Journal of Cell Science, Vol 112, Issue 11 1785-1795, Copyright © 1999 by Company of Biologists

JOURNAL ARTICLES

Phosphorylation of a major GPI-anchored surface protein of Trypanosoma brucei during transport to the plasma membrane

P Butikofer, E Vassella, S Ruepp, M Boschung, G Civenni, T Seebeck, A Hemphill, N Mookherjee, TW Pearson and I Roditi
Institutes of Biochemistry and Molecular Biology, General Microbiology, and Parasitology, University of Bern, Bern, Switzerland. peter.buetikofer@mci.unibe.ch

The surface coat of procyclic forms of Trypanosoma brucei consists of related, internally repetitive glycoproteins known as EP and GPEET procyclins. Previously we showed that the extracellular domain of GPEET is phosphorylated. We now show that phosphorylation of this glycosylphosphatidylinositol-anchored surface protein can be induced in vitro using a procyclic membrane extract. Using antibodies that recognize either the phosphorylated or unphosphorylated form of GPEET, we analyzed their expression during differentiation of bloodstream forms to procyclic forms. Unphosphorylated GPEET, together with EP, was detected in cell lysates 2-4 hours after initiating differentiation whereas phosphorylated GPEET only appeared after 24 hours. Surface expression of EP and both forms of GPEET occurred after 24-48 hours and correlated with the detection of phosphorylated GPEET on immuno-blots. Electron micrographs showed that unphosphorylated GPEET was predominantly in the flagellar pocket whereas the phosphorylated form was distributed over the cell surface. In contrast, expression of a membrane-bound human placental alkaline phosphatase in procyclic forms caused the accumulation of dephosphorylated GPEET on the cell surface, while the phosphorylated form was restricted to the flagellar pocket. A GPEET-Fc fusion protein, which was retained intracellularly, was not phosphorylated. We propose that unphosphorylated GPEET procyclin is transported to a location close to or at the cell surface, most probably the flagellar pocket, where it becomes phosphorylated. To the best of our knowledge, this study represents the first localization of phosphorylated and unphosphorylated forms of a GPI-anchored protein within a cell.

This article has been cited by other articles:

				A. Signorell, J. Jelk, M. Rauch, and P. Butikofer Phosphatidylethanolamine Is the Precursor of the Ethanolamine Phosphoglycerol Moiety Bound to Eukaryotic Elongation Factor 1A J. Biol. Chem., July 18, 2008; 283(29): 20320 - 20329. [Abstract] [Full Text] [PDF]

				S. Manthri, M L. S Guther, L. Izquierdo, A. Acosta-Serrano, and M. A J Ferguson Deletion of the TbALG3 gene demonstrates site-specific N-glycosylation and N-glycan processing in Trypanosoma brucei Glycobiology, May 1, 2008; 18(5): 367 - 383. [Abstract] [Full Text] [PDF]

				M. L. S. Guther, S. Lee, L. Tetley, A. Acosta-Serrano, and M. A.J. Ferguson GPI-anchored Proteins and Free GPI Glycolipids of Procyclic Form Trypanosoma brucei Are Nonessential for Growth, Are Required for Colonization of the Tsetse Fly, and Are Not the Only Components of the Surface Coat Mol. Biol. Cell, December 1, 2006; 17(12): 5265 - 5274. [Abstract] [Full Text] [PDF]

				J. R. Roper, M. L. S. Guther, J. I. MacRae, A. R. Prescott, I. Hallyburton, A. Acosta-Serrano, and M. A. J. Ferguson The Suppression of Galactose Metabolism in Procylic Form Trypanosoma brucei Causes Cessation of Cell Growth and Alters Procyclin Glycoprotein Structure and Copy Number J. Biol. Chem., May 20, 2005; 280(20): 19728 - 19736. [Abstract] [Full Text] [PDF]

				N. Fankhauser and P. Maser Identification of GPI anchor attachment signals by a Kohonen self-organizing map Bioinformatics, May 1, 2005; 21(9): 1846 - 1852. [Abstract] [Full Text] [PDF]

