Phylogenetic analysis of carboxylate carriers. The maximum likelihood tree was generated by IQ-TREE with the LG + F + R7 model suggested by ModelFinder. The support was calculated using ultrafast bootstrap analysis. The unrooted phylogenetic tree shows the uniqueness of the identified putative stramenopile glycolytic intermediate carriers amongst eukaryotes. The stramenopile-specific glycolytic intermediate carriers (GIC - pink) are most closely related to di/tricarboxylate carriers (DTC – green), and to a lesser extend to oxoglutarate carriers (OGC - blue) and dicarboxylate carriers (DIC - yellow). Stramenopile proteins are in red, proteins included in this study are in bold. Note the absence of any non-stramenopile sequences in this clade. Bootstrap values less than 90 and a value for the GIC group are indicated.

A) Localisation of GIC carriers in Blastocystis ST7-B cells using specific antibodies (orange). The signal colocalises with the mitochondrial protein TPI-GAPDH (green) (Bártulos et al., 2018), demonstrating its mitochondrial localisation. DNA is stained with Hoechst 33342 (blue). Inserts top left: corresponding brightfield image. The scale bar is 5 μm. B) Western blots of Blastocystis OGC, GIC-1, and GIC-2 antibodies against purified protein confirm that there is no cross reactivity.

Expression, purification and biophysical characterization of Blastocystis carriers. A) Instant-blue stained SDS-PAGE gel of purified bOGC, bGIC-1 and bGIC-2. B) Typical unfolding curves of bOGC (black trace), bGIC-1 (red trace), and bGIC-2 (blue trace) using nanoDSF. C) The peak in the derivative of the unfolding curve (dR/dT) is the apparent melting temperature (Tm). D) [14C]-malate uptake curve of bOGC reconstituted into proteoliposomes loaded with (white circles) or without (black circles) 5 mM malate. Transport was initiated by the external addition of 2.5 μM [14C]-malate. E) Initial transport rates, calculated from panel D) from the linear part of the uptake curve (first 60 seconds). The data represent the mean and standard deviation is of n=4 (two biological repeats, each with two technical repeats).

Thermostability screening of detergent-solubilized, purified protein using nanoDSF (Prometheus, Nanotemper). A) hOGC, B) hDIC, C) bOGC, D) bGIC-1, and E) bGIC-2 against 10 mM di- and tricarboxylates, glycolytic intermediates and inorganic ions. The temperature shift (ΔTm) is the apparent melting temperature in the presence of compound minus the apparent melting temperature in the absence of compound. The data are represented by the mean and standard deviation of two biological repeats, each in triplicate (n=6 in total). G6P, glucose-6-phosphate; F6P, fructose-6-phosphate; F16BP, fructose 1-6 bisphosphate; DHAP, dihydroxyacetone phosphate; G3P, glyceraldehyde-3-phosphate; 3PG, 3-phosphoglycerate; PEP, phosphoenolpyruvate.

Proteoliposomes containing Blastocystis putative glycolytic intermediate carriers 1 (bGIC-1; A, C and E) and 2 (bGIC-2; B, D and F) were loaded with either (A and B) 5 mM malate, (C and D) 10 mM phosphate or (E and F) 5 mM sodium sulphate, and transport initiated by the external addition of (A and B) 2.5 μM [14C]-malate, (C and D) 200 μM [33P]-phosphate or (E and F) 25 μM [35S]-sulphate. Controls with no internalized substrate were also tested (black circles). Initial transport rates were calculated from the linear part of the uptake curve (60 seconds). The data represent the mean and standard deviation of two biological repeats, each with two technical repeats (n=4 in total).

Hetero-exchange reactions catalysed by reconstituted carriers. A) Proteoliposomes containing human oxoglutarate carrier (hOGC, SLC25A11), B) human dicarboxylate carrier (hDIC, SLC25A10), C) Blastocystis oxoglutarate carrier (bOGC) and D) Blastocystis putative glycolytic intermediate carrier 2 (bGIC-2) were loaded with 5 mM compound, and transport initiated by the external addition of 2.5 μM [14C]-malate (hOGC and bOGC) or 25 μM [35S]-sulphate (hDIC and bGIC-2). Initial transport rates were calculated from the linear part of the uptake curve (60 seconds). The data represent the mean and standard deviation of two biological repeats, each with two technical repeats (n=4 in total). G6P, glucose-6-phosphate; F6P, fructose-6-phosphate; F16BP, fructose 1-6 bisphosphate; DHAP, dihydroxyacetone phosphate; G3P, glyceraldehyde-3-phosphate; 3PG, 3-phosphoglycerate; PEP, phosphoenolpyruvate.

Comparing glycolysis in humans and Blastocystis.