Stage-specific expression and targeting of cyst wall protein-green fluorescent protein chimeras in Giardia.
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.
Hehl
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AB
Institute of Parasitology, University of Zürich, 8057 Zürich, Switzerland.
Marti
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M
Köhler
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P
D020413:3' Untranslated Regions; D000818:Animals; D001692:Biological Transport; D002451:Cell Compartmentation; D002473:Cell Wall / Q000378:metabolism; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D005872:Giardia / Q000502:physiology*; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D015800:Protozoan Proteins / Q000235:genetics* / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012045:Regulatory Sequences, Nucleic Acid
D020413:3' Untranslated Regions; C094333:CWP1 protein, Giardia lamblia; D008164:Luminescent Proteins; D015800:Protozoan Proteins; D012333:RNA, Messenger; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins