Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 1998 Mar 1;330(Pt 2):959–966. doi: 10.1042/bj3300959

High-efficiency expression and characterization of the synaptic-vesicle monoamine transporter from baculovirus-infected insect cells.

M K Sievert 1, D S Thiriot 1, R H Edwards 1, A E Ruoho 1
PMCID: PMC1219231  PMID: 9480916

Abstract

The full-length cDNA for the rat synaptic-vesicle monoamine transporter (VMAT2) containing a C-terminal polyhistidine epitope has been engineered into baculovirus DNA for expression in Spodoptera frugiperda (Sf9) insect cells. Using this recombinant baculovirus and cultured Sf9 cells, rVMAT2 has been expressed at levels of 7.8x10(6) transporters per cell, as assessed by [3H]dihydrotetrabenazine binding. A 1l culture of infected cells produced approx. 15 nmol (900 microg) of transporter. rVMAT2 expressed in the Sf9 cells bound [3H]dihydrotetrabenazine with a KD of 31.2 nM and a Bmax of 19.9 pmol/mg. Two polypeptides of 55 and 63 kDa were identified using the photolabel, 7-azido-8-[125I]iodoketanserin ([125I]AZIK). Photoaffinity labelling of rVMAT2 by 1 nM [125I]AZIK was protectable by 10 microM tetrabenazine and 10 microM 7-aminoketanserin. Digitonin-solubilized VMAT2 was purified to greater than 95% homogeneity using immobilized Ni2+-affinity chromatography, followed by lectin (Concanavalin A) chromatography. The purified transporter migrates as a single broad band with a molecular mass of approx. 63kDa, as analyzed by SDS/PAGE. The purified transporter retained the ability to bind ligands ([125I]AZIK and [3H]dihydrotetrabenazine). The purified VMAT2 bound [3H]dihydrotetrabenazine with a KD of 86.2 nM. As is the case with the monoamine transporter from bovine chromaffin granule membranes, purified VMAT2 is covalently modified by dicyclohexylcarbodi-imide (DCCD) and is specifically labelled by [14C]DCCD. This labelling is inhibited by tetrabenazine and ketanserin. These data indicate that VMAT2 can be overexpressed using the baculovirus expression system and purified.

