Fig. 5: SMIM4 is required for SLC25A1-mediated mitochondrial malate/citrate transportation. | Nature Communications

Fig. 5: SMIM4 is required for SLC25A1-mediated mitochondrial malate/citrate transportation.

From: The integral membrane protein smim4 modulates redox balance via malate compartmentalization in pancreatic cancer

Fig. 5: SMIM4 is required for SLC25A1-mediated mitochondrial malate/citrate transportation.The alternative text for this image may have been generated using AI.

A Mitochondrial extracts from WT and SMIM4-FLAG HEK-293T cells analyzed by native IP and quantitative LC-MS/MS. Black: published data41, Gray: our experimental results (n = 3 biologically independent samples). B 3%–12.5% BN-PAGE/immunoblot analysis of SLC25A1 in WT, SMIM4-depleted, and SLC25A1-knockout MIA PaCa-2 cells. C WB of SMIM4 and SLC25A1 in MIA PaCa-2 cells with or without SMIM4 depletion. D, E FLAG or V5-immunoisolation of HEK-293T cells expressing SMIM4 FLAG, SLC25A1 V5, or both. F Molecular docking of SMIM4 with SLC25A1. G. Predicted ΔΔG_bind changes for interface mutations. H, I FLAG- and SLC25A1-immunoisolation in SMIM4 knockout MIA PaCa-2 cells rescued with WT or mutant SMIM4 variants. J Schematic of SLC25A1 sublocalization and sequence features and in vitro Co-IP of GST-SMIM4 with His-SLC25A1-IS. K–N Cell number (K, N) and lipid peroxidation (L, M) in WT, sgSMIM4, and sgSMIM4 cells rescued with SMIM4 variants under 24 h glucose deprivation ± 10 µM CTPI-2 (n = 3 biologically independent samples). O 3%–12.5% BN-PAGE/immunoblot analysis of SLC25A1-containing complexes in sgSMIM4 cells rescued with various SMIM4 variants in MIA PaCa-2 cells. P, Q Cellular (P) and cytosolic (Q) malate levels in MIA PaCa-2 cells with or without SMIM4 depletion, under 24 h glucose deprivation ± 10 µM CTPI-2 (n = 3 biologically independent samples). R–V Schematics illustrate potential fates of citrate derived from glutamine (R) or glucose (U). Fractional enrichment ratios (S, T, V) in MIA PaCa-2 cells with or without SMIM4 depletion and treatments as indicated (n = 5 biologically independent samples). W WB of SMIM4 and SLC25A1 in MIA PaCa-2 cells with or without SMIM4-KO and SLC25A1 depletion. X Malate uptake in isolated mitochondria from MIA PaCa-2 cells with or without SMIM4-KO or SLC25A1 depletion or CTPI-2 treatment (n = 3 biologically independent samples). Data are presented as means ± SEM for bar graphs from at least three independent experiments. Representative images for (B–E, H–J, O, and W) are from three independent biological replicates. Statistical significance was determined by unpaired two-tailed Student’s t-test for (A, K–N, P, Q, S, T, V, and X). ns not significant. Source data are provided as a Source Data file.

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