Membrane Topology Mapping of Vitamin K Epoxide Reductase by in Vitro Translation/Cotranslocation

Vitamin K epoxide reductase (VKOR) catalyzes the conversion of vitamin K 2,3-epoxide into vitamin K in the vitamin K redox cycle. Recently, the gene encoding the catalytic subunit of VKOR was identified as a 163-amino acid integral membrane protein. In this study we report the experimentally derived membrane topology of VKOR. Our results show that four hydrophobic regions predicted as the potential transmembrane domains in VKOR can individually insert across the endoplasmic reticulum membrane in vitro. However, in the intact enzyme there are only three transmembrane domains, residues 10–29, 101–123, and 127–149, and membrane-integration of residues 75–97 appears to be suppressed by the surrounding sequence. Results of N-linked glycosylation-tagged full-length VKOR shows that the N terminus of VKOR is located in the endoplasmic reticulum lumen, and the C terminus is located in the cytoplasm. Further evidence for this topological model of VKOR was obtained with freshly prepared intact microsomes from insect cells expressing HPC4-tagged full-length VKOR. In these experiments an HPC4 tag at the N terminus was protected from proteinase K digestion, whereas an HPC4 tag at the C terminus was susceptible. Altogether, our results suggest that VKOR is a type III membrane protein with three transmembrane domains, which agrees well with the prediction by the topology prediction program TMHMM. Vitamin K epoxide reductase (VKOR) catalyzes the conversion of vitamin K 2,3-epoxide into vitamin K in the vitamin K redox cycle. Recently, the gene encoding the catalytic subunit of VKOR was identified as a 163-amino acid integral membrane protein. In this study we report the experimentally derived membrane topology of VKOR. Our results show that four hydrophobic regions predicted as the potential transmembrane domains in VKOR can individually insert across the endoplasmic reticulum membrane in vitro. However, in the intact enzyme there are only three transmembrane domains, residues 10–29, 101–123, and 127–149, and membrane-integration of residues 75–97 appears to be suppressed by the surrounding sequence. Results of N-linked glycosylation-tagged full-length VKOR shows that the N terminus of VKOR is located in the endoplasmic reticulum lumen, and the C terminus is located in the cytoplasm. Further evidence for this topological model of VKOR was obtained with freshly prepared intact microsomes from insect cells expressing HPC4-tagged full-length VKOR. In these experiments an HPC4 tag at the N terminus was protected from proteinase K digestion, whereas an HPC4 tag at the C terminus was susceptible. Altogether, our results suggest that VKOR is a type III membrane protein with three transmembrane domains, which agrees well with the prediction by the topology prediction program TMHMM. The K vitamins, phylloquinone (K1), menaquinones (K2), and menadione (K3), are a family of structurally similar, fatsoluble, 2-methyl-1,4-naphthoquinones. The main function of vitamin K is to act as a co-factor for the γ-glutamyl carboxylase that catalyzes the post-translational carboxylation of specific glutamic acid to γ-carboxyglutamic acid (Gla) 1The abbreviations used are: Gla, γ-carboxyglutamic acid; VKOR, vitamin K epoxide reductase; VKORC1, vitamin K epoxide reductase complex subunit 1; TM, transmembrane; HPC4, peptide epitope comprising residues; EDQVDPRLIDGK, ER endoplasmic reticulum; RM, canine rough microsomes; MES, 2-(N-morpholino)ethanesulfonic acid, hemisodium salt; CHAPS, (3-[(3-cholamidopropyl)di-methylammonio]-1-propane sulfonate); Endo H, endoglycosidase H; PVDF, polyvinylidene difluoride. of variety of vitamin K-dependent proteins (1Presnell S.R. Stafford D.W. Thromb. Haemostasis. 