Research Article 5603 Re-defining the Golgi complex in Plasmodium falciparum using the novel Golgi marker PfGRASP Nicole S. Struck1, Suzana de Souza Dias1, Christine Langer1, Matthias Marti2, J. Andrew Pearce2, Alan F. Cowman2 and Tim W. Gilberger1,* 1Bernhard Nocht Institute for Tropical Medicine, Malaria II, Bernhard-Nocht-Str. 74, 20359 Hamburg, Germany 2The Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Melbourne 3050, Australia *Author for correspondence (e-mail: [email protected]) Accepted 2 September 2005 Journal of Cell Science 118, 5603-5613 Published by The Company of Biologists 2005 doi:10.1242/jcs.02673 Summary Plasmodium falciparum, the causative agent of malaria, within the boundaries of the parasite and colocalises relies on a sophisticated protein secretion system for host with the cis-Golgi marker ERD2. Correct subcellular cell invasion and transformation. Although the parasite localisation of this Golgi matrix protein depends on a cross- displays a secretory pathway similar to those of all species conserved functional myristoylation motif and is eukaryotic organisms, a classical Golgi apparatus has never insensitive to Brefeldin A. Taken together our results define been described. We identified and characterised the the Golgi apparatus in Plasmodium and depict the putative Golgi matrix protein PfGRASP, a homologue of morphological organisation of the organelle throughout the the Golgi re-assembly stacking protein (GRASP) family. asexual life cycle of the parasite. We show that PfGRASP is expressed as a 70 kDa protein throughout the asexual life cycle of the parasite. We Supplementary material available online at generated PfGRASP-GFP-expressing transgenic parasites http://jcs.biologists.org/cgi/content/full/118/23/5603/DC1 and showed that this protein is localised to a single, juxtanuclear compartment in ring-stage parasites. The Key words: Golgi, GRASP, Plasmodium, Secretory pathway, PfGRASP compartment is distinct from the ER, restricted Transfection Introduction of this organelle is believed to reflect the requirement for the Journal of Cell Science The intracellular parasite Plasmodium falciparum is the processing machinery to be compartmentalised for a causative agent of malaria and responsible for over two sequential series of modification and sorting events (Farquhar million deaths each year (Butler, 2002). After an initial and Palade, 1998). multiplication step in liver cells, the parasite invades and The Golgi of protists offers an interesting insight into multiplies within red blood cells. To survive, the parasite variations of the structure and organisation of this organelle. extensively modifies the host cell by exporting parasitic For example, representatives of two early-diverged proteins to the host cell cytoplasm and to its cell surface. It eukaryotic lineages exhibit entirely distinct Golgi has recently been shown that protein transport from the morphology and function: the diplomonad parasite Giardia parasite via the surrounding vacuole and into the host cell intestinalis appears to have no stable Golgi compartment and involves a pentameric motif conserved across the genus there is no evidence for abundant protein modifications (Marti Plasmodium (Marti et al., 2004; Hiller et al., 2004). Secreted et al., 2003), whereas Trypanosoma brucei (kinetoplastids), proteins are synthesised in the parasite cytoplasm and the causative agent of sleeping sickness in humans, shows a translocated into the ER. This is mediated through a classical classical stacked Golgi and high abundance of N- hydrophobic leader sequence, although some can be glycosylation in surface proteins (He et al., 2004). Further, considerably recessed (Papakrivos et al., 2005). In addition, a Toxoplasma gondii, an apicomplexan parasite related to functional retrieval mechanism for soluble proteins such as Plasmodium, has a single stacked Golgi apparatus as part of PfBiP via the KDEL receptor PfERD2 has been demonstrated a highly polarised secretory pathway (Hager et al., 1999; in this parasite (Elmendorf and Haldar, 1993a; Van Dooren et Joiner and Roos, 2002; Pelletier et al., 2002). By contrast, al., 2005). However, morphological and functional evidence morphology, organisation, function and even localisation of for a Golgi apparatus and post-Golgi transport pathways in the Golgi in Plasmodium are still controversially discussed Plasmodium is still rather sparse. The Golgi apparatus is the (Lingelbach, 1993; Elmendorf and Haldar, 1993b; Benting central hub of the eukaryotic secretory machinery and plays a et al., 1994; Mattei et al., 1999; Bannister et al., 2000). pivotal role in protein modification, processing and sorting. In Immunofluorescence and electron microscopy have not as yet general, the Golgi is