Journal: Life Science Alliance

Article Title: Structural and biochemical comparison of the FLVCR and CTL membrane protein families in eukaryotes

doi: 10.26508/lsa.202503583

Figure Lengend Snippet: (A) Schematic depicting the principle of using frog oocytes to assay protein activity with radioactive substrates. (B) Representative confocal microscopy images of GFP signal from recombinantly expressed CTL1, CherI, PNS1, FLVCR1, and FLVCR2 on the plasma membrane of oocytes (scale bar: 200 μm). (C) Time-dependent, linear uptake of choline by FLVCR1 and FLVCR2 (n = 3–5). A simple linear regression was applied to fit the data. (D) Titration curve of FLVCR1 and FLVCR2 with H 3 -ethanolamine (n = 4–6). Saturation was not achieved, and a K m value could not be determined when applying a Michaelis–Menten least squares fit. The plots appear to be fitted with a simple linear regression fit but is in fact a Michaelis–Menten least squares fit. (E) Uptake of 0.5 μM C 14 -choline by CTL1, PNS1, and CherI under different pH conditions (n = 3–6). Each data point represents an average of two to six oocytes and the SD as error bars. No fit was applied to the data. (F) Uptake of 2 μM C 14 -choline by CTL1, PNS1, and CherI under different pH conditions (n = 4–5). Each data point represents an average of four to five oocytes and the SD as error bars. No fit was applied to the data.

Article Snippet: The gene encoding H. sapiens FLVCR2 (UniProt: Q9UPI3 ) was commercially sequence-optimized (for yeast expression) and synthesized by Twist Bioscience.

Techniques: Activity Assay, Confocal Microscopy, Clinical Proteomics, Membrane, Titration

Journal: Life Science Alliance

Article Title: Structural and biochemical comparison of the FLVCR and CTL membrane protein families in eukaryotes

doi: 10.26508/lsa.202503583

Figure Lengend Snippet: Statistical significance is denoted by asterisks that correspond to certain probabilities ( P -values). Not significant (ns; P > 0.05), significant (* P < 0.05), highly significant (** P < 0.01), very high significance (*** P < 0.001), and extremely high significance (**** P < 0.0001). (A) Uptake of 100 μM H 3 -choline by CTL1, PNS1, CherI, FLVCR1, and FLVCR2 (n = 4–6). Compared with the negative control of water, choline uptake is not observed for CTL1 ( P = 0.7376), PNS1 ( P = 0.8621), or CherI ( P = 0.9875). Choline uptake is observed for FLVCR1 ( P = 0.0208) and FLVCR2 ( P < 0.0001). Statistics were calculated from a one-way ANOVA with Dunnett’s multiple comparisons test. (B) Uptake of 100 μM H 3 -ethanolamine by CTL1, PNS1, CherI, FLVCR1, and FLVCR2 (n = 6). Compared with the negative control of water, ethanolamine uptake is not observed for CTL1 ( P > 0.9999), PNS1 ( P = 0.9977), or CherI ( P > 0.9999). Ethanolamine uptake is observed for FLVCR1 ( P < 0.0001) and FLVCR2 ( P < 0.0001). Statistics were calculated from a one-way ANOVA with Dunnett’s multiple comparisons test. (C) Titration curve of FLVCR1 with H 3 -choline (n = 4–6). Measurements were corrected by subtracting the average of counts obtained from water for each concentration. A Michaelis–Menten fit was applied to obtain the apparent affinity ( K m ) for FLVCR1 as 26 ± 8 μM. (D) Titration curve of FLVCR2 with H 3 -choline (n = 3–6). Measurements were corrected by subtracting the average of counts obtained from water for each concentration. A Michaelis–Menten fit was applied to obtain the apparent affinity ( K m ) for FLVCR2 as 164 ± 21 μM. (E) Evaluating the substrate scope of FLVCR1 and FLVCR2 with choline, ethanolamine, carnitine, and histamine (n = 2–6). When comparing water with H 3 -choline, FLVCR1 ( P < 0.0001) and FLVCR2 ( P < 0.0001) exhibited transport. For water compared with H 3 -ethanolamine, FLVCR1 ( P < 0.0001) and FLVCR2 ( P < 0.0001) showed transport. When comparing water with H 3 -carnitine, FLVCR1 ( P = 0.9090) and FLVCR2 ( P = 0.7424) did not exhibit transport. For water compared with H 3 -histamine, FLVCR1 ( P > 0.9999) and FLVCR2 ( P > 0.9999) did not show transport. When comparing water to C 14 -choline, FLVCR1 ( P < 0.0001) and FLVCR2 ( P < 0.0001) exhibited transport. Statistics were calculated from a one-way ANOVA with Sidak’s multiple comparisons test. (F) Transport of H 3 -choline by FLVCR1 and FLVCR2 under different pH conditions (n = 3–4). Measurements were corrected by subtracting the average of counts obtained from water for each pH condition. (G) Influence of protons and the membrane potential on the transport of choline by FLVCR1 and FLVCR2 (n = 2–4). When comparing FLVCR1 without and with CCCP ( P = 0.2283), no significant dependency on protons was observed. For FLVCR2 with and without CCCP ( P = 0.0011), a highly significant dependency on protons was observed. Statistics were calculated from a one-way ANOVA with Sidak’s multiple comparisons test. (H) Transport of H 3 -choline (C) and H 3 -ethanolamine (E) by FLVCR1 and FLVCR2 in the presence (Na) of 96 mM sodium or absence (K) of sodium (using 96 mM potassium; n = 3–5). Neither FLVCR1 nor FLVCR2 seems to depend on sodium to transport choline or ethanolamine. For negative controls, water and choline with sodium and with potassium, P = 0.9801, as well as water and ethanolamine with sodium or potassium, P = 0.9996 were obtained. For FLVCR1 with choline and sodium or potassium, P = 0.9999. For FLVCR1 and ethanolamine and sodium or potassium, P = 0.9152. For FLVCR2 with choline and sodium or potassium, P = 0.1761. For FLVCR1 and ethanolamine and sodium or potassium, P = 0.9795. Statistics were calculated from a one-way ANOVA with Sidak’s multiple comparisons test.