2002; 87: 937-946Crossref PubMed Scopus (83) Google Scholar). Members of the vitamin K-dependent protein family include coagulation factors (factor II, VII, IX, X) as well as several other proteins that function in bone metabolism (2Price P.A. Annu. Rev. Nutr. 1988; 8: 565-583Crossref PubMed Scopus (151) Google Scholar) and signal transduction (3Manfioletti G. Brancolini C. Avanzi G. Schneider C. Mol. Cell. Biol. 1993; 13: 4976-4985Crossref PubMed Scopus (532) Google Scholar). Concomitant with γ-glutamyl carboxylation, the reduced form of vitamin K (vitamin K hydroquinone) is converted to vitamin K 2,3-epoxide, which must be converted back to vitamin K hydroquinone for the reaction to continue because of limited vitamin K amounts in vivo (4Furie B. Bouchard B.A. Furie B.C. Blood. 1999; 93: 1798-1808Crossref PubMed Google Scholar). This cyclic conversion of vitamin K establishes a redox cycle known as the vitamin K cycle (5Saxena S.P. Israels E.D. Israels L.G. Apoptosis. 2001; 6: 57-68Crossref PubMed Scopus (29) Google Scholar). VKOR is responsible for the conversion of vitamin K 2,3-epoxide into vitamin K and is highly sensitive to inhibition by coumarin drugs, such as R,S-warfarin (4-hydroxy-3-(3-oxo-1-phenylbutyl-coumarin)), the most commonly prescribed oral anticoagulant. Warfarin inhibition of VKOR reduces the availability of reduced vitamin K, which reduces the rate of carboxylation and leads to partially carboxylated, inactive vitamin K-dependent proteins. Since its discovery in 1970 (6Bell R.G. Matschiner J.T. Arch. Biochem. Biophys. 1970; 141: 473-476Crossref PubMed Scopus (61) Google Scholar), numerous futile attempts to purify the enzyme were reported (7Mukharji I. Silverman R.B. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 2713-2717Crossref PubMed Scopus (25) Google Scholar, 8Lee J.J. Principe L.M. Fasco M.J. Biochemistry. 1985; 24: 7063-7070Crossref PubMed Scopus (17) Google Scholar, 9Wallin R. Guenthner T.M. Methods Enzymol. 1997; 282: 395-408Crossref PubMed Scopus (12) Google Scholar, 10Cain D. 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Recently, the gene encoding VKOR was identified independently by our laboratory (16Li T. Chang C.Y. Jin D.Y. Lin P.J. Khvorova A. Stafford D.W. Nature. 2004; 427: 541-544Crossref PubMed Scopus (598) Google Scholar) and that of Johannes Oldenburg (17Rost S. Fregin A. Ivaskevicius V. Conzelmann E. Hortnagel K. Pelz H.J. Lappegard K. Seifried E. Scharrer I. Tuddenham E.G. Muller C.R. Strom T.M. Oldenburg J. Nature. 2004; 427: 537-541Crossref PubMed Scopus (995) Google Scholar). The enzyme was designated VKOR by us and VKORC1 by Oldenburg and co-workers, who assume that there is another sub-unit still to be identified (either view may be correct). VKOR is a 163-amino acid integral membrane protein with a mass of 18.2 kDa. To understand the structure/function relationships that define VKOR activity, it is necessary to understand its membrane topology, i.e. its specific number of TM segments and their location relative to the cytoplasm or endoplasmic reticulum. Several computer programs to predict the topology of membrane proteins are available (18Moller S. Croning M.D. Apweiler R. Bioinformatics. 2001; 17: 646-653Crossref PubMed Scopus (860) Google Scholar, 19Nilsson J. Persson B. von Heijne G. FEBS Lett. 2000; 486: 267-269Crossref PubMed Scopus (93) Google Scholar, 20Melen K. Krogh A. von Heijne G. J. Mol. Biol. 2003; 327: 735-744Crossref PubMed Scopus (174) Google Scholar, 21Taylor P.D. Attwood T.K. Flower D.R. Nucleic Acids Res. 2003; 31: 3698-3700Crossref PubMed Scopus (34) Google Scholar). If the location of one of the termini is known, the best current program correctly predicts the topology of 65–70% of those membrane proteins with known crystal structure (20Melen K. Krogh A. von Heijne G. J. Mol. Biol. 2003; 327: 735-744Crossref PubMed Scopus (174) Google Scholar). The reliability of a given topology prediction is increased if the predictions from a number of different programs agree (19Nilsson J. Persson B. von Heijne G. FEBS Lett. 2000; 486: 267-269Crossref PubMed Scopus (93) Google Scholar, 20Melen K. Krogh A. von Heijne G. J. Mol. Biol. 2003; 327: 735-744Crossref PubMed Scopus (174) Google Scholar). Moreover, using C-terminal reporter fusions, experimental examination of the topology is reliable (22Kim H. Melen K. von Heijne G. J. Biol. Chem. 2003; 278: 10208-10213Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). In this study, we report an experimental determination of the membrane topology of VKOR. We first determined the topology of VKOR using multiple prediction programs. These predictions were then tested by in vitro translation/cotranslocation of a series of VKOR truncations containing an N-linked glycosylation reporter tag. Our results suggest that VKOR is a type III membrane protein with three transmembrane domains. The N terminus of VKOR resides in the lumen of the endoplasmic reticulum, and the C terminus resides in the cytoplasm. The N-terminal domain of VKOR, like many type III membrane proteins, is relatively short, which facilitates its transfer into the lumen (23Goder V. Spiess M. FEBS Lett. 2001; 504: 87-93Crossref PubMed Scopus (139) Google Scholar). The main features of our in vitro experiments were confirmed by experiments with microsomes derived from insect cells expressing full-length VKOR. Materials—All oligonucleotide primers, the NuPAGE pre-cast gel, and the insect cell expression vector pVL1392 were from Invitrogen. The in vitro translation vector pSPUTK was obtained from Stratagene (San Diego, CA). The SP6 mMESSAGE mMACHINE capped mRNA transcription kit and MEGAClear RNA purification kit were obtained from Ambion (Austin, TX). Rabbit reticulocyte lysate, amino acid master mixture, proteinase K, and ribonuclease inhibitor were obtained from Promega (Madison, WI). [35S]Methionine, [35S]cysteine, and the enhanced chemiluminescence Western blotting detection reagents were from Amersham Biosciences. MES, HEPES, CHAPS, and phenylmethylsulfonyl fluoride were from Sigma. BacVector-3000 DNA kit was from Novagen (Madison, WI). Taq polymerase and Endo H were from Roche Applied Science. Trans-Blot transfer medium PVDF membrane (0.2 μm) and biotinylated protein size markers were from Bio-Rad. All the restriction endonucleases and T4 DNA ligase were from New England BioLabs (Beverly, MA). Horseradish peroxidase-conjugated secondary antibody was from Jackson Laboratories (West Grove, PA). Dog pancreatic microsomal membranes were prepared as described (24Walter P. Blobel G. Methods Enzymol. 1983; 96: 84-93Crossref PubMed Scopus (480) Google Scholar). Construction of Full-length VKOR Fusions with the NST Reporter Tag at the N or C Terminus—The gene encoding VKOR was amplified by PCR using pVL1392-VKOR (16Li T. Chang C.Y. Jin D.Y. Lin P.J. Khvorova A. Stafford D.W. Nature. 2004; 427: 541-544Crossref PubMed Scopus (598) Google Scholar) as the template. A 5′-NcoI site and 3′-PstI site was introduced into the PCR fragment, and the PCR product was cloned into the in vitro translation vector pSPUTK by NcoI/PstI to yield pSPUTK-VKOR. The N-terminal glycosylation acceptor tag with the amino acid sequence of MGGNSTGGSGGSGGSG was obtained by annealing the oligonucleotides 5′-CATGGGTGGGAACAGCACCGGTGGGAGCGGGGGCAGCGGGGGCAGCGG-3′ and 5′-CATGCCGCTGCCCCCGCTGCCCCCGCTCCCACCGGTGCTGTTCCCACC-3′ followed by ligation to NcoI-digested pSPUTK-VKOR. Joining the vector and the insert by ligation destroys the NcoI site at the 3′ end of the insert. This plasmid was named pSPUTK-NST-VKOR. A C-terminal glycosylation acceptor tag with the amino acid sequence of SGGSGGSNSTGGSG was obtained by annealing the oligonucleotides 5′-AAGCGGGGGCAGCGGTGGGAGCAACAGCACCGGGGGTAGCGGTCTGCA-3′ and 5′-GACCGCTACCCCCGGTGCTGTTGCTCCCACCGCTGCCCCCGCTTTGCA-3′ followed by ligation to PstI-digested pSPUTK-VKOR. In this case ligation of the PstI-cleaved pSPUTK-VKOR to the insert results in the destruction of the PstI site at the 5′ end of the insert. This plasmid is named pSPUTK-VKOR-NST. A dimer C-terminal glycosylation tag was engineered by first ligating the insert itself and then ligating the product to PstI-digested pSPUTK-VKOR plasmid to yield pSPUTK-VKOR-NST2. Construction of VKOR Truncations with NST Reporter Tag at the N Terminus—Site-directed mutagenesis was performed to remove the endogenous BamHI site (without affecting the amino acid sequence) at 365 nucleotides of the VKOR cDNA from pSPUTK-NST-VKOR. The resulting plasmid was used as the PCR template to generate various C-terminal truncations of VKOR. A sense oligonucleotide comprising a NcoI site