A mitochondrial carrier transports glycolytic intermediates to link cytosolic and mitochondrial glycolysis in the human gut parasite Blastocystis

Reviewer #1 (Public Review):

Summary:
This study identifies a family of solute transports in the enteric protist, Blastocystis, that may mediate the transport of glycolytic intermediates across the mitochondrial membrane. The study builds on previous observations suggesting that Blastocystis (and other Stramenopiles) are unusual in having a compartmentalized glycolytic pathway with enzymes involved in upper and lower glycolysis being located in the cytosol and mitochondria, respectively. In this study, the authors identified two putative Stamenopile metabolite transporters that are related to plant di/tricarboxylic acid transporters that might mediate the transport of glycolytic intermediates across the mitochondrial membrane. These GIC-transporters were localized to the Blastocystis mitochondrion using specific rabbit antibodies and shown to bind several glycolytic intermediates (including GAP, DHAP, and PEP) based on thermostability shift assays. Direct evidence for transport activity was obtained by reconstituting native proteins in proteoliposomes and measuring the uptake of 14C-malate or 35S-sulphate against unlabelled substrates. This assay showed that GIC-2 transported DHAP, GAP, and PEP. However, significant transport activity was not observed for bGIC-2. Overall, the study provides strong, but not conclusive evidence that bGIC-1 is involved in transporting glycolytic intermediates across the inner membrane of the mitochondria, while the function of GIC-2 remains unclear, despite exhibiting the same metabolite binding properties as bGIC-2 in thermostability assays.

Strengths:
Overall, the findings are of interest in the context of understanding the diversity of core metabolic pathways in evolutionarily diverse eukaryotes, as well as the process by which cytosolic glycolysis evolved in most eukaryotes. The experiments are carefully performed and clearly described.

Weaknesses:
The main weakness of the study is the lack of direct evidence that either bGIC-1 and/or bGIC2 are active in vivo. While it is appreciated that the genetic tools for disrupting GIC genes in Blastocystis are limited/lacking, are there opportunities to ectopically express or delete these genes in other Stamenopiles, such as Phaeodactylum triconuteum, to demonstrate function in vivo?

The authors demonstrate that both bGIC-1 and bGIC-2 are targeted to the mitochondrion, based on immunofluorescence studies. However, the precise localization and topology of these carriers in the inner or outer membrane are not defined. The conclusions of the study would be strengthened if the authors could show that one/both transporters are present in the inner membrane using protease protection experiments following differential solubilization of the outer and inner mitochondrial membranes.

It is not clear why hetero-exchange reactions were not performed for bGIC-1 (only for bGIC-2).

The summary slide depicted in Fig 7 is somewhat simplified and inaccurate. First, the authors show that TPI is located in the mitochondria in this study, while in the summary figure, TPI is shown to be present in both the cytosol and mitochondrial matrix. A cytosolic localization for TPI provides a functional rationale for having a triose-P carrier in the inner membrane - however, this is not supported by the data shown here. Second, if bGIC1/2 uses PEP as a counter ion to import GA3P and DHAP into the mitochondrion, as proposed in Fig 7, the lower glycolytic pathway would be effectively truncated at PEP, removing substrate for pyruvate kinase and formation of pyruvate/ATP. Third, the authors suggest that DHAP may have other functions in the mitochondria although these are not shown in the figure.

Reviewer #2 (Public Review):

In this manuscript, the authors set out to identify transporters that must exist in Stramenophiles due to the fact that the second half of glycolysis appears to be conducted in the mitochondria. They hypothesize that a Stramenophile-specific clade of transporters related to the dicarboxylate carriers is likely the relevant family and then go on to test two proteins from Blastocystis due to the infectious disease relevance of this organism. They show rather convincingly that these two proteins are expressed and are localized to the mitochondria in the native organism. The purified proteins bind to glycolytic intermediates and one of them, GIC-2, transports several glycolytic intermediates in vitro. This is a very solid and well-executed study that clearly demonstrates that bCIC-2 can transport glycolytic intermediates.

1. The major weakness is that the authors aren't able to show that this protein actually has this function in the native organism. This could be impossible due to the lack of genetic tools in Blastocystis, but it leaves us without absolute confidence that bGIC-2 is the important glycolytic intermediate mitochondrial transporter (or even that it has this function in vivo).

2. It's atypical that the figures and figure panels don't really follow the order of their citation in the text. It's not a big deal, but mildly annoying to have to skip around in the figures (e.g. Figure 3D-E are described in the same paragraph as Figure 5). In addition, to facilitate the flow and a proper understanding I would encourage a reordering between figures 5D and 6 since Figure 6 is needed to understand the results shown in panel 5D, which may lead to confusion.

3. My impression is that the authors under-emphasize the fact that the hDIC also binds (and is stabilized by) glycolytic intermediates (G3P and 3PG). In the opinion of this reviewer, this might change the interpretation about the uniqueness of the bGIC proteins. They act on additional glycolytic intermediates, but it's not unique.

Reviewer #3 (Public Review):

Summary:
Unlike most eukaryotes, Blastocystis has a branched glycolysis pathway, which is split between the cytoplasm and the mitochondrial matrix. An outstanding question was how the glycolytic intermediates generated in the 'preparatory' phase' are transported into the mitochondrial matrix for the 'pay off' phase. Here, the authors use bioinformatic analysis to identify two candidate solute carrier genes, bGIC-1, and bGIC-2, and use biochemical and biophysical methods to characterise their substrate specificity and transport properties. The authors demonstrate that bGIC-2 can transport dihydroxyacetone phosphate, glyceraldehyde-3-phosphate, 3-phosphoglycerate, and phosphoenolpyruvate, establishing this protein as the 'missing link' connecting the two split branches of glycolysis in this branch of single-celled eukaryotes. The authors also present their data on bGIC-1, which suggests a role in anion transport and bOGC, which is a close functional homologue of the human oxoglutarate carrier (hOGC, SLC25A11) and human dicarboxylate carrier (hDIC, SLC25A10).

Strengths:
The results are presented in a clear and logical arrangement, which nicely leads the reader through the process of gene identification and subsequent ligand screening and functional reconstitution. The results are compelling and well supported - the thermal stabilisation data is supported by the exchange studies. Caveats, where apparent, are discussed and rational explanations are given.

Weaknesses:
The study does not contain any significant weaknesses in my view. I would like to see the authors include the initial rate plots used in the main figures (possibly as insets), so we can observe the data points used for these calculations. It would also have been interesting to include the AlphaFold models for bGIC-1 and bGIC-2 and a discussion/rationalisation for the substrate specificity discussed in the study.