Skip to main content
NCBI home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1987 Aug 1;105(2):659–668. doi: 10.1083/jcb.105.2.659

In vitro mutagenesis of trypsinogen: role of the amino terminus in intracellular protein targeting to secretory granules

PMCID: PMC2114750  PMID: 3040770

Abstract

The mouse anterior pituitary tumor cell line, AtT-20, targets secretory proteins into two distinct intracellular pathways. When the DNA that encodes trypsinogen is introduced into AtT-20 cells, the protein is sorted into the regulated secretory pathway as efficiently as the endogenous peptide hormone ACTH. In this study we have used double- label immunoelectron microscopy to demonstrate that trypsinogen colocalizes in the same secretory granules as ACTH. In vitro mutagenesis was used to test whether the information for targeting trypsinogen to the secretory granules resides at the amino (NH2) terminus of the protein. Mutations were made in the DNA that encodes trypsinogen, and the mutant proteins were expressed in AtT-20 cells to determine whether intracellular targeting could be altered. Replacing the trypsinogen signal peptide with that of the kappa-immunoglobulin light chain, a constitutively secreted protein, does not alter targeting to the regulated secretory pathway. In addition, deletion of the NH2-terminal "pro" sequence of trypsinogen has virtually no effect on protein targeting. However, this deletion does affect the signal peptidase cleavage site, and as a result the enzymatic activity of the truncated trypsin protein is abolished. We conclude that neither the signal peptide nor the 12 NH2-terminal amino acids of trypsinogen are essential for sorting to the regulated secretory pathway of AtT-20 cells.

Full Text

The Full Text of this article is available as a PDF (1.7 MB).

