Journal: The EMBO Journal

Article Title: Human mitochondrial carriers of the SLC25 family function as monomers exchanging substrates with a ping-pong kinetic mechanism

doi: 10.1038/s44318-024-00150-0

Figure Lengend Snippet: Instant-blue stained SDS-PAGE gel of purified protein ( A ) oxoglutarate carrier (OGC), ( E ) citrate carrier (CIC), ( I ) dicarboxylate carrier (DIC), ( M ) aspartate/glutamate carrier (AGC2). Typical unfolding curves of ~10 μg protein without compound (black trace), or with 10 mM compound (red trace) ( B ) OGC + 10 mM malate, ( F ) CIC + 10 mM citrate, ( J ) DIC + 10 mM malate, or ( N ) AGC2 + 10 mM aspartate, using nano differential scanning fluorimetry (Nanotemper Prometheus). The peak in the derivative of the unfolding curve (dR/dT) is at the apparent melting temperature (Tm). ( C ) [ 14 C]-malate uptake curves of OGC reconstituted into proteoliposomes loaded with (white circles) or without (black circles) 1 mM malate. Transport was initiated by the external addition of 2.5 μM [ 14 C]-malate. ( G ) [ 14 C]-citrate uptake curves of CIC reconstituted into proteoliposomes loaded with (white circles) or without (black circles) 1 mM citrate. Transport was initiated by the external addition of 2.5 μM [ 14 C]-citrate. ( K ) [ 14 C]-malate uptake curves of DIC reconstituted into proteoliposomes loaded with (white circles) or without (black circles) 1 mM malate. Transport was initiated by the external addition of 2.5 μM [ 14 C]-malate. ( O ) [ 14 C]-aspartate uptake curves of AGC2 reconstituted into proteoliposomes loaded with (white circles) or without (black circles) 1 mM aspartate. Transport was initiated by the external addition of 2.5 μM [ 14 C]-aspartate. ( D , H , L , P ) Initial rates were estimated by fitting the uptake curves to Eq. . The data represent the average and standard deviation of two biological repeats, each with three technical repeats except AGC2, which is the average of three technical repeats. .

Article Snippet: Genes encoding the human oxoglutarate carrier (OGC, SLC25A11, UniProt accession code: Q02978 ), the human citrate carrier (CIC, SLC25A1, UniProt accession code: P53007 ), the human dicarboxylate carrier (DIC, SLC25A10, UniProt accession code: Q9UBX3 ) and the human aspartate/glutamate carrier 2 (AGC2, SLC25A13, UniProt accession code: Q9UJS0 ) were codon-optimized with an N-terminal eight histidine tag and either Xa (OGC, CIC and DIC) or TEV (AGC2) protease cleavage sites to aid purification (OGC, ∆2-13; CIC, ∆2-12; DIC, GKP added (positions 2–4)) (synthesized by GenScript).

Techniques: Staining, SDS Page, Purification, Nano Differential Scanning Fluorimetry, Standard Deviation

Journal: The EMBO Journal

Article Title: Human mitochondrial carriers of the SLC25 family function as monomers exchanging substrates with a ping-pong kinetic mechanism

doi: 10.1038/s44318-024-00150-0

Figure Lengend Snippet: ( A ) Structural models of the human dicarboxylate carrier (DIC), oxoglutarate carrier (OGC), citrate carrier (CIC), ADP/ATP carrier (AAC1), and aspartate/glutamate carrier (AGC2) were all determined by the Alphafold 3.0 server (Abramson et al, ), except for the experimentally determined structure of an uncoupling protein (UCP1) (pdb entry: 8G8W) (Jones et al, ). ( B ) Determination of the molecular weights in LMNG/TOCL by size exclusion chromatography. The normalized absorbance traces for monomeric AAC1 (blue), DIC (red), OGC (green), CIC (cyan), UCP1 (brown), and dimeric AGC2 (purple) are shown. The standards used for sizing were ferritin (440 kDa), aldolase (158 kDa), conalbumin (76 kDa), and ovalbumin (43 kDa). The dotted line indicates the elution volume of a hypothetical dimer peak for DIC, OGC, and CIC. .

Article Snippet: Genes encoding the human oxoglutarate carrier (OGC, SLC25A11, UniProt accession code: Q02978 ), the human citrate carrier (CIC, SLC25A1, UniProt accession code: P53007 ), the human dicarboxylate carrier (DIC, SLC25A10, UniProt accession code: Q9UBX3 ) and the human aspartate/glutamate carrier 2 (AGC2, SLC25A13, UniProt accession code: Q9UJS0 ) were codon-optimized with an N-terminal eight histidine tag and either Xa (OGC, CIC and DIC) or TEV (AGC2) protease cleavage sites to aid purification (OGC, ∆2-13; CIC, ∆2-12; DIC, GKP added (positions 2–4)) (synthesized by GenScript).

Techniques: Size-exclusion Chromatography

Journal: The EMBO Journal

Article Title: Human mitochondrial carriers of the SLC25 family function as monomers exchanging substrates with a ping-pong kinetic mechanism

doi: 10.1038/s44318-024-00150-0

Figure Lengend Snippet: Proteoliposomes containing human dicarboxylate carrier were loaded with either 0.10 mM (red traces), 0.25 mM (orange traces), 0.50 mM (green traces), or 1.00 mM (blue traces) malate int , and transport was initiated by the externally added radiolabeled malate at either 1.0 μM (filled circles), 1.5 μM (filled squares), 2.5 μM (filled upward triangles), 5 μM (filled downward triangles), 10 μM (open circles), 15 μM (open squares), 20 μM (open upward triangles), 25 μM (open downward triangles), or 50 μM (crosses) [ 14 C]-malate (malate ext ). Initial rates were estimated by fitting the uptake data to Eq. . The data represent the average and standard deviation of n = 6 (two independent experiments, each with three technical repeats, except the 1 and 50 μM external [ 14 C]-malate datasets, which are the average of three technical repeats). .

Article Snippet: Genes encoding the human oxoglutarate carrier (OGC, SLC25A11, UniProt accession code: Q02978 ), the human citrate carrier (CIC, SLC25A1, UniProt accession code: P53007 ), the human dicarboxylate carrier (DIC, SLC25A10, UniProt accession code: Q9UBX3 ) and the human aspartate/glutamate carrier 2 (AGC2, SLC25A13, UniProt accession code: Q9UJS0 ) were codon-optimized with an N-terminal eight histidine tag and either Xa (OGC, CIC and DIC) or TEV (AGC2) protease cleavage sites to aid purification (OGC, ∆2-13; CIC, ∆2-12; DIC, GKP added (positions 2–4)) (synthesized by GenScript).

Techniques: Standard Deviation

Journal: bioRxiv

Article Title: Evolutionary divergence of anaphase spindle mechanics in nematode embryos constrained by antagonistic pulling and viscous forces

doi: 10.1101/2021.08.10.455863

Figure Lengend Snippet: The diversity in spindle movement in single celled embryos of related nematode species is seen in the DIC images. These images were used to estimate the change in position of the spindle mid-plane along the AP axis (black lines) and the transverse movements of the spindle poles (blue: anterior, red: posterior). Some of the species display oscillations of spindle poles in the transverse direction, while some even display AP oscillations.

Article Snippet: Embryo images were partitioned into anterior, middle and posterior regions and over 1,000 granules in the anterior posterior portion of each embryo were tracked ( ) using a previously developed MATLAB code for single particle tracking in DIC images ( ).

Techniques:

Journal: bioRxiv

Article Title: Evolutionary divergence of anaphase spindle mechanics in nematode embryos constrained by antagonistic pulling and viscous forces

doi: 10.1101/2021.08.10.455863

Figure Lengend Snippet: (A) Representative tracked granules from WT are compared to embryos treated with RNAi targeting ATP2 and CYC-1 are overlaid on DIC images of the embryo in the anterior (blue), mid-cell (magenta) and posterior (red) regions. (B) The MSD (red, ) with time is plotted for whole embryos corresponding to (A) . The grey area is the s.d. The data was fit using the anomalous diffusion model, (blue) to estimate the effective diffusion coefficient (D eff ) and anomaly coefficient (α). (C) The mean D eff (error bar: 2 SEM) and (D) effective viscosity are compared between untreated and treated embryos. The mean values were compared using a KS test; ns: not significant.

Article Snippet: Embryo images were partitioned into anterior, middle and posterior regions and over 1,000 granules in the anterior posterior portion of each embryo were tracked ( ) using a previously developed MATLAB code for single particle tracking in DIC images ( ).

Techniques: Diffusion-based Assay, Viscosity