Proteins with Glycosylphosphatidylinositol (GPI) Signal Sequences Have Divergent Fates during a GPI Deficiency
GPIs ARE ESSENTIAL FOR NUCLEAR DIVISION IN TRYPANOSOMA CRUZI*
Abstract
Glycosylphosphatidylinositols (GPIs) are membrane anchors for cell surface proteins of several major protozoan parasites of humans, including Trypanosoma cruzi, the causative agent of Chagas’ disease. To investigate the general role of GPIs inT. cruzi, we generated GPI-deficient parasites by heterologous expression of T. bruceiGPI-phospholipase C. Putative protein-GPI intermediates were depleted, causing the biochemical equivalent of a dominant-negative loss of function mutation in the GPI pathway. Cell surface expression of major GPI-anchored proteins was diminished in GPI-deficient T. cruzi. Four proteins that are normally GPI-anchored in T. cruzi exhibited different fates during the GPI shortage; Ssp-4 and p75 were secreted prematurely, while protease gp50/55 and p60 were degraded intracellularly. These observations demonstrate that secretion and intracellular degradation of GPI-anchored proteins may occur in the same genetic background during a GPI deficiency. We postulate that the interaction between a protein-GPI transamidase and the COOH-terminal GPI signal sequence plays a pivotal role in determining the fate of these proteins.
At a nonpermissive GPI deficiency, T. cruzi amastigotes inside mammalian cells replicated their single kinetoplast but failed at mitosis. Hence, in these protozoans, GPIs appear to be essential for nuclear division, but not for mitochondrial duplication.
Footnotes
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↵* This work was supported in part by National Institutes of Health Grants AI 33383 and AI 33106 (to K. M.-W and R. L. T, respectively), by World Health Organization/TDR Grant 940352, and by a biotechnology grant from the University of Georgia Research Foundation (to R. L. T. and K. M.-W.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵‡ Recipient of a Burroughs Wellcome Fund Scholar Award in Molecular Parasitology.
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↵§ Burroughs Wellcome Fund New Investigator in Molecular Parasitology. To whom correspondence should be addressed: Department of Cellular Biology, 724, Biological Sciences Bldg., University of Georgia, Athens, GA 30602. Tel.: 706-542-3355; Fax: 706-542-4271; E-mail:mensawil{at}cellmate.cb.uga.edu.
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↵1 The abbreviations used are: GPI, glycosylphosphatidylinositol; AHM, 2,5-anhydromannitol; EtN, ethanolamine; GPI-PLC, glycosylphosphatidylinositol phospholipase C; GlcN, glucosamine; HPTLC, high performance thin layer chromatography; Man, mannose; Man3-GlcN-PI, Manα1–2Manα1–6Manα1–4GlcNα1–6inositol-1-phosphoglycerolipid; PGTase, protein-GPI transamidase; PI, phosphatidylinositol; PI-PLC, phosphatidylinositol-specific phospholipase C from Bacillus cereus; PAGE, polyacrylamide gel electrophoresis; VSG, variant surface glycoprotein of T. brucei; FBS, fetal bovine serum; JBAM, jack bean α-mannosidase; PBS, phosphate-buffered saline; ER, endoplasmic reticulum; PLA2, phospholipase A2; SAPA, shed acute phase antigen.
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↵2 M. A. M. Santos and R. L. Tarleton, unpublished data.
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↵3 N. Garg, K. Mensa-Wilmot, and R. L. Tarleton, submitted for publication.
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↵4 J. C. Morris and K. Mensa-Wilmot, submitted for publication.
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- Received January 16, 1997.
- Revision received March 6, 1997.