Selected References

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

  1. Bassetti M., Spada A., Arosio M., Vallar L., Brina M., Giannattasio G. Morphological studies on mixed growth hormone (GH)- and prolactin (PRL)-secreting human pituitary adenomas. Coexistence of GH and PRL in the same secretory granule. J Clin Endocrinol Metab. 1986 Jun;62(6):1093–1100. doi: 10.1210/jcem-62-6-1093. [DOI] [PubMed] [Google Scholar]
  2. Bendayan M. Double immunocytochemical labeling applying the protein A-gold technique. J Histochem Cytochem. 1982 Jan;30(1):81–85. doi: 10.1177/30.1.6172469. [DOI] [PubMed] [Google Scholar]
  3. Bergman L. W., Kuehl W. M. Formation of an intrachain disulfide bond on nascent immunoglobulin light chains. J Biol Chem. 1979 Sep 25;254(18):8869–8876. [PubMed] [Google Scholar]
  4. Blobel G. Intracellular protein topogenesis. Proc Natl Acad Sci U S A. 1980 Mar;77(3):1496–1500. doi: 10.1073/pnas.77.3.1496. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bode W., Huber R. Induction of the bovine trypsinogen-trypsin transition by peptides sequentially similar to the N-terminus of trypsin. FEBS Lett. 1976 Oct 1;68(2):231–236. doi: 10.1016/0014-5793(76)80443-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bode W., Schwager P., Huber R. The transition of bovine trypsinogen to a trypsin-like state upon strong ligand binding. The refined crystal structures of the bovine trypsinogen-pancreatic trypsin inhibitor complex and of its ternary complex with Ile-Val at 1.9 A resolution. J Mol Biol. 1978 Jan 5;118(1):99–112. doi: 10.1016/0022-2836(78)90246-2. [DOI] [PubMed] [Google Scholar]
  7. Burgess T. L., Craik C. S., Kelly R. B. The exocrine protein trypsinogen is targeted into the secretory granules of an endocrine cell line: studies by gene transfer. J Cell Biol. 1985 Aug;101(2):639–645. doi: 10.1083/jcb.101.2.639. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Burgess T. L., Kelly R. B. Sorting and secretion of adrenocorticotropin in a pituitary tumor cell line after perturbation of the level of a secretory granule-specific proteoglycan. J Cell Biol. 1984 Dec;99(6):2223–2230. doi: 10.1083/jcb.99.6.2223. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Burnette W. N. "Western blotting": electrophoretic transfer of proteins from sodium dodecyl sulfate--polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Anal Biochem. 1981 Apr;112(2):195–203. doi: 10.1016/0003-2697(81)90281-5. [DOI] [PubMed] [Google Scholar]
  10. Comb M., Liston D., Martin M., Rosen H., Herbert E. Expression of the human proenkephalin gene in mouse pituitary cells: accurate and efficient mRNA production and proteolytic processing. EMBO J. 1985 Dec 1;4(12):3115–3122. doi: 10.1002/j.1460-2075.1985.tb04053.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Copeland C. S., Doms R. W., Bolzau E. M., Webster R. G., Helenius A. Assembly of influenza hemagglutinin trimers and its role in intracellular transport. J Cell Biol. 1986 Oct;103(4):1179–1191. doi: 10.1083/jcb.103.4.1179. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Craik C. S., Largman C., Fletcher T., Roczniak S., Barr P. J., Fletterick R., Rutter W. J. Redesigning trypsin: alteration of substrate specificity. Science. 1985 Apr 19;228(4697):291–297. doi: 10.1126/science.3838593. [DOI] [PubMed] [Google Scholar]
  13. Davey J., Dimmock N. J., Colman A. Identification of the sequence responsible for the nuclear accumulation of the influenza virus nucleoprotein in Xenopus oocytes. Cell. 1985 Mar;40(3):667–675. doi: 10.1016/0092-8674(85)90215-6. [DOI] [PubMed] [Google Scholar]
  14. Dingwall C., Laskey R. A. Protein import into the cell nucleus. Annu Rev Cell Biol. 1986;2:367–390. doi: 10.1146/annurev.cb.02.110186.002055. [DOI] [PubMed] [Google Scholar]
  15. Douglas M. G., Geller B. L., Emr S. D. Intracellular targeting and import of an F1-ATPase beta-subunit-beta-galactosidase hybrid protein into yeast mitochondria. Proc Natl Acad Sci U S A. 1984 Jul;81(13):3983–3987. doi: 10.1073/pnas.81.13.3983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Evans E. A., Gilmore R., Blobel G. Purification of microsomal signal peptidase as a complex. Proc Natl Acad Sci U S A. 1986 Feb;83(3):581–585. doi: 10.1073/pnas.83.3.581. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Fehlhammer H., Bode W., Huber R. Crystal structure of bovine trypsinogen at 1-8 A resolution. II. Crystallographic refinement, refined crystal structure and comparison with bovine trypsin. J Mol Biol. 1977 Apr 25;111(4):415–438. doi: 10.1016/s0022-2836(77)80062-4. [DOI] [PubMed] [Google Scholar]
  18. Fersht A. R. Conformational equilibria in -and -chymotrypsin. The energetics and importance of the salt bridge. J Mol Biol. 1972 Mar 14;64(2):497–509. doi: 10.1016/0022-2836(72)90513-x. [DOI] [PubMed] [Google Scholar]
  19. Fumagalli G., Zanini A. In cow anterior pituitary, growth hormone and prolactin can be packed in separate granules of the same cell. J Cell Biol. 1985 Jun;100(6):2019–2024. doi: 10.1083/jcb.100.6.2019. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Garoff H. Using recombinant DNA techniques to study protein targeting in the eucaryotic cell. Annu Rev Cell Biol. 1985;1:403–445. doi: 10.1146/annurev.cb.01.110185.002155. [DOI] [PubMed] [Google Scholar]
  21. Gething M. J., McCammon K., Sambrook J. Expression of wild-type and mutant forms of influenza hemagglutinin: the role of folding in intracellular transport. Cell. 1986 Sep 12;46(6):939–950. doi: 10.1016/0092-8674(86)90076-0. [DOI] [PubMed] [Google Scholar]
  22. Glabe C. G., Hanover J. A., Lennarz W. J. Glycosylation of ovalbumin nascent chains. The spatial relationship between translation and glycosylation. J Biol Chem. 1980 Oct 10;255(19):9236–9242. [PubMed] [Google Scholar]
  23. Gorman C. M., Merlino G. T., Willingham M. C., Pastan I., Howard B. H. The Rous sarcoma virus long terminal repeat is a strong promoter when introduced into a variety of eukaryotic cells by DNA-mediated transfection. Proc Natl Acad Sci U S A. 1982 Nov;79(22):6777–6781. doi: 10.1073/pnas.79.22.6777. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Guillet J. G., Lai M. Z., Briner T. J., Smith J. A., Gefter M. L. Interaction of peptide antigens and class II major histocompatibility complex antigens. Nature. 1986 Nov 20;324(6094):260–262. doi: 10.1038/324260a0. [DOI] [PubMed] [Google Scholar]
  25. Gumbiner B., Kelly R. B. Two distinct intracellular pathways transport secretory and membrane glycoproteins to the surface of pituitary tumor cells. Cell. 1982 Jan;28(1):51–59. doi: 10.1016/0092-8674(82)90374-9. [DOI] [PubMed] [Google Scholar]
  26. HARTLEY B. S. AMINO-ACID SEQUENCE OF BOVINE CHYMOTRYPSINOGEN-A. Nature. 1964 Mar 28;201:1284–1287. doi: 10.1038/2011284a0. [DOI] [PubMed] [Google Scholar]
  27. Hager D. A., Burgess R. R. Elution of proteins from sodium dodecyl sulfate-polyacrylamide gels, removal of sodium dodecyl sulfate, and renaturation of enzymatic activity: results with sigma subunit of Escherichia coli RNA polymerase, wheat germ DNA topoisomerase, and other enzymes. Anal Biochem. 1980 Nov 15;109(1):76–86. doi: 10.1016/0003-2697(80)90013-5. [DOI] [PubMed] [Google Scholar]
  28. Hall M. N., Hereford L., Herskowitz I. Targeting of E. coli beta-galactosidase to the nucleus in yeast. Cell. 1984 Apr;36(4):1057–1065. doi: 10.1016/0092-8674(84)90055-2. [DOI] [PubMed] [Google Scholar]
  29. Hanahan D. Studies on transformation of Escherichia coli with plasmids. J Mol Biol. 1983 Jun 5;166(4):557–580. doi: 10.1016/s0022-2836(83)80284-8. [DOI] [PubMed] [Google Scholar]
  30. Hase T., Müller U., Riezman H., Schatz G. A 70-kd protein of the yeast mitochondrial outer membrane is targeted and anchored via its extreme amino terminus. EMBO J. 1984 Dec 20;3(13):3157–3164. doi: 10.1002/j.1460-2075.1984.tb02274.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Hellerman J. G., Cone R. C., Potts J. T., Jr, Rich A., Mulligan R. C., Kronenberg H. M. Secretion of human parathyroid hormone from rat pituitary cells infected with a recombinant retrovirus encoding preproparathyroid hormone. Proc Natl Acad Sci U S A. 1984 Sep;81(17):5340–5344. doi: 10.1073/pnas.81.17.5340. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Horwich A. L., Kalousek F., Mellman I., Rosenberg L. E. A leader peptide is sufficient to direct mitochondrial import of a chimeric protein. EMBO J. 1985 May;4(5):1129–1135. doi: 10.1002/j.1460-2075.1985.tb03750.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Huber R., Kukla D., Bode W., Schwager P., Bartels K., Deisenhofer J., Steigemann W. Structure of the complex formed by bovine trypsin and bovine pancreatic trypsin inhibitor. II. Crystallographic refinement at 1.9 A resolution. J Mol Biol. 1974 Oct 15;89(1):73–101. doi: 10.1016/0022-2836(74)90163-6. [DOI] [PubMed] [Google Scholar]