and part of the NST tag-coding sequence was used as 5′ primer for all the PCR reactions. Oligonucleotides containing a BamHI site and various antisense sequences corresponding to the C terminus of the predicted TM domains were used as 3′ primers for the PCR reaction. PCR-amplified fragments containing each of the predicted TM domains and the N-terminal glycosylation reporter tag were cloned into the NcoI/BamHI-cleaved pSPUTK vector. Construction of VKOR Truncations with NST Reporter Tag at the C Terminus—The C-terminal glycosylation acceptor tag with the sequence of SGGSGGSNSTGGSG was amplified by PCR using the above pSPUTK-VKOR-NST plasmid as template. A 5′-BamHI site and a 3′-EcoRI site were introduced into the PCR fragment, and the resulting PCR product was cloned to pSPUTK by BamHI/EcoRI to yield pSPUTK-NST. Various VKOR truncations (containing each of the predicted TM segments) were then amplified by PCR from the pSPUKT-NST-VKOR plasmid with the deleted endogenous BamHI site. The 5′ primer encoding the start sequence of VKOR includes a NcoI site. All the 3′ primers were the same as above. PCR products were cloned into NcoI/BamHI-cleaved pSPUTK-NST to yield different VKOR truncations with the glycosylation reporter tag at the C terminus. The 3′-BamHI (GGATCC) site introduced two amino acid residues (glycine and serine) between the VKOR truncations and the NST tag. Construct 1–74/98–130-NST was obtained by connecting the PCR fragment of 1–74 and 98–130. Fragment 1–74 was amplified by using the above 5′ primer and a 3′ primer containing part of the antisense sequence of VKOR ending at amino acid residue 74 with a XbaI site. Fragment 98–130 was amplified by a 5′ primer containing a XbaI site with part of the sense coding sequence of VKOR starting from residue 98 and the above 3′ primer used for VKOR truncation 1–130. The two obtained PCR fragments were ligated simultaneously with NcoI/BamHI-cleaved pSPUTK-NST vector to yield the construct of pSPUTK-NST-1–74/98–130. A C-terminal glycosylation tag with a sequence of NSTGGGS, which does not contain the flexible extension linker, was fused to the VKOR truncation of 1–130 by PCR to yield pSPUTK-1–130-NST′. The 5′ primer used is the same as above, and the 3′ primer contains part of the C-terminal antisense sequence of 1–130 VKOR truncation and the antisense sequence of the tag with a BamHI site. The PCR product was cloned to pSPUTK by NcoI/BamHI. All the constructs used for in vitro transcription/translation are shown in Fig. 2. Construction of the Full-length VKOR Fusions with HPC4 Tag at the N or C Terminus in Insect Cell Expression Vector—A 12-amino acid peptide (EDQVDPRLIDGK) of HPC4 epitope (25Stearns D.J. Kurosawa S. Sims P.J. Esmon N.L. Esmon C.T. J. Biol. Chem. 1988; 263: 826-832Abstract Full Text PDF PubMed Google Scholar) was introduced to either the N or C terminus of full-length VKOR. The HPC4-tagged VKOR cDNA was subcloned into the EcoRI site of the insect cell expression vector of pVL1392. The nucleotide sequences of all the constructs were confirmed by the DNA sequencing facility at the University of North Carolina at Chapel Hill. In Vitro Transcription and Translation/Cotranslocation—Before in vitro transcription the recombinant pSPUTK vectors were linearized at the BamHI site immediately 3′ to the C terminus of all the N-terminal NST-tagged VKOR truncation fusions or at the EcoRI site immediately 3′ to the C terminus of all the C-terminal NST-tagged VKOR truncation fusions. Capped mRNA was synthesized by SP6 RNA polymerase and purified by MEGAClear RNA purification kit according to the manufacturer's instructions. The transcription reaction was incubated at 37 °C for at least 5 h to increase the yield of mRNA. In vitro translations using rabbit reticulocyte lysate, and cotranslocations using RM were performed as described previously (26Tie J. Wu S.M. Jin D. Nicchitta C.V. Stafford D.W. Blood. 