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First published online 3 June 2008
doi: 10.1242/jcs.021154

Journal of Cell Science 121, 2123-2129 (2008)
Published by The Company of Biologists 2008
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Plasmodium falciparum possesses two GRASP proteins that are differentially targeted to the Golgi complex via a higher- and lower-eukaryote-like mechanism

Nicole S. Struck1, Susann Herrmann1, Christine Langer1, Andreas Krueger2, Bernardo J. Foth3, Klemens Engelberg1, Ana L. Cabrera1, Silvia Haase1, Moritz Treeck1, Matthias Marti4, Alan F. Cowman5, Tobias Spielmann1 and Tim W. Gilberger1,*

1 Bernhard Nocht Institute for Tropical Medicine, Malaria II, Bernhard-Nocht-Strasse 74, 20359 Hamburg, Germany
2 German Armed Forces, Bernhard Nocht Institute for Tropical Medicine, 20359 Hamburg, Germany
3 School of Biological Sciences, Nanyang Technological University, 637551, Singapore
4 Department of Immunology and Infectious Diseases, Harvard School of Public Health, Boston, MA, USA
5 The Walter and Eliza Hall Institute of Medical Research, Melbourne 3050, Australia


Figure 1
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  		Fig. 1. Identification of two GRASP populations in P. falciparum. (A) Northern blot analysis using total RNA of wild-type parasites. A P32 labelled grasp-specific probe detects two transcripts of approximately 3.8 kb and 4 kb. (B) RT-PCR analysis. Two PCR products with a size difference of approximately 150 bp are amplified on cDNA (lane 1) using grasp-specific oligonucleotides. Genomic DNA (gDNA) was used as a positive control (lane 2). To exclude non-specific amplification reactions without template were run as negative controls (lanes 3 and 6). To exclude contaminations of the cDNA preparation with gDNA, erd2-specific oligonucleotides were used (lane 4-6). Consistent with the two-intron structure of the erd2 gene (PF13_0280), a size difference is visible between cDNA (660 bp, lane 5) and gDNA (960 bp, lane 6). (C) Detection of two GRASP proteins in parasite extract. Maximum separation of parasite proteins and subsequent western blotting with GRASP-specific antibodies reveal two translation products of ~70 kDa. (D) Schematic representation of the genomic grasp gene and transcript heterogeneity. (Top) Exon 1 (red) encompasses 33 bp and is separated by a 148 bp intron from exon 2 (grey, 1689 bp). The intron possesses a putative start ATG and 63 bp ORF (yellow) in-frame with exon 2. (Middle and bottom) RT-PCR products were cloned and sequenced. Two different cDNA populations were identified and named cDNA grasp1 and cDNA grasp2, representing a spliced and unspliced version of the grasp gene. The deduced N-terminal amino acid sequence of GRASP2 is displayed in one-letter code in yellow and grey. Vertical line represents stop codons within the intron. Relative positions of oligonucleotides used in RT-PCR are presented as red and grey bars.

 

Figure 2
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  		Fig. 2. Phylogenetic analysis of GRASP proteins across the eukaryotic tree of life. This unrooted neighbour-joining tree is based on 53 protein sequences and was estimated from an alignment of GRASP N-terminal domains (~370 amino acids). Numbers at branches indicate statistical support (bootstrap of 300 replicates) of >50% in the corresponding consensus tree. The insects and deuterostomia branch is shown at a magnified scale.

 

Figure 3
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  		Fig. 3. Expression and localisation of GRASP2 (A) Schematic representation of the transfection vector pARL-GRASP1*/2-GFP. The complete genomic grasp sequence (exon 1: red; alternative start ATG and hydrophobic stretch: orange; exon 2: grey) was cloned into the pARL transfection vector in frame with GFP (green). A mutation was introduced into the 5' splice site (*). The human Dhfr selection cassette is displayed in black. Relative positions of oligonucleotides used in RT-PCR are presented as bars. (B) Transcriptional analysis using appropriate oligonucleotides and either cDNA or genomic DNA (gDNA) derived from pARL-GRASP1*/2-GFP-expressing parasites. RT-PCR reveals a single PCR product using cDNA (lane 1) but does not amplify a product on gDNA (lane 2). The absence of any contamination of the cDNA preparation with gDNA was confirmed using erd2-specific oligonucleotides (lane 4-5). (C) Disruption of the functional 5' splice site (GRASP1*/2-GFP) leads to translation of a grasp transgene encoding GRASP2-GFP. Expression of the GFP fusion protein was confirmed with either anti-GRASP or anti-GFP specific antibodies. (D) In the double transgenic parasite line GRASP2-GFP/GRASP1-HA simultaneous expression of the fusion proteins was confirmed with either GFP- or HA-specific antibodies. (E) Localisation of GRASP2-GFP in unfixed parasites. Free merozoites display GFP fluorescence in one tightly defined compartment (a). A duplication of this compartment takes place in ring-stage parasites prior to nuclear division (b). As the parasite matures from trophozoite (c) to schizont (d) the organelle further multiplies until one compartment can be equally distributed among the forming progeny. (F) Colocalisation of the two GRASP proteins in the double transgenic parasite line. Immunofluorescence assay of GRASP2-GFP (green) with HA-specific antibodies representing GRASP1 (red). The merged image shows colocalisation of the two compartments. All images show the nucleus in blue (DAPI).

 

Figure 4
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  		Fig. 4. Distribution of GRASP2 depends on its hydrophobic N-terminus. (A) Expression of the N-terminal deletion mutants GRASP2{Delta}SA-GFP and GRASP2{Delta}SA2-GFP was confirmed using GFP-specific antibodies. A single band of ~100 kDa resembling the GFP-fusion protein is recognised in all transgenic parasite lines. (B) Fluorescence microscopy on live parasites that express either GRASP2-GFP or the N-terminal deletion mutants GRASP2{Delta}SA-GFP and GRASP2{Delta}SA2. Whereas GRASP2-GFP is restricted to tightly defined compartments (a), mutation of the N-terminus either by complete deletion of the hydrophobic stretch (b) or partial removal of its proximal part leaving the extreme N-terminus intact (c) abolishes Golgi targeting and results in a cytoplasmic distribution of the fusion protein. All images show the nucleus in blue (DAPI).

 

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