  34. Hurt E. C., Pesold-Hurt B., Schatz G. The amino-terminal region of an imported mitochondrial precursor polypeptide can direct cytoplasmic dihydrofolate reductase into the mitochondrial matrix. EMBO J. 1984 Dec 20;3(13):3149–3156. doi: 10.1002/j.1460-2075.1984.tb02272.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Jackson R. C., Blobel G. Post-translational cleavage of presecretory proteins with an extract of rough microsomes from dog pancreas containing signal peptidase activity. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5598–5602. doi: 10.1073/pnas.74.12.5598. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Johnson L. M., Bankaitis V. A., Emr S. D. Distinct sequence determinants direct intracellular sorting and modification of a yeast vacuolar protease. Cell. 1987 Mar 13;48(5):875–885. doi: 10.1016/0092-8674(87)90084-5. [DOI] [PubMed] [Google Scholar]
  37. Kelley D. E., Coleclough C., Perry R. P. Functional significance and evolutionary development of the 5'-terminal regions of immunoglobulin variable-region genes. Cell. 1982 Jun;29(2):681–689. doi: 10.1016/0092-8674(82)90184-2. [DOI] [PubMed] [Google Scholar]
  38. Kelly R. B. Pathways of protein secretion in eukaryotes. Science. 1985 Oct 4;230(4721):25–32. doi: 10.1126/science.2994224. [DOI] [PubMed] [Google Scholar]
  39. Kossiakoff A. A., Chambers J. L., Kay L. M., Stroud R. M. Structure of bovine trypsinogen at 1.9 A resolution. Biochemistry. 1977 Feb 22;16(4):654–664. doi: 10.1021/bi00623a016. [DOI] [PubMed] [Google Scholar]
  40. Kreis T. E., Lodish H. F. Oligomerization is essential for transport of vesicular stomatitis viral glycoprotein to the cell surface. Cell. 1986 Sep 12;46(6):929–937. doi: 10.1016/0092-8674(86)90075-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Mains R. E., Eipper B. A. Biosynthesis of adrenocorticotropic hormone in mouse pituitary tumor cells. J Biol Chem. 1976 Jul 10;251(13):4115–4120. [PubMed] [Google Scholar]
  42. Mains R. E., Eipper B. A. Coordinate, equimolar secretion of smaller peptide products derived from pro-ACTH/endorphin by mouse pituitary tumor cells. J Cell Biol. 1981 Apr;89(1):21–28. doi: 10.1083/jcb.89.1.21. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Maroux S., Baratti J., Desnuelle P. Purification and specificity of porcine enterokinase. J Biol Chem. 1971 Aug 25;246(16):5031–5039. [PubMed] [Google Scholar]
  44. Mollay C., Vilas U., Kreil G. Cleavage of honeybee prepromelittin by an endoprotease from rat liver microsomes: identification of intact signal peptide. Proc Natl Acad Sci U S A. 1982 Apr;79(7):2260–2263. doi: 10.1073/pnas.79.7.2260. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Moore H. H., Kelly R. B. Re-routing of a secretory protein by fusion with human growth hormone sequences. Nature. 1986 May 22;321(6068):443–446. doi: 10.1038/321443a0. [DOI] [PubMed] [Google Scholar]
  46. Moore H. P., Kelly R. B. Secretory protein targeting in a pituitary cell line: differential transport of foreign secretory proteins to distinct secretory pathways. J Cell Biol. 1985 Nov;101(5 Pt 1):1773–1781. doi: 10.1083/jcb.101.5.1773. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Moore H. P., Walker M. D., Lee F., Kelly R. B. Expressing a human proinsulin cDNA in a mouse ACTH-secreting cell. Intracellular storage, proteolytic processing, and secretion on stimulation. Cell. 1983 Dec;35(2 Pt 1):531–538. doi: 10.1016/0092-8674(83)90187-3. [DOI] [PubMed] [Google Scholar]
  48. Moreland R. B., Nam H. G., Hereford L. M., Fried H. M. Identification of a nuclear localization signal of a yeast ribosomal protein. Proc Natl Acad Sci U S A. 1985 Oct;82(19):6561–6565. doi: 10.1073/pnas.82.19.6561. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Palade G. Intracellular aspects of the process of protein synthesis. Science. 1975 Aug 1;189(4200):347–358. doi: 10.1126/science.1096303. [DOI] [PubMed] [Google Scholar]
  50. Robinson N. C., Neurath H., Walsh K. A. The relation of the -amino group of trypsin to enzyme function and zymogen activation. Biochemistry. 1973 Jan 30;12(3):420–426. doi: 10.1021/bi00727a010. [DOI] [PubMed] [Google Scholar]
