Stage-specific expression and targeting of cyst wall protein-green fluorescent protein chimeras in Giardia

Hehl, A. B., Marti, M. and Köhler, P. (2000) Stage-specific expression and targeting of cyst wall protein-green fluorescent protein chimeras in Giardia. Molecular Biology of the Cell, 11(5), pp. 1789-1800. (doi:10.1091/mbc.11.5.1789) (PMID:10793152) (PMCID:PMC14884)

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Abstract

In preparation for being shed into the environment as infectious cysts, trophozoites of Giardia spp. synthesize and deposit large amounts of extracellular matrix into a resistant extracellular cyst wall. Functional aspects of this developmentally regulated process were investigated by expressing a series of chimeric cyst wall protein 1 (CWP1)–green fluorescent protein (GFP) reporter proteins. It was demonstrated that a short 110 bp 5′ flanking region of the CWP1 gene harbors all necessary cis-DNA elements for strictly encystation-specific expression of a reporter during in vitro encystation, whereas sequences in the 3′ flanking region are involved in modulation of steady-state levels of its mRNA during encystation. Encysting Giardia expressing CWP1–GFP chimeras showed formation and maturation of labeled dense granule-like vesicles and subsequent incorporation of GFP-tagged protein into the cyst wall, dependent on which domains of CWP1 were included. The N-terminal domain of CWP1 was required for targeting GFP to regulated compartments of the secretory apparatus, whereas a central domain containing leucine-rich repeats mediated association of the chimera with the extracellular cyst wall. We show that analysis of protein transport using GFP-tagged molecules is feasible in an anaerobic organism and provides a useful tool for investigating the organization of primitive eukaryotic vesicular transport.

Item Type:Articles
Status:Published
Refereed:Yes
Glasgow Author(s) Enlighten ID:Marti, Professor Matthias
Authors: Hehl, A. B., Marti, M., and Köhler, P.
College/School:College of Medical Veterinary and Life Sciences > Institute of Infection Immunity and Inflammation
Journal Name:Molecular Biology of the Cell
Publisher:American Society for Cell Biology
ISSN:1059-1524
ISSN (Online):1939-4586
Copyright Holders:Copyright © 2000 The American Society for Cell Biology
First Published:First published in Molecular Biology of the Cell 11(5):1789-1800
Publisher Policy:Reproduced under a Creative Commons License

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