organised into three functionally distinct provided unambiguous results as to the nature of the Golgi regions: the cis-Golgi network (entry face), the Golgi stack (Aikawa, 1971; Elmendorf and Haldar, 1993a; Van Wye et and the trans-Golgi network (exit face). The ordered structure al., 1996; Bannister et al., 1990; Bannister et al., 2000; 5604 Journal of Cell Science 118 (23) Bannister et al., 2003). For instance, it was shown that CTG-3Ј and PfERD2-AS, 5Ј-GCGCCCTAGGTTTTACTTCACCA- PfERD2, a Golgi marker protein that in mammalian systems TTAAATG-3Ј. is concentrated in the cis-Golgi, is localised to the perinuclear To generate transfection vectors expressing C-terminal GFP fusion proteins, PCR products were digested with KpnI and AvrII (bold) and region of the parasite (Elmendorf and Haldar, 1993a; Van – Wye et al., 1996; Noe et al., 2000). It was also shown that the cloned into pARL1a (Crabb et al., 2004). GFP was previously Xho – Ј Avr distribution of the trans-Golgi marker PfRab6 (Novick and inserted into the I site of pARL1a with an additional 5 II site. To alter the putative myristoylation site in PfGRASP we changed the Brennwald, 1993) in the parasite is distinct from PfERD2 (De amino acid glycine at position 2 to alanine by using the sense primer Castro et al., 1996; Van Wye et al., 1996). It was concluded (PfGRASP-G2/A-GFP: 5Ј-GCGCGGTACCATGGCAGCAGGAC- that in early blood stages the parasite displays a functional AAACG-3Ј) and fused the fragment in-frame with GFP as described but primitive, unstacked Golgi with distinct compartments above. (Van Wye et al., 1996). This data was partially supported by electron microscopy. A typically stacked Golgi apparatus has Antisera and immunoblotting not been identified in the parasite, but a discoid cisterna close Mouse antisera were raised against synthetic polypeptides of to the nucleus has been described that was provisionally PfGRASP (N SSKMDNITKGTYIN ) and PfRab6 (PlasmoDB, specified as a minimal Golgi apparatus (Bannister et al., 529 543 PF11_0461, N181EANVVDIQLTNNSNKND197). Other antibodies 2000; Bannister et al., 2003). Previous fixation methods for used in immunodetection were rabbit anti-PfERD2 (obtained through Plasmodium have resulted in very poor preservation for the Malaria Research and Reference Center, NIH, MRA-72; accession all membranous structures and organelles, so the number NP705420) (Elmendorf and Haldar, 1993a), monoclonal anti- characterisation of the Golgi in fixed Plasmodium parasites GFP (Roche) and anti-PfBiP (Malaria Research and Reference Center, must be treated with caution. To overcome the problem of MR-19; AAA29501) (Kumar et al., 1991). For immunoblots, parasite poor organellar fixation, we used live-cell microscopy to proteins from a synchronised culture were separated on 10% SDS- visualise the Golgi apparatus. PAGE gels and transferred to nitrocellulose membranes (Schleicher In the present study, we characterised the P. falciparum & Schuell). Anti-PfGRASP was diluted 1:1500 in phosphate-buffered saline (PBS). The secondary antibody was sheep anti-rabbit IgG orthologue of the Golgi re-assembly stacking protein horseradish peroxidase (Sigma) and used at a 1:3000 dilution. The (GRASP). GRASP proteins are peripheral membrane proteins immunoblots were developed by chemiluminescence using ECL involved in stacking of Golgi cisternae (Barr et al., 1997; Barr (Amersham). et al., 1998; Shorter et al., 1999). They are conserved from yeast to mammals and are Golgi-defining matrix proteins. Here Immunofluorescence and analysis of GFP-expressing we show that PfGRASP is expressed in blood stage parasites parasites as a 70 kDa protein. It is localised to a compartment juxtaposed Green fluorescence of GFP-expressing transfectant cell lines was to the nucleus. Using green fluorescence protein (GFP)-tagged observed and captured in live cells through the erythrocytic life cycle chimeric proteins we analysed morphology and development every 8 hours using a Leica Axioskop 2 and OpenLab software of the Golgi apparatus in vivo during the life cycle of the (Improvision) or a Leica confocal microscope (TCS SP2) and Leica parasite. We also show that targeting of PfGRASP depends on confocal software (LCS). Immunofluorescence assays were a functional N-terminal myristoylation motif. In addition we performed on fixed parasites as previously described (Tonkin et al., Journal of Cell Science used indirect immunofluorescence microscopy and specific 2004). The primary antibody dilutions used in 3% BSA: rabbit anti- antibodies against other Golgi marker proteins to analyse the PfERD2 (1:500), rabbit anti-PfBiP (1:1000), mouse anti-PfGRASP spatial organisation and compartmentalisation of the Golgi (1:1000) and mouse anti-PfRab6 (1:1000). The cells were incubated apparatus