Tsp-GFP fusion constructs co-localize with the proton pump on contractile vacuoles.

<p>(A) Fixed amoebae expressing TspC-GFP show a delicate green network with occasional bladders at the bottom of the cell that does not co-localize with a marker of the ER (PDI, red) in indirect immunofluorescence. OL = overlay. (B) In a more medial plane of the same cell, the GFP signal is found within the plasma membrane in addition to having a cloudy appearance (arrowheads). This is indicative of the Golgi-apparatus lying close to the ER-rings that represent the nuclear envelopes. (C) Using the V-H⁺ ATPase (or proton pump, PP, red) as a stain for the CV, the cisternae (arrows) and tubular elements (arrowheads) strongly overlap with TspC-GFP in the overlay (OL). Some vesicular structures only labeled by the V-H⁺ ATPase antibody may be components of the endolysosomal system that is known to be populated by the V-H⁺ ATPases [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0162065#pone.0162065.ref060" target="_blank">60</a>]. N-terminal (D–F) as well as C-terminal (G–H) GFP fusions to TspA, TspC and TspD proteins all localize to bladders and tubules of the CV system (merge yellow) counterstained as in (C). Scale bars = 5 μm.</p

Structural and functional analysis of cell-free produced PfAQP without a GFP fusion.

<p><b>A</b>. PfAQP in the presence of Brij35 (blue curve) and Brij78 (red curve) was analyzed by circular dichroism. The left panel shows the mean residue molar ellipticity [θ] in the range of 200–250 nm. Thermal unfolding was monitored at 222 nm from 20°C to 95°C (middle panel). A plot of the photomultiplyer dynode voltage versus temperature (right panel) indicates an increase in turbidity at 80°C in the sample with Brij35 solubilized PfAQP suggesting protein agglomeration. <b>B</b>. Reconstitution of PfAQP, produced in the prescence of Brij78, into proteoliposomes was controlled by sucrose density gradient centrifugation and Western blot using an anti-His₅ antiserum. The fractions with 15% and 20% sucrose contained reconstituted liposomal PfAQP; the 30% sucrose fraction displays precipitated, non-integrated PfAQP protein. PfAQP monomers (≈25 kDa) and dimers are visible. <b>C</b>. For functional analysis, PfAQP-proteoliposomes (red traces) and empty control liposomes (blue traces) were subjected to an outward osmotic gradient of 300 mosm kg⁻¹ (left panel) and an inward isotonic glycerol gradient of 300 mM (right panel). Changes in the light scattering intensity reflect liposome shrinkage due to water efflux (increase in light scattering) and liposome swelling due to glycerol plus secondary water influx (decrease in light scattering). Note the difference of the scale of the abscissae as a consequence of lower glycerol permeability by at least one order of magnitude. The slow glycerol flux across the plain lipid liposome membrane did not reach a plateau, hence, the photomultiplyer signal was plotted without normalization. For each experiment nine traces were averaged and fitted to single exponential functions.</p

Schematic representation of human and <i>D</i>. <i>discoideum</i> Tsp topologies.

<p>A generic topology of human Tsp is shown on the left (adapted from [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0162065#pone.0162065.ref033" target="_blank">33</a>] and [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0162065#pone.0162065.ref005" target="_blank">5</a>]), the proposed <i>Dictyostelium</i> Tsp topology on the right, as inferred from protein structure predictions and protein sequence alignments. Numbers in blue indicate the range of amino acids (aa) in the N-terminus, the small (EC1) and large (EC2) extracellular loops, the inner cytoplasmic loop (ICL) and the C-terminus. Blue and yellow shadings represent the variable (protein-protein interactions) and conserved domain of EC2, respectively. Cysteines in yellow are 100% conserved, red circles represent the “CCG”-motif that is altered (X) in four of five <i>D</i>. <i>discoideum</i> Tsps. Conserved residues in the transmembrane regions are indicated in italic letters. Potential disulfide bridges are represented by blue lines. Palmitoylation sites as predicted by CSS Palm 1.0 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0162065#pone.0162065.ref058" target="_blank">58</a>] are marked by pink wavy lines. Black pins show potential <i>N</i>-glycosylation sites (N x T/S). Please note that not all Tsps may undergo these modifications to the same extent.</p

Expression of <i>TspA-E</i> in <i>D</i>. <i>discoideum</i>.

<p>Total RNA was isolated from growth phase cells (amoebae) and from cells after 16 h of development (slugs), cDNA was prepared and PCR reactions were performed with sequence-specific primers (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0162065#pone.0162065.s003" target="_blank">S1 Table</a>). PCR products had the predicted sizes of approximately 700 bp.</p

The C Isoform of <i>Dictyostelium</i> Tetraspanins Localizes to the Contractile Vacuole and Contributes to Resistance against Osmotic Stress

<div><p>Tetraspanins (Tsps) are membrane proteins that are widely expressed in eukaryotic organisms. Only recently, Tsps have started to acquire relevance as potential new drug targets as they contribute, via protein-protein interactions, to numerous pathophysiological processes including infectious diseases and cancer. However, due to a high number of isoforms and functional redundancy, knowledge on specific functions of most Tsps is still scarce. We set out to characterize five previously annotated Tsps, TspA-E, from <i>Dictyostelium discoideum</i>, a model for studying proteins that have human orthologues. Using reverse transcriptase PCRs, we found mRNAs for <i>TspA-E</i> in the multicellular slug stage, whereas vegetative cells expressed only <i>TspA</i>, <i>TspC</i> and, to a lesser extent, <i>TspD</i>. We raised antibodies against TspA, TspC and TspD and detected endogenous TspA, as well as heterologously expressed TspA and TspC by Western blot. <i>N</i>-deglycosylation assays and mutational analyses showed glycosylation of TspA and TspC <i>in vivo</i>. GFP-tagged Tsps co-localized with the proton pump on the contractile vacuole network. Deletion strains of <i>TspC</i> and <i>TspD</i> exibited unaltered growth, adhesion, random motility and development. Yet, <i>tspC</i>^<i>−</i> cells showed a defect in coping with hypo-osmotic stress, due to accumulation of contractile vacuoles, but heterologous expression of <i>TspC</i> rescued their phenotype. In conclusion, our data fill a gap in <i>Dictyostelium</i> research and open up the possibility that Tsps in contractile vacuoles of e.g. <i>Trypanosoma</i> may one day constitute a valuable drug target for treating sleeping sickness, one of the most threatening tropical diseases.</p></div

Overview of Tsps in <i>D</i>. <i>discoideum</i>.

<p>Overview of Tsps in <i>D</i>. <i>discoideum</i>.</p

Generation of TspA, TspC and TspD antibodies, Western blots of Tsp-GFP fusion proteins and specification of the TspA antibody.

<p>(A) Partial sequence alignment of <i>D</i>. <i>discoideum</i> TspA-E and human CD9 (the plot was generated using TEXshade [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0162065#pone.0162065.ref059" target="_blank">59</a>]). Peptide sequences that were used for antibody production and conserved Cys residues are labeled in white. The conserved “CCG”-motif is indicated. (B) Western blots of cell lysates (30 μg protein per lane) of <i>D</i>. <i>discoideum</i> vegetative cells transformed with the control plasmid pDM323 (−) or <i>Tsp-GFP</i> using a polyclonal α-GFP antibody as well as the affinity purified antibodies α-TspA and α-TspC. The specificity of the anti-TspA antiserum (preblock) was tested by a 1 h preincubation with 1 μg ml^-1 of the immunizing peptide corresponding to a 10-fold excess. (C) Western blot of cell lysates (88 μg protein per lane) of <i>D</i>. <i>discoideum</i> vegetative cells (−) and of cells transformed with <i>TspA-His</i> using the affinity purified α-TspA antibody as well as a monoclonal α-His antibody. * = unspecific band.</p

Cell-free synthesis of the <i>Plasmodium falciparum</i> aquaglyceroporin, PfAQP, with a C-terminal GFP shows detergent-dependent signal patterns.

<p><b>A</b>. The left panel displays a Western blot of PfAQP-GFP produced in the presence of various non-ionic detergents of the Brij family and digitonin (Digit.). A polyclonal anti-GFP antiserum and a secondary horseradish peroxidase-labeled antibody were used for detection. Luminol chemiluminescence was monitored using a CCD-camera. PfAQP-GFP was obtained as two folding species with apparent molecular weights of 45 and 48 kDa. A signal representing the 24 kDa GFP domain alone was also visible. In the right panel, excitation of in-gel GFP fluorescence yielded emission signals of the 45 kDa PfAQP-GFP protein species and the 24 kDa GFP-band. <b>B</b>. PfAQP-GFP band intensities were determined semi-quantitatively by integration from the Western blot using arbitrary units (BLU – biomedical light units). The height of the bars in the left panel represents the total protein yield as a sum of the signals from the upper 48 kDa band (black) and the lower 45 kDa band (green). The plot in the right panel confirms correlation of the in-gel GFP fluorescence signal with GFP fluorometry of the crude cell-free reaction mixture. Intensity units given by the fluorometer are arbitrary (AU). The symbols and error bars denote mean values and the data range from two independent synthesis reactions and fluorometric evaluations. <b>C</b>. Cell-free synthesis, SDS-PAGE, and Western blot of non-fused PfAQP yielded signals for the monomer (≈25 kDa) and the dimer using an anti-His₅ antiserum. A semi-quantitative representation of the protein yield is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042186#pone.0042186.s003" target="_blank">Fig. S3</a>. Most stable dimers were obtained with Brij58 and Brij78, which also led to highest in-gel fluorescence intensity.</p

Twenty-four hour development.

<p>Cells were starved on KK2 agar plates and incubated at 22°C in a moist chamber. Pictures were taken at the indicated time points illustrating aggregation (4 h), streaming (8 h), culmination (16 h) and spore-head formation (24 h). Development of <i>tspC</i>^<i>−</i>, as well as of <i>tspD</i>^<i>−</i> mutant was unaltered compared to wildtype cells. Scale bars = 100 μm.</p

Disruption of the <i>TspC</i> and <i>TspD</i> genes.

<p>(A) Genomic 5´ and 3´ fragments of <i>TspC</i> and <i>TspD</i> were amplified by PCR, ligated into a blasticidin-resistance cassette and each construct was transferred into AX2 wildtype cells by electroporation. The adjacent genes of <i>TspC</i> (<i>lipocalin</i>, DDB_G0269882; <i>pseudogene samkB</i>, DDB_G0270988) and <i>TspD</i> (<i>TspA</i>; DDB_G0269884) are shown. (B) For each gene, two independent blasticidin resistant clones were compared with the wildtype (wt) for correct genomic 5´ integration (primers 1/2), insertion of the blasticidin-resistance gene (primers 3/4), and 3´ integration (primers 5/6).</p