2000; 96: 973-978Crossref PubMed Google Scholar). The reactions were performed at 25 °C for 30 min in a final volume of 20 μl containing 10 μl of rabbit reticulocyte lysate, 0.5 μl of ribonuclease inhibitor (40 units/μl), 0.4 μl of a 1 mmol/liter amino acid mixture without methionine and cysteine, 1.6 μl of [35S]methionine (3.7 × 1013 Bq (1000 Ci/mmol)) at 370 MBq (10 mCi/ml), 1.6 μl of [35S]cysteine (3.7 × 1013 Bq (1000 Ci/mmol)) at 370 MBq (10 mCi/ml), and 1 μg of capped mRNA with or without 1 eq of RM. The translation products were chilled on ice and mixed with 30 μl of buffer A containing 110 mmol/liter potassium acetate, 2.5 mmol/liter magnesium acetate, and 25 mmol/liter potassium-HEPES (pH 7.4). For deglycosylation, 1 μl of Endo H (1 milliunits/μl) and 1 μl of 5% CHAPS were added to a 10-μl aliquot of the diluted translation products and incubated for 1 h at 37 °C. Before being subjected to SDS-PAGE analysis, the samples were precipitated with 2 volumes of saturated (NH4)2SO4, washed with 5% trichloroacetic acid, and redissolved in 10 μl of SDS-PAGE sample buffer. Expression of HPC4-tagged VKOR in Insect Cells and Protease Digestion of VKOR Microsomes—Full-length VKOR molecules with a HPC4 tag at either the N or C termini were expressed in insect cells. Microsomes from cells expressing HPC4-VKOR or VKOR-HPC4 were prepared as described (27Mutucumarana V.P. Stafford D.W. Stanley T.B. Jin D.Y. Solera J. Brenner B. Azerad R. Wu S.M. J. Biol. Chem. 2000; 275: 32572-32577Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). Microsome protein concentrations were adjusted to 10 mg/ml. For protease digestion 2 μl of proteinase K (1 mg/ml) was added to a 10-μl aliquot of freshly prepared microsome in the presence or absence of 0.5% CHAPS as the final concentration. The reactions were carried out on ice for 2 h and terminated by adding phenylmethylsulfonyl fluoride to a final concentration of 3 mmol/liter. After protease digestion, the microsomes were washed 3 times with buffer A containing 3 mmol/liter phenylmethylsulfonyl fluoride, and the pellets were dissolved in SDS sample buffer and subjected to SDS-PAGE analysis. NuPAGE and Western Blot Analysis—NuPAGE analysis was performed according to the manufacturer's instructions under reducing conditions using 1× MES running buffer (50 mm MES, 50 mm Tris, 1 mm EDTA, and 0.1% SDS (pH 7.3). Samples from the in vitro translation were subjected to 4–12% gradient NuPAGE. After electrophoresis the proteins were transferred to a PVDF membrane (0.2 μm), and autoradiography was performed (Amersham Biosciences Storm 840 PhosphorImager). Samples of recombinant HPC4-taged VKOR microsome and protease-digested microsome were subjected to 12% Nu-PAGE and transferred to a PVDF membrane as above. Protein bands were probed with anti-HPC4 monoclonal antibody followed by a horseradish peroxidase-conjugated secondary antibody, and the protein bands were detected using the enhanced chemiluminescence Western blot reagents. Prediction of the Membrane Topology of VKOR by Different Computer-prediction Algorithms—The membrane topology of VKOR was predicted using seven different topology prediction programs (us.expasy.org/tools/#ptm) with the default parameters. The results are listed in Table I. As can be seen, five of the seven programs predict three TM domains in VKOR, PHD predicts two, and MEMSAT predicts four TM domains. The five programs that predict the location of the C terminus all agree that the C terminus of VKOR is located in the cytoplasm. All programs except PHD predict that the first TM domain comprises residues 10 through 29. The remaining TM domain predictions are somewhat variable for the different prediction programs. The combined candidate TM domains in VKOR predicted by all the programs used are 10–29, 75–97, 101–123, and 127–149. It has been reported that the reliability of topology predictions is greatly increased if different topology prediction methods give the same prediction (19Nilsson J. Persson B. von Heijne G. FEBS Lett. 2000; 486: 267-269Crossref PubMed Scopus (93) Google Scholar, 20Melen K. Krogh A. von Heijne G. J. Mol. Biol. 2003; 327: 735-744Crossref PubMed Scopus (174) Google Scholar). Therefore, it is likely that VKOR has three TM domains with the C terminus located in the cytoplasm and N terminus located in the ER lumen. Fig. 1 shows the membrane