  51. Schauer I., Emr S., Gross C., Schekman R. Invertase signal and mature sequence substitutions that delay intercompartmental transport of active enzyme. J Cell Biol. 1985 May;100(5):1664–1675. doi: 10.1083/jcb.100.5.1664. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Schreier P. H., Seftor E. A., Schell J., Bohnert H. J. The use of nuclear-encoded sequences to direct the light-regulated synthesis and transport of a foreign protein into plant chloroplasts. EMBO J. 1985 Jan;4(1):25–32. doi: 10.1002/j.1460-2075.1985.tb02312.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Scrimger S. T., Hofmann T. The involvement of the amino-terminal amino acid in the activity of pancreatic proteases. II. The effects of nitrous acid on trypsin. J Biol Chem. 1967 May 25;242(10):2528–2533. [PubMed] [Google Scholar]
  54. Silver P. A., Keegan L. P., Ptashne M. Amino terminus of the yeast GAL4 gene product is sufficient for nuclear localization. Proc Natl Acad Sci U S A. 1984 Oct;81(19):5951–5955. doi: 10.1073/pnas.81.19.5951. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Smeekens S., Bauerle C., Hageman J., Keegstra K., Weisbeek P. The role of the transit peptide in the routing of precursors toward different chloroplast compartments. Cell. 1986 Aug 1;46(3):365–375. doi: 10.1016/0092-8674(86)90657-4. [DOI] [PubMed] [Google Scholar]
  56. Southern P. J., Berg P. Transformation of mammalian cells to antibiotic resistance with a bacterial gene under control of the SV40 early region promoter. J Mol Appl Genet. 1982;1(4):327–341. [PubMed] [Google Scholar]
  57. Stroud R. M., Kay L. M., Dickerson R. E. The structure of bovine trypsin: electron density maps of the inhibited enzyme at 5 Angstrom and at 2-7 Angstron resolution. J Mol Biol. 1974 Feb 25;83(2):185–208. doi: 10.1016/0022-2836(74)90387-8. [DOI] [PubMed] [Google Scholar]
  58. Tartakoff A. M. Mutations that influence the secretory path in animal cells. Biochem J. 1983 Oct 15;216(1):1–9. doi: 10.1042/bj2160001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Tartakoff A., Vassalli P., Détraz M. Comparative studies of intracellular transport of secretory proteins. J Cell Biol. 1978 Dec;79(3):694–707. doi: 10.1083/jcb.79.3.694. [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. Valentino K. L., Crumrine D. A., Reichardt L. F. Lowicryl K4M embedding of brain tissue for immunogold electron microscopy. J Histochem Cytochem. 1985 Sep;33(9):969–973. doi: 10.1177/33.9.2991364. [DOI] [PubMed] [Google Scholar]
  61. Valls L. A., Hunter C. P., Rothman J. H., Stevens T. H. Protein sorting in yeast: the localization determinant of yeast vacuolar carboxypeptidase Y resides in the propeptide. Cell. 1987 Mar 13;48(5):887–897. doi: 10.1016/0092-8674(87)90085-7. [DOI] [PubMed] [Google Scholar]
  62. Van den Broeck G., Timko M. P., Kausch A. P., Cashmore A. R., Van Montagu M., Herrera-Estrella L. Targeting of a foreign protein to chloroplasts by fusion to the transit peptide from the small subunit of ribulose 1,5-bisphosphate carboxylase. 1985 Jan 31-Feb 6Nature. 313(6001):358–363. doi: 10.1038/313358a0. [DOI] [PubMed] [Google Scholar]
  63. Walter P., Gilmore R., Blobel G. Protein translocation across the endoplasmic reticulum. Cell. 1984 Aug;38(1):5–8. doi: 10.1016/0092-8674(84)90520-8. [DOI] [PubMed] [Google Scholar]
  64. Walter P., Jackson R. C., Marcus M. M., Lingappa V. R., Blobel G. Tryptic dissection and reconstitution of translocation activity for nascent presecretory proteins across microsomal membranes. Proc Natl Acad Sci U S A. 1979 Apr;76(4):1795–1799. doi: 10.1073/pnas.76.4.1795. [DOI] [PMC free article] [PubMed] [Google Scholar]
  65. Wychowski C., Benichou D., Girard M. A domain of SV40 capsid polypeptide VP1 that specifies migration into the cell nucleus. EMBO J. 1986 Oct;5(10):2569–2576. doi: 10.1002/j.1460-2075.1986.tb04536.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  66. Zoller M. J., Smith M. Oligonucleotide-directed mutagenesis: a simple method using two oligonucleotide primers and a single-stranded DNA template. DNA. 1984 Dec;3(6):479–488. doi: 10.1089/dna.1.1984.3.479. [DOI] [PubMed] [Google Scholar]
  67. von Heijne G. A new method for predicting signal sequence cleavage sites. Nucleic Acids Res. 1986 Jun 11;14(11):4683–4690. doi: 10.1093/nar/14.11.4683. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from The Journal of Cell Biology are provided here courtesy of The Rockefeller University Press

RESOURCES