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
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![Instant-blue stained SDS-PAGE gel of purified protein ( A ) oxoglutarate carrier (OGC), ( E ) citrate carrier (CIC), ( I ) <t>dicarboxylate</t> 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. .](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_9753/pmc11329753/pmc11329753__44318_2024_150_Fig1_HTML.jpg)
![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). .](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_9753/pmc11329753/pmc11329753__44318_2024_150_Fig7_ESM.jpg)