topology prediction and the probability profile of VKOR by the most commonly used prediction program TMHMM. It predicts that VKOR has 3 TM domains, 10–29, 101–123, and 127–149, with the orientation of Nexoplasmic/Ccytoplasmic. One candidate TM domain (75–97), predicted by other programs but not by TMHMM, is indicated in Fig. 1.Table IPrediction of membrane topology of VKOR by different membrane topology prediction programs Membrane topology of VKOR was predicted by different predication programs using the default parameters. The combined four candidate TM domains are listed. Numbers under the TM correspond to the amino acid residue of VKOR.ProgramsTM no.C terminusTM1TM2TM3TM4PHD2InaIn, cytoplasmic location of ER85–109 (1.0)bNumbers in the parentheses are the probability of the predicted TM segment servers as the stop-transfer sequence119–143 (0.87)TMHMM 2.03In10–29 (1.0)101–123 (0.90)127–149 (0.93)TopPred 23In9–29 (0.65)78–98 (0.67)109–129 (1.0)TMPred3In9–29 (0.81)75–97 (0.57)101–129 (1.0)DAS312–2783–96102–146SOSUI311–31 (primary)75–97 (secondary)116–138 (primary)MEMSAT4In13–29 (0.76)81–97 (0.60)104–124 (1.0)131–148 (0.68)a In, cytoplasmic location of ERb Numbers in the parentheses are the probability of the predicted TM segment servers as the stop-transfer sequence Open table in a new tab VKOR Fusions with the N-Linked Glycosylation Reporter Tag—The glycosylation mapping technique (28Nilsson I.M. von Heijne G. J. Biol. Chem. 1993; 268: 5798-5801Abstract Full Text PDF PubMed Google Scholar) was used in this study to experimentally determine the membrane topology of VKOR. A conserved N-linked glycosylation acceptor site (NST) was introduced at either the N or C termini of the full-length VKOR or various VKOR truncations. According to the predicted membrane topology of VKOR, only nine amino acid residues precede the first candidate TM domain. Moreover, if the last predicted TM domain ends at residue149, as predicted by TMHMM and MEMSAT, only 13 C-terminal amino acid residues protrude from the membrane. It has been reported that half-maximal glycosylation occurs when the acceptor Asn is 10∼14 residues away from the membrane (28Nilsson I.M. von Heijne G. J. Biol. Chem. 1993; 268: 5798-5801Abstract Full Text PDF PubMed Google Scholar, 29Nilsson I. Saaf A. Whitley P. Gafvelin G. Waller C. von Heijne G. J. Mol. Biol. 1998; 284: 1165-1175Crossref PubMed Scopus (120) Google Scholar). Therefore, we added flexible amino acid linkers to extend the Asn glycosylation acceptor site away from the membrane. The amino acid sequence of the N-terminal NST tag is MGGNSTGGSGGSGGSG, and the C-terminal NST tag sequence is SGGSGGSNSTGGSG. The above extended NST tags were joined to the N or C terminus of full-length VKOR and to all of the VKOR truncations except when otherwise stated. In addition, we fused a dimer C-terminal NST tag at the C terminus of full-length VKOR (VKOR-NST2). To examine whether the predicted TM region of 75–97 or 101–123 is the authentic TM domain, we deleted the predicted TM region of 75–97 in the fusion of 1–130-NST (1–75/98–130-NST). In addition, we fused a NST glycosylation tag without the flexible linker (NST-GGGS) immediately to the C terminus of VKOR truncation 1–130. All the constructs used are shown in Fig. 2. In Vitro Translation/Cotranslocation of the NST-tagged Fulllength VKOR Fusions—Fig. 3 shows the autoradiograph of the in vitro translation of the full-length VKOR and NST-tagged full-length VKOR in the presence and absence of RM. As can be seen, N-terminal tagged VKOR, NST-VKOR, is glycosylated in the presence of RM, and the glycosylation product is sensitive to Endo H treatment, which specifically cleaves N-linked core sugars. This result indicates that the N terminus of VKOR is located in the ER lumen. In contrast, neither C-terminal tagged VKOR (VKOR-NST or VKOR-NST2) is glycosylated (Fig. 3), suggesting that the C terminus of VKOR is located in the cytoplasm. These results together suggest that VKOR is a type III membrane protein with the orientation of Nexoplasmic/Ccytoplasmic, and it has an odd number of TM domains. This agrees with the topology model predicted