				E. Vassella, M. Probst, A. Schneider, E. Studer, C. K. Renggli, and I. Roditi Expression of a Major Surface Protein of Trypanosoma brucei Insect Forms Is Controlled by the Activity of Mitochondrial Enzymes Mol. Biol. Cell, September 1, 2004; 15(9): 3986 - 3993. [Abstract] [Full Text] [PDF]

				A.-C. Schlaeppi, T. Malherbe, and P. Butikofer Coordinate Expression of GPEET Procyclin and Its Membrane-associated Kinase in Trypanosoma brucei Procyclic Forms J. Biol. Chem., December 12, 2003; 278(50): 49980 - 49987. [Abstract] [Full Text] [PDF]

				S. Lillico, M. C. Field, P. Blundell, G. H. Coombs, and J. C. Mottram Essential Roles for GPI-anchored Proteins in African Trypanosomes Revealed Using Mutants Deficient in GPI8 Mol. Biol. Cell, March 1, 2003; 14(3): 1182 - 1194. [Abstract] [Full Text] [PDF]

				L. M. Thomson, D. J. Lamont, A. Mehlert, J. D. Barry, and M. A. J. Ferguson Partial Structure of Glutamic Acid and Alanine-rich Protein, a Major Surface Glycoprotein of the Insect Stages of Trypanosoma congolense J. Biol. Chem., December 6, 2002; 277(50): 48899 - 48904. [Abstract] [Full Text] [PDF]

				M. J. McConville, K. A. Mullin, S. C. Ilgoutz, and R. D. Teasdale Secretory Pathway of Trypanosomatid Parasites Microbiol. Mol. Biol. Rev., March 1, 2002; 66(1): 122 - 154. [Abstract] [Full Text] [PDF]

				A. Guha-Niyogi, D. R. Sullivan, and S. J. Turco Glycoconjugate structures of parasitic protozoa Glycobiology, April 1, 2001; 11(4): 45R - 59R. [Abstract] [Full Text] [PDF]

				P. BUTIKOFER, T. MALHERBE, M. BOSCHUNG, and I. RODITI GPI-anchored proteins: now you see 'em, now you don't FASEB J, February 1, 2001; 15(2): 545 - 548. [Abstract] [Full Text] [PDF]

				E. Vassella, J. V. Den Abbeele, P. Bütikofer, C. K. Renggli, A. Furger, R. Brun, and I. Roditi A major surface glycoprotein of Trypanosoma brucei is expressed transiently during development and can be regulated post-transcriptionally by glycerol or hypoxia Genes & Dev., March 1, 2000; 14(5): 615 - 626. [Abstract] [Full Text]

				A. Acosta-Serrano, R. N. Cole, A. Mehlert, M. G.-S. Lee, M. A. J. Ferguson, and P. T. Englund The Procyclin Repertoire of Trypanosoma brucei. IDENTIFICATION AND STRUCTURAL CHARACTERIZATION OF THE GLU-PRO-RICH POLYPEPTIDES J. Biol. Chem., October 15, 1999; 274(42): 29763 - 29771. [Abstract] [Full Text] [PDF]

				M. H. Le Du, T. Stigbrand, M. J. Taussig, A. Menez, and E. A. Stura Crystal Structure of Alkaline Phosphatase from Human Placenta at 1.8 A Resolution. IMPLICATION FOR A SUBSTRATE SPECIFICITY J. Biol. Chem., March 16, 2001; 276(12): 9158 - 9165. [Abstract] [Full Text] [PDF]

				A. Acosta-Serrano, E. Vassella, M. Liniger, C. K. Renggli, R. Brun, I. Roditi, and P. T. Englund The surface coat of procyclic Trypanosoma brucei: Programmed expression and proteolytic cleavage of procyclin in the tsetse fly PNAS, February 13, 2001; 98(4): 1513 - 1518. [Abstract] [Full Text] [PDF]