Full Text

The Full Text of this article is available as a PDF (489.0 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Apps D. K., Pryde J. G., Phillips J. H. Both the transmembrane pH gradient and the membrane potential are important in the accumulation of amines by resealed chromaffin-granule 'ghosts'. FEBS Lett. 1980 Mar 10;111(2):386–390. doi: 10.1016/0014-5793(80)80833-7. [DOI] [PubMed] [Google Scholar]
  2. Calamia J., Manoil C. lac permease of Escherichia coli: topology and sequence elements promoting membrane insertion. Proc Natl Acad Sci U S A. 1990 Jul;87(13):4937–4941. doi: 10.1073/pnas.87.13.4937. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Daniels A. J., Reinhard J. F., Jr Energy-driven uptake of the neurotoxin 1-methyl-4-phenylpyridinium into chromaffin granules via the catecholamine transporter. J Biol Chem. 1988 Apr 15;263(11):5034–5036. [PubMed] [Google Scholar]
  4. Darchen F., Scherman D., Laduron P. M., Henry J. P. Ketanserin binds to the monoamine transporter of chromaffin granules and of synaptic vesicles. Mol Pharmacol. 1988 Jun;33(6):672–677. [PubMed] [Google Scholar]
  5. Edwards R. H. The transport of neurotransmitters into synaptic vesicles. Curr Opin Neurobiol. 1992 Oct;2(5):586–594. doi: 10.1016/0959-4388(92)90023-e. [DOI] [PubMed] [Google Scholar]
  6. Erickson J. D., Eiden L. E. Functional identification and molecular cloning of a human brain vesicle monoamine transporter. J Neurochem. 1993 Dec;61(6):2314–2317. doi: 10.1111/j.1471-4159.1993.tb07476.x. [DOI] [PubMed] [Google Scholar]
  7. Erickson J. D., Eiden L. E., Hoffman B. J. Expression cloning of a reserpine-sensitive vesicular monoamine transporter. Proc Natl Acad Sci U S A. 1992 Nov 15;89(22):10993–10997. doi: 10.1073/pnas.89.22.10993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. FREIS E. D. Mental depression in hypertensive patients treated for long periods with large doses of reserpine. N Engl J Med. 1954 Dec 16;251(25):1006–1008. doi: 10.1056/NEJM195412162512504. [DOI] [PubMed] [Google Scholar]
  9. Ford C. F., Suominen I., Glatz C. E. Fusion tails for the recovery and purification of recombinant proteins. Protein Expr Purif. 1991 Apr-Jun;2(2-3):95–107. doi: 10.1016/1046-5928(91)90057-p. [DOI] [PubMed] [Google Scholar]
  10. Gasnier B., Roisin M. P., Scherman D., Coornaert S., Desplanches G., Henry J. P. Uptake of meta-iodobenzylguanidine by bovine chromaffin granule membranes. Mol Pharmacol. 1986 Mar;29(3):275–280. [PubMed] [Google Scholar]
  11. Gasnier B., Scherman D., Henry J. P. Dicyclohexylcarbodiimide inhibits the monoamine carrier of bovine chromaffin granule membrane. Biochemistry. 1985 Feb 26;24(5):1239–1244. doi: 10.1021/bi00326a028. [DOI] [PubMed] [Google Scholar]
  12. George S. T., Arbabian M. A., Ruoho A. E., Kiely J., Malbon C. C. High-efficiency expression of mammalian beta-adrenergic receptors in baculovirus-infected insect cells. Biochem Biophys Res Commun. 1989 Sep 29;163(3):1265–1269. doi: 10.1016/0006-291x(89)91114-5. [DOI] [PubMed] [Google Scholar]
  13. Hassinen I. E., Vuokila P. T. Reaction of dicyclohexylcarbodiimide with mitochondrial proteins. Biochim Biophys Acta. 1993 Sep 13;1144(2):107–124. doi: 10.1016/0005-2728(93)90164-b. [DOI] [PubMed] [Google Scholar]
  14. Henry J. P., Scherman D. Radioligands of the vesicular monoamine transporter and their use as markers of monoamine storage vesicles. Biochem Pharmacol. 1989 Aug 1;38(15):2395–2404. doi: 10.1016/0006-2952(89)90082-8. [DOI] [PubMed] [Google Scholar]
  15. Isambert M. F., Gasnier B., Botton D., Henry J. P. Characterization and purification of the monoamine transporter of bovine chromaffin granules. Biochemistry. 1992 Feb 25;31(7):1980–1986. doi: 10.1021/bi00122a012. [DOI] [PubMed] [Google Scholar]
  16. Isambert M. F., Gasnier B., Laduron P. M., Henry J. P. Photoaffinity labeling of the monoamine transporter of bovine chromaffin granules and other monoamine storage vesicles using 7-azido-8-[125I]iodoketanserin. Biochemistry. 1989 Mar 7;28(5):2265–2270. doi: 10.1021/bi00431a044. [DOI] [PubMed] [Google Scholar]
  17. Isambert M. F., Henry J. P. Photoaffinity labeling of the tetrabenazine binding sites of bovine chromaffin granule membranes. Biochemistry. 1985 Jul 2;24(14):3660–3667. doi: 10.1021/bi00335a039. [DOI] [PubMed] [Google Scholar]
  18. Johnson R. G., Jr Accumulation of biological amines into chromaffin granules: a model for hormone and neurotransmitter transport. Physiol Rev. 1988 Jan;68(1):232–307. doi: 10.1152/physrev.1988.68.1.232. [DOI] [PubMed] [Google Scholar]
  19. Johnson R. G., Scarpa A. Protonmotive force and catecholamine transport in isolated chromaffin granules. J Biol Chem. 1979 May 25;254(10):3750–3760. [PubMed] [Google Scholar]