by five of the seven topology prediction programs listed in Table I. In addition, neither of the two natural potential glycosylation sites in the VKOR sequence, Asn-77 and Asn-142, is glycosylated; this is consistent with the prediction of TMHMM, which indicates that Asn-77 is located on the cytoplasm side of the membrane, and Asn-142 is located within the membrane. Determination of the Signal-anchor Potential of the Predicted TM Domain by in Vitro Translation/Cotranslocation—According to the computer-generated predictions, there are four potential TM segments in VKOR sequence. Therefore, C-terminal truncations of VKOR were made after each of the predicted potential TM segments. The N-linked glycosylation reporter tag was fused to either the N terminus or the C terminus of these truncations (Fig. 2). Fig. 4 shows the in vitro translation/cotranslocation results obtained from these NST-tagged VKOR truncations in the presence and absence of RM and the proposed topological orientation. As can be seen, NST-1–40 was glycosylated, but 1–40-NST was not, indicating that the first predicted TM segment, 10–29, can function as a reverse signalanchor sequence, characteristic of type III membrane protein. Both fusions of NST-1–100 and 1–100-NST were glycosylated, indicating that both the N and C termini are located in the ER lumen with the hairpin structure. Together with the result of fusion 1–40, this result suggests that the second predicted TM domain, 75–97, can function as a signal-anchor sequence. However, both fusions of NST-1–130 and 1–130-NST, which has one more predicted TM domain than fusion 1–100, are also glycosylated, indicating that they have the same topological orientation as fusion 1–100. This result raises the question of whether the additional predicted TM domain of 101–123 in the fusion of 1–130 functions as the signal anchor sequence. Predicted TM Region of 101–123 Functions as a Genuine TM Domain—To examine whether the predicted TM region of 101–123 functions as a genuine TM domain, we deleted the potential TM region of 75–97 in the fusion of 1–130-NST (1–75/98–130-NST) (Fig. 2). As shown in Fig. 5A, the C-terminal glycosylation tag can be glycosylated in the presence of RM, indicating that the C terminus is located in the lumen of ER. Together with the result of fusion 1–40, this result suggests that the predicted TM region of 101–123 functions as a genuine TM domain in the fusion 1–75/98–130-NST. In a further attempt to determine which of the potential TM domains, 75–97 or 101–123, functions as the genuine TM domain in the VKOR truncation 1–130, a glycosylation reporter tag with the sequence of NSTGGGS was fused to the C terminus of VKOR-1–130 (1–130-NST′). This places the Asn glycosylation acceptor site immediately after the predicted TM segment 101–123. According to the "minimum glycosylation distance" rule (28Nilsson I.M. von Heijne G. J. Biol. Chem. 1993; 268: 5798-5801Abstract Full Text PDF PubMed Google Scholar, 29Nilsson I. Saaf A. Whitley P. Gafvelin G. Waller C. von Heijne G. J. Mol. Biol. 1998; 284: 1165-1175Crossref PubMed Scopus (120) Google Scholar), this tag should not be glycosylated if 101–123 functions as the stop-transfer sequence in this construct. On the other hand, if 75–97 serves as a TM domain, this tag should be glycosylated since there is enough extension sequence between the Asn acceptor site and the TM segment. As shown in Fig. 5B, no glycosylated product was detected in the presence of RM. This result suggests that the predicted TM segment 101–123 is the functional TM domain in the VKOR truncation 1–130, but 75–97 does not. Independent Determination of the Topological Orientation of N and C Termini of VKOR in Vivo—To determine the topological orientation of the N and C termini of VKOR in vivo, we constructed two full-length recombinant VKOR molecules tagged with a HPC4 epitope at either end, HPC4-VKOR and VKOR-HPC4. Both VKOR constructs expressed in insect (Sf9) cells displayed kinetic parameters characteristic of the nontagged VKOR, indicating correct folding of the recombinanttagged enzymes (data not shown). Freshly prepared microsomes fro