Hostname: page-component-cd9895bd7-7cvxr Total loading time: 0 Render date: 2024-12-27T18:45:07.341Z Has data issue: false hasContentIssue false
  • English
  • Français

Identification of a 44 kDa protein localized within the endoplasmic reticulum of Trypanosoma brucei brucei

Published online by Cambridge University Press:  06 April 2009

D. Nandan ,
C. W. Wells ,
D. Ndegwa  and
T. W. Pearson
D. Nandan
Affiliation:
International Laboratory for Research on Animal Diseases, P.O. Box 30709, Nairobi, Kenya
C. W. Wells
Affiliation:
International Laboratory for Research on Animal Diseases, P.O. Box 30709, Nairobi, Kenya
D. Ndegwa
Affiliation:
International Laboratory for Research on Animal Diseases, P.O. Box 30709, Nairobi, Kenya
T. W. Pearson
Affiliation:
Department of Biochemistry and Microbiology, University of Victoria, Victoria, British Columbia, CanadaV8W 3P6
Get access Rights & Permissions [Opens in a new window]

Summary

Immunoaffinity chromatography and gel electrophoresis were used to isolate a 44 kDa protein that was bound to a 72 kDa chaperone in Trypanosoma brucei brucei. A polyclonal antiserum to the 44 kDa protein was raised in rats and employed in conjunction with chromatography using DEAE-cellulose, Sephacryl S-300, and hydroxyapatite to purify the protein from membranes of bloodstream forms of the trypanosomes. Immunoblot analysis using this antiserum revealed a protein doublet of 44/45 kDa in T. b. brucei and a single protein band of 53 kDa in almost equivalent amounts throughout the life-cycle stages of T. congolense. Indirect immunofluorescence using affinity-purified antibodies specific for the 44 kDa protein showed labelling of the perinuclear area and reticular system extending throughout the parasites, suggesting that this protein was located in the endoplasmic reticulum. Localization of the 44 kDa molecule in the endoplasmic reticulum was confirmed by immunoelectron microscopy. Protease protection experiments demonstrated that the epitopes bound by antibody were buried within the membrane or towards the lumenal face of the endoplasmic reticulum. Ruthenium Red overlay of nitrocellulose blots containing the 44/45 kDa doublet suggested that the molecules have the potential to bind calcium. The N-terminal amino acid sequence of the 44 kDa protein showed no sequence similarity to any proteins in the database.

Keywords

African trypanosomesendoplasmic reticulumcalcium bindingmembrane proteinchaperones
Type
Research Article
Information
Parasitology , Volume 111 , Issue 3 , September 1995 , pp. 313 - 323
Copyright
Copyright © Cambridge University Press 1995

Access options

Get access to the full version of this content by using one of the access options below. (Log in options will check for institutional or personal access. Content may require purchase if you do not have access.)

