alone are not sufficient for ER targeting. The fusion protein of GFP and the last 29 Rdh1 amino acid residues, GFP/Rdh1(289-317), also showed a diffuse cytoplasmic and nuclear signal with an intense band in the nuclear membrane. These data show that the 22 N-terminal residues, but not the last 29 C-terminal residues, of Rdh1 are sufficient to direct and anchor the soluble protein GFP to the ER. Rdh1 Faces the Cytoplasm—We assessed access of antibodies to Rdh1 expressed in COS cells treated with SLO, digitonin, or Triton X-100. SLO selectively permeabilizes the plasma membrane by binding to cholesterol and polymerizing to create pores ≥12 nm, permitting uptake of large molecules (38Palmer M. Vulicevic I. Saweljew P. Valeva A. Kehoe M. Bhakdi S. Biochemistry. 1998; 37: 2378-2383Crossref PubMed Scopus (46) Google Scholar). Digitonin also creates pores in plasma membranes by interacting with cholesterol. To place the Rdh1 results into context with a luminal facing SDR, we made a chimera of the first 41 N-terminal residues of h11β-HSD1 fused to the N terminus of GFP. These residues contain a signaling sequence, bounded near their N terminus by two lysine residues and bounded on their C terminus by two glutamate residues, that direct h11β-HSD1 into the ER facing the lumen (39Ozols J. J. Biol. Chem. 1995; 270: 2305-2312Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 40Odermatt A. Arnold P. Stauffer A. Fr" @default.
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• W2092504384 title "Elements in the N-terminal Signaling Sequence That Determine Cytosolic Topology of Short-chain Dehydrogenases/Reductases" @default.
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