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Fig. S1. RT-PCR using multiple genes to exclude gDNA contaminations of the cDNA preparation. The following intron containing genes were used for this experiment: GRASP (cDNA 651 bp/gDNA 799 bp) PF13_0280 (cDNA 666 bp/gDNA 996 bp), PF11_0164 (588/924), PFB0570w (753/1071), PF14_0119 (804/957) and PF13_0082 (621/1029). Consistent with the exon-intron structures of the control genes a size difference is visible; no gDNA fragments are detectable in the cDNA preparation.
Fig. S2. Schematic representation of various grasp gene structures within the Alveolata branch. The two exon structure is well conserved within Plasmodium spp. Additional open reading frames are marked with an asterisk. Pf, Plasmodium falciparum GRASP1 (chr10.phat_187); Pv, Plasmodium vivax (Pv_6638); Pb, Plasmodium berghei (PB001215.00.0); Py, Plasmodium yoelii (PY00200); Pg, Plasmodium gallinaceum (Pg_c000318647.Contig1); Pk, Plasmodium knowlesi (PK10_1650c); Ta, Theileria annulata (TA03555); Cp, Cryptosporidium parvum (AAEE01000001-5-82885-80690 cgd7_340); Tg: Toxoplasma gondii (55.m04966).
Fig. S3. Multiple sequence alignment of the GRASP N-terminus of various taxa. Sequences of various GRASP proteins were retrieved from Joint Genome Institute (http://genome.jgi-psf.org), PlasmoDB (www.plasmoDB.org) or GenBankTM. The conserved, N-terminal GRASP domain of a total of 53 taxonomic units was used for the phylogenetic analysis. Sequences were entered into the alignment program ClustalX (Higgins et al., 1996; Thompson et al., 1997) using pairwise parameter settings for the gap opening penalty of 15.0 and gap extension penalty of 0.30. Other parameters were set on default. Myristoylation motifs were analyzed using the NMT-Predictor tool (http://mendel.imp.ac.at/myristate). N-terminal myristoylation sites predicted as “reliable” are marked in red, sites that have less complete concordance with the myristoylation pattern implemented in the predictor are marked in grey.
Plasmodium chabaudi (Pc_3589), Theileria parva (EAN30984), Cryptosporidium hominis (XP_668364), Trypanosoma cruzi (EAN86064), Trypanosoma brucei (XP_828349), Leishmania major (CAJ06649), Leishmania infantum (xP_001466876), Leishmania braziliensis (CAM40796), Tetrahymena thermophila (XP_001031869), Paramecium tetraurelia (XP_001427243), Dictyostelium discoideum (XP_629220), Aspergillus fumigatus (XP_750751), Cryptococcus neoformans (XP_571967), Ustilago maydis (EAK82035), Schizosaccharomyces pombe (NP_593015), Phaeosphaeria nodorum (EAT80636), Chaetomium globosum (XP_001220168), Yarrowia lipolytica (XP_503480), Caenorhabditis elegans (NP_501354), Schistosoma japonicum (AAX26880), Anopheles gambiae (EAA04452), Aedes aegypti (EAT40259), Drosophila melanogaster (NP_649160), Apis mellifera (XP_393076), Gallus gallus GRASP 65 (NP_001026134), Gallus gallus GRASP 55 (NP_001012612), Monodelphis domestica GRASP 55 (XP_001367823), Monodelphis domestica GRASP 65 (XP_001373335), Rattus norvegicus GRASP 65 (O35254), Rattus norvegicus GRASP 55 (Q9R064), Mus musculus GRASP 55 (Q99JX3), Homo sapiens GRASP 65 (Q9BQQ3), Homo sapiens GRASP 55 (Q9H8Y8), Danio rerio GRASP 55 (NP_956997), Danio rerio GRASP 65 (NP_001007412), Xenopus laevis (AAH97604), Xenopus tropicalis (CAJ82834), Tetraodon nigroviridis (CAF97646), Strongylocentrotus purpuratus (XP_782738), Phytophthora ramorum (Physo1_1|158405), Phytophthora sojae (Phyra1_1|93616), Thalassiosira pseudonana (Thaps3|7103) and Encephalitozoon cuniculi (XP_965887).
Fig. S4. Episomal grasp gene expression leads to GRASP heterogeneity. (A) Schematic representation of a transfection vector used to test transcriptional and translational heterogeneity. The complete genomic grasp sequence was cloned into the pARL transfection vector. A double cMyc-tag (blue) was introduced into exon I (red) and a HA-tag (yellow) into the ORF within the intron downstream of the first alternative start ATG and hydrophobic stretch (orange) producing the pARL-GRASP1/2 transfection vector. Exon 2 is presented in grey and the human dhfr selection cassette in black. Putative transcriptional initiation sites are marked with coloured arrows (red and orange). Relative positions of the oligonucleotides used are displayed as black arrows. (B) Transcriptional analysis of GRASP1/2 expressing parasites. Using appropriate oligonucleotides and cDNA derived from the GRASP1/2 transgenic parasite line two PCR products with a size difference of approximately 150 bp were amplified. Genomic DNA and a PCR without template were used as a control to exclude non-specific amplification. (C) Parasite extract of WT and transgenic parasites were separated on a 10% SDS-PAGE and subjected to Western Blot analysis using GRASP, HA or cMyc- specific antibodies. Using GRASP specific antibodies the endogenous protein of approximately 70 kDa is detected in both WT and transgenic parasites. A second signal with slightly larger molecular weight representing the HA and Myc-tagged fusion proteins is recognized exclusively in transgenic parasites. HA- and Myc-specific antibodies recognize the differentially tagged GRASP proteins, GRASP1-cMyc and GRASP2-HA, confirming the expression of both variants in vivo. Notice, although this result confirms the presence of two translated grasp transcripts encoding two proteins with different N-termini sequencing of the spliced product revealed a cryptic 3splice site within the HA-tag that was used as a splice acceptor site. This lead to a spliced transcript coding for a double tagged GRASP1 with a cMyc-tag and HA-tag (data not shown). As a result HA-specific antibodies do not exclusively detect GRASP2 but also GRASP1.
Fig. S5. GRASP2-GFP lies in close proximity to the ER and co-localizes with ERD2. (A) Co-localization of GRASP2-GFP (green) with the ER-marker BiP (red) in fixed parasites. The GRASP2-defined compartments are in close proximity to but clearly distinct from the ER. (B) Immunofluorescence assay of GRASP2-GFP (green) with antibodies against the cis-Golgi marker ERD2 (red). The merged image shows co-localization of the two compartments. All images show the nucleus in blue (DAPI).
Fig. S6. Expression of GRASP2 as a TY1-tagged protein does not alter its subcellular distribution. (A) Expression of GRASP2 with a C-terminal TY1 epitope tag. Anti-TY1 antibodies exclusively recognize a protein of approximately 70 kDa in GRASP2-TY1 expressing parasites but not in the WT parasite line. (B) Immunofluorescence assay of GRASP2-TY1 (green) reveals a similar fluorescence distribution compared to GRASP2-GFP expressing parasites.
Fig. S7. GRASP variants show differential solubility characteristics. (A) Immunoblot with anti-GFP specific antibodies and saponin lysed parasites expressing either GRASP2-GFP or GRASP1-GFP that were hypotonically lysed (Hypo), and separated into supernatant (SN) and pellet fraction (P). Equal amounts of the insoluble pellet were treated with 0.1M carbonate (Carb), 6M Urea (Urea) or 1% Triton-X-100 (T-x-100), respectively, and separated into soluble (SN) and pellet (P) fractions. Equal amounts of pellet and supernatant fractions were loaded. Minimal amounts of GRASP2-GFP were released by hypotonic buffer conditions, carbonate or urea treatment. This is in contrast to GRASP1-GFP where a significant proportion was solubilised by carbonate treatment and more than 50% was released by urea treatment. In addition, both isoforms were only partially solubilized by treatment with Triton-x-100 at 4°C, suggesting interactions to structural components other than membranes. (B) Although the two GRASP-GFP isoforms utilize differential membrane attachments, they show a similar perinuclear distribution.
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