Article Snippet: The gene encoding H. sapiens FLVCR2 (UniProt: Q9UPI3 ) was commercially sequence-optimized (for yeast expression) and synthesized by Twist Bioscience.

Techniques: Negative Control, Titration, Concentration Assay, Membrane

Journal: Life Science Alliance

Article Title: Structural and biochemical comparison of the FLVCR and CTL membrane protein families in eukaryotes

doi: 10.26508/lsa.202503583

Figure Lengend Snippet: (A) Diagram of the major facilitator topology exhibited by FLVCR2, divided into an N domain (rosy brown) and C domain (sand). The twofold pseudosymmetry point is indicated by the black oval. (B) Model and map of FLVCR2 (PDB: 9QU4 ) in a schematic of its localization in the plasma membrane. The map shows where the Protein A repeats (gray) were linked to the N terminus, but this fiducial was not built in the model. (C) Map of the DDM:CHS micelle, shown as a gray surface, with our model of FLVCR2 inside in black. The AlphaFold2 prediction of Protein A fused to FLVCR2, colored according to the predicted local distance difference test (pLDDT) scale, is superposed onto our model. (D) Unmodeled, elongated map regions, possibly of sterols and retained lipids, form belts around FLVCR2, particularly on the face where the membrane would be in contact with the extracellular environment. (E) Model of FLVCR2 at 3.4 Å adopts the inward-facing conformation. Choline (brown) is bound within a pocket located at a central twofold pseudosymmetry point. Residues involved in coordinating choline are shown as sticks and annotated in the map (gray). (F) Hydrophobicity of the binding pocket with choline inside. The slices were made through the front and the top of FLVCR2. (G) Electrostatics of the binding pocket with choline inside. The slices were made through the front and the top of FLVCR2. (H) Hydrophobicity coloring of FLVCR2 from the front and the back. (I) Electrostatic coloring of FLVCR2 from the front and the back.

Article Snippet: The gene encoding H. sapiens FLVCR2 (UniProt: Q9UPI3 ) was commercially sequence-optimized (for yeast expression) and synthesized by Twist Bioscience.

Techniques: Clinical Proteomics, Membrane, Binding Assay

Journal: Life Science Alliance

Article Title: Structural and biochemical comparison of the FLVCR and CTL membrane protein families in eukaryotes

doi: 10.26508/lsa.202503583

Figure Lengend Snippet: (A) Schematic of the construct of FLVCR2 used for structure determination. The three repeats of Protein A served as a fiducial to facilitate particle alignment during data processing. The thrombin site was used after Ni-NTA purification to remove the 10× His-Tag. (B) SDS–PAGE gel of fractions taken during the purification of FLVCR2. Lanes 1–7: Ni-NTA elution fractions; lane 8: molecular weight marker; lanes 9–15: fractions of SEC. The asterisk (*) denotes the sample used to prepare grids for cryo-EM. (C) Size-exclusion chromatography trace of FLVCR2 from an S200 Superdex Increase 10/300 column. The asterisk (*) shows the fraction used to prepare grids for cryo-EM. (D) Representative micrograph from the data collected on the copper-support grid with R1.2/1.3 300 mesh. Particles were evenly distributed within the ice. (E) Representative 2D classes obtained from picked particles. Multiple orientations were sampled of FLVCR2, with the Protein A fiducial protruding from the micelle. (F) Summary of the data processing steps performed in cryoSPARC.

Article Snippet: The gene encoding H. sapiens FLVCR2 (UniProt: Q9UPI3 ) was commercially sequence-optimized (for yeast expression) and synthesized by Twist Bioscience.

Techniques: Construct, Purification, SDS Page, Molecular Weight, Marker, Cryo-EM Sample Prep, Size-exclusion Chromatography

Journal: Life Science Alliance

Article Title: Structural and biochemical comparison of the FLVCR and CTL membrane protein families in eukaryotes

doi: 10.26508/lsa.202503583

Figure Lengend Snippet: (A) Superposition of FLVCR2 solved in the current study (PDB: 9QU4 in sand) to other structures in the inward-facing conformation and a close-up view of the substrate binding pocket. All the structures, except the model and labels in pink, are of human FLVCR2. When superposing the three models to PDB: 9QU4 , the RMSD Ca is 0.7 Å for all the structures. (B) Overlay of CTL1 determined in the current study (teal color) and in previous work (gray). Regions that were modeled to various degrees are indicated in colors corresponding to the models.

Article Snippet: The gene encoding H. sapiens FLVCR2 (UniProt: Q9UPI3 ) was commercially sequence-optimized (for yeast expression) and synthesized by Twist Bioscience.

Techniques: Binding Assay