References

REFERENCES

Bangs, J. D., Andrews, N., Hart, G. W. & Englund, P. T. (1986). Posttranslation modification and intracellular transport of a trypanosome variant surface glycoprotein. Journal of Cell Biology 103, 255–63.CrossRefGoogle ScholarPubMed
Bangs, J. D., Uyetake, L. M. J., Balber, A. E. & Boothroyd, J. C. (1993). Molecular cloning and cellular localization of a BiP homologue in Trypanosoma brucei. Journal of Cell Science 105, 1101–13.CrossRefGoogle ScholarPubMed
Bienen, E. J., Webster, P. & Fish, W. R. (1991). Trypanosoma (Nannomonas) congolense: changes in respiratory metabolism in the life cycle. Experimental Parasitology 73, 403–12.CrossRefGoogle ScholarPubMed
Boulange, A. & Authie, E. (1994). A 69 kDa immunodominant antigen of Trypanosoma (Nannomonas) congolense is homologous to immunoglobulin heavy chain binding protein (BiP). Parasitology 109, 163–73.CrossRefGoogle ScholarPubMed
Brun, R. & Schonenberger, M. (1979). Cultivation and in vitro cloning of procyclic culture forms of Trypanosoma brucei in a semi-defined medium. Acta Tropica 36, 289–92.Google Scholar
Burleigh, B. A., Wells, C. W., Clark, M. W. & Gardiner, P. R. (1993). An integral membrane glycoprotein associated with an endocytic compartment of Trypanosoma vivax: identification and partial characterization. Journal of Cell Biology 120, 339–52.CrossRefGoogle ScholarPubMed
Charuk, J. H. M., Pirroglia, C. A. & Reithmeier, R. A. F. (1990). Interaction of ruthenium red with Ca2+-binding proteins. Analytical Biochemistry 188, 123–31.CrossRefGoogle ScholarPubMed
Edman, J. C., Ellis, L., Blacher, R. W., Roth, R. A. & Rutter, W. J. (1985). Sequence of protein disulphide isomerase and implications of its relationship to thioredoxin. Nature, London 317, 267–70.CrossRefGoogle ScholarPubMed
Fish, W. R., Muriuki, C. W., Muthiani, A. M., Grab, D. J. & Lonsdale-Eccles, J. D. (1989). Disulfide bond involvement in the maintenance of the cryptic nature of the cross-reacting determinants of metacyclic forms of Trypanosoma congolense. Biochemistry 28, 5415–21.CrossRefGoogle ScholarPubMed
Gething, M. J. & Sambrook, J. (1992). Protein folding in the cell. Nature, London 355, 33–45.CrossRefGoogle ScholarPubMed
Grab, D. J. & Bwayo, J. J. (1982). Isopyenic isolation of African trypanosomes on percoll gradients formed in situ. Acta Tropica 39, 363–6.Google Scholar
Grab, D. J., Shaw, M. K., Wells, C. W., Russo, D. C. W., Verjee, Y., Webster, P., Naessens, J. & Fish, W. R. (1993). The transferrin receptor in African trypanosomes: identification, partial characterization and subcellular localisation. European Journal of Cell Biology 62, 114–26.Google Scholar
Grab, D. J., Webster, P., Ito, S., Fish, W. R., Verjee, Y. & Lonsdale-Eccles, J. D. (1987). Subcellular localisation of a variable surface glycoprotein phosphotidylinositol-specific phospholipase-C in African trypanosomes. Journal of Cell Biology 105, 737–46.CrossRefGoogle Scholar
Haghighat, N. & Ruben, L. (1992). Purification of novel calcium binding proteins from Trypanosoma brucei: properties of 22-, 24- and 38-kiloDalton proteins. Molecular and Biochemical Parasitology 51, 99–110.CrossRefGoogle ScholarPubMed
Hirumi, H., Doyle, J. J. & Hirumi, K. (1977). African trypanosomes. Cultivation of animal infective Trypanosoma brucei brucei in vitro. Science 196, 992–4.CrossRefGoogle ScholarPubMed
Hsu, M. P., Muhich, M. & Boothroyd, J. C. (1989). A developmentally regulated gene of trypanosomes encodes a homologue of rat protein disulfide isomerase and phosphoinositol-phospholipase C. Biochemistry 28, 6440–6.CrossRefGoogle ScholarPubMed
Hurtely, S. M. & Helenius, A. (1989). Protein oligomerization in the endoplasma: reticulum. Annual Review of Cell Biology 5, 277–307.CrossRefGoogle Scholar
Kivirikko, K. I. & Myllyla, R. (1982). Posttranslational enzymes in the biosynthesis of collagen: intracellular enzymes. In Methods in Enzymology (ed. Cunningham, L. W. & Frederiksen, D. W.), Vol. 82A, pp. 245–304. New York: Academic Press.Google Scholar
Kornfeld, R. & Kornfeld, S. (1985). Assembly of asparagine-linked oligosaccharides. Annual Review of Biochemistry 54, 631–64.CrossRefGoogle ScholarPubMed
Laemmli, U. K. (1970). Cleavage of the structural proteins during the assembly of the head of bapteriophage T4. Nature, London 227, 680–5.CrossRefGoogle ScholarPubMed
Lanham, S. M. & Godfrey, D. G. (1970). Isolation of salivarian trypanosomes from man and other mammals using DEAE-cellulose. Experimental Parasitology 28, 521–34.CrossRefGoogle ScholarPubMed
Lowry, O. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. (1951). Protein measurement with the Folin phenol reagent: Journal of Biological Chemistry 193, 265–75CrossRefGoogle ScholarPubMed
Milner, R. E., Baksh, S., Shemanko, C., Carpenter, M. R., Smillie, L., Vance, J. E. & Michalak, M. (1991). Calreticulin, and not calsequestrin, is the major calcium binding protein of smooth muscle sarcoplasmic reticulum and liver endoplasmic reticulum. Journal of Biological Chemistry 266, 7155–65.CrossRefGoogle Scholar
Munro, S. & Pelham, H. R. B. (1986). An Hsp 70-like protein in the ER: identity with the 78 kDa glucose regulated protein and immunoglobulin heavy chain binding protein. Cell 46, 291–300.CrossRefGoogle Scholar
Nandan, D., Ball, E. H. & Sanwal, B. D. (1990). Two stress proteins of the endoplasmic reticulum bind denatured collagen. Biochemistry and Cell Biology 68, 1057–61.CrossRefGoogle ScholarPubMed
Nandan, D., Daubenberger, C., Mpimbza, G. & Pearson, T. W. (1994). A rapid, single step purification method for immunogenic members of the hsp 70 family: validation and application. Journal of Immunological Methods 176, 255–63.CrossRefGoogle Scholar
Parsell, D. A. & Lindquist, S. (1993). The function of heat shock proteins in stress tolerance: degradation and reactivation of damaged proteins. Annual Review of Genetics 27, 437–96CrossRefGoogle ScholarPubMed
Rawlings, D. J. & Kaslow, D. C. (1992). A novel membrane-associated EF-hand calcium binding protein in Plasmodium falciparttm. Journal of Biological Chemistry 267, 3976–82.CrossRefGoogle Scholar
Ruben, L., Hutchinson, A. & Moehlman, H. (1991). Calcium homeostasis in Trypanosoma brucei. Journal of Biological Chemistry 266, 24351–8.CrossRefGoogle ScholarPubMed
Sorger, P. K. & Pelham, H. R. B. (1988). The glucoseregulated protein grp 94 is related to heat shock protein hsp 90. Journal of Molecular Biology 194, 341–4.CrossRefGoogle Scholar
Terasaki, M. & Sarget, C. (1991). Demonstration of calcium uptake and release by sea urchin egg cortical endoplasmic reticulum. Journal of Cell Biology 115, 1031–7.CrossRefGoogle ScholarPubMed
Towbin, H., Staehelin, T. & Gordon, J. (1979). Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proceedings of the National Academy of Sciences, USA 76, 4350–4.CrossRefGoogle ScholarPubMed
Wada, I., Rindress, D., Cameron, P. H., Ou, W. J., Doherty, J. J., Louvard, D., Bell, A. W., Dignard, D., Thomas, D. Y. & Bergeron, J. J. M. (1991). SSR and associated calnexin are major calcium binding proteins of the endoplasmic reticulum membrane. Journal of Biological Chemistry 226, 19599–610.CrossRefGoogle Scholar
Wu, Y., Haghighat, N. G. & Ruben, L. (1992). The prominent calcimedins from Trypanosoma brucei comprise a family of flagellar EF-hand calciumbinding proteins. Biochemical Journal 287, 187–93.CrossRefGoogle Scholar