  20. Kaback H. R., Bibi E., Roepe P. D. Beta-galactoside transport in E. coli: a functional dissection of lac permease. Trends Biochem Sci. 1990 Aug;15(8):309–314. doi: 10.1016/0968-0004(90)90020-c. [DOI] [PubMed] [Google Scholar]
  21. Knoth J., Zallakian M., Njus D. Stoichiometry of H+-linked dopamine transport in chromaffin granule ghosts. Biochemistry. 1981 Nov 10;20(23):6625–6629. doi: 10.1021/bi00526a016. [DOI] [PubMed] [Google Scholar]
  22. Krejci E., Gasnier B., Botton D., Isambert M. F., Sagné C., Gagnon J., Massoulié J., Henry J. P. Expression and regulation of the bovine vesicular monoamine transporter gene. FEBS Lett. 1993 Nov 29;335(1):27–32. doi: 10.1016/0014-5793(93)80432-t. [DOI] [PubMed] [Google Scholar]
  23. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  24. Liu Y., Peter D., Roghani A., Schuldiner S., Privé G. G., Eisenberg D., Brecha N., Edwards R. H. A cDNA that suppresses MPP+ toxicity encodes a vesicular amine transporter. Cell. 1992 Aug 21;70(4):539–551. doi: 10.1016/0092-8674(92)90425-c. [DOI] [PubMed] [Google Scholar]
  25. Near J. A., Mahler H. R. Reserpine labels the catecholamine transporter in synaptic vesicles from bovine caudate nucleus. FEBS Lett. 1983 Jul 11;158(1):31–35. doi: 10.1016/0014-5793(83)80670-x. [DOI] [PubMed] [Google Scholar]
  26. Neyfakh A. A., Bidnenko V. E., Chen L. B. Efflux-mediated multidrug resistance in Bacillus subtilis: similarities and dissimilarities with the mammalian system. Proc Natl Acad Sci U S A. 1991 Jun 1;88(11):4781–4785. doi: 10.1073/pnas.88.11.4781. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Njus D., Kelley P. M., Harnadek G. J. Bioenergetics of secretory vesicles. Biochim Biophys Acta. 1986;853(3-4):237–265. doi: 10.1016/0304-4173(87)90003-6. [DOI] [PubMed] [Google Scholar]
  28. Peter D., Finn J. P., Klisak I., Liu Y., Kojis T., Heinzmann C., Roghani A., Sparkes R. S., Edwards R. H. Chromosomal localization of the human vesicular amine transporter genes. Genomics. 1993 Dec;18(3):720–723. doi: 10.1016/s0888-7543(05)80383-0. [DOI] [PubMed] [Google Scholar]
  29. Porath J. Immobilized metal ion affinity chromatography. Protein Expr Purif. 1992 Aug;3(4):263–281. doi: 10.1016/1046-5928(92)90001-d. [DOI] [PubMed] [Google Scholar]
  30. Scherman D., Darchen F., Desnos C., Henry J. P. 1-Methyl-4-phenylpyridinium is a substrate of the vesicular monoamine uptake system of chromaffin granules. Eur J Pharmacol. 1988 Feb 9;146(2-3):359–360. doi: 10.1016/0014-2999(88)90317-2. [DOI] [PubMed] [Google Scholar]
  31. Scherman D., Henry J. P. Reserpine binding to bovine chromaffin granule membranes. Characterization and comparison with dihydrotetrabenazine binding. Mol Pharmacol. 1984 Jan;25(1):113–122. [PubMed] [Google Scholar]
  32. Scherman D., Henry J. P. Role of the proton electrochemical gradient in monoamine transport by bovine chromaffin granules. Biochim Biophys Acta. 1980 Oct 2;601(3):664–677. doi: 10.1016/0005-2736(80)90567-2. [DOI] [PubMed] [Google Scholar]
  33. Scherman D., Jaudon P., Henry J. P. Liaison d'un dérivé de la tétrabénazine au transporteur des monoamines de la membrane des granules chromaffines. C R Seances Acad Sci III. 1981 Oct 5;293(4):221–224. [PubMed] [Google Scholar]
  34. Scherman D., Raisman R., Ploska A., Agid Y. [3H]dihydrotetrabenazine, a new in vitro monoaminergic probe for human brain. J Neurochem. 1988 Apr;50(4):1131–1136. doi: 10.1111/j.1471-4159.1988.tb10583.x. [DOI] [PubMed] [Google Scholar]
  35. Schuldiner S. A molecular glimpse of vesicular monoamine transporters. J Neurochem. 1994 Jun;62(6):2067–2078. doi: 10.1046/j.1471-4159.1994.62062067.x. [DOI] [PubMed] [Google Scholar]
  36. Schägger H., von Jagow G. Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal Biochem. 1987 Nov 1;166(2):368–379. doi: 10.1016/0003-2697(87)90587-2. [DOI] [PubMed] [Google Scholar]
  37. Stern-Bach Y., Greenberg-Ofrath N., Flechner I., Schuldiner S. Identification and purification of a functional amine transporter from bovine chromaffin granules. J Biol Chem. 1990 Mar 5;265(7):3961–3966. [PubMed] [Google Scholar]
  38. Suchi R., Stern-Bach Y., Gabay T., Schuldiner S. Covalent modification of the amine transporter with N,N'-dicyclohexylcarbodiimide. Biochemistry. 1991 Jul 2;30(26):6490–6494. doi: 10.1021/bi00240a020. [DOI] [PubMed] [Google Scholar]
  39. Surratt C. K., Persico A. M., Yang X. D., Edgar S. R., Bird G. S., Hawkins A. L., Griffin C. A., Li X., Jabs E. W., Uhl G. R. A human synaptic vesicle monoamine transporter cDNA predicts posttranslational modifications, reveals chromosome 10 gene localization and identifies TaqI RFLPs. FEBS Lett. 1993 Mar 8;318(3):325–330. doi: 10.1016/0014-5793(93)80539-7. [DOI] [PubMed] [Google Scholar]
  40. Vincent M. S., Near J. A. Purification of a [3H]dihydrotetrabenazine-binding protein from bovine adrenal medulla. Mol Pharmacol. 1991 Dec;40(6):889–894. [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES