slc25a46  (Proteintech)

Journal: The Journal of Biological Chemistry

Article Title: Identification of SLC25A46 interaction interfaces with mitochondrial membrane fusogens Opa1 and Mfn2

doi: 10.1016/j.jbc.2024.107740

Figure Lengend Snippet: SLC25A46 interacts with the bundle signaling element (BSE) of Opa1. A , domain diagrams of SLC25A46 and Opa1. s-Opa1 (amino acids 253-960) and Opa1(MGD) (minimal GTPase domain comprising the GTPase domain and three BSE helices) are shown as line diagrams below full length Opa1 sequence. B , GDN extracts prepared from Pichia pastoris expressing SLC25A46-His, Strep-Opa1, or coexpressing SLC25A46-His and Strep-Opa1 subjected to StrepTactin column binding and elution. C , cross-links identified in BSE helices 2 and 3 of Opa1. Circular map of SLC25A46 and Opa1 with identified lysine-lysine crosslinks ( black arcs ). Crosslinker used to generate the crosslink is indicated. D , AlphaFold 2 model of the SLC25A46-Opa1 interaction. N-terminal region of SLC25A46 comprising residues 1 to 83 (disordered) not displayed, residues 1 to 263 of Opa1 not modeled. Box region displaying identified cross-links and the calculated Cɑ-Cɑ distances based on the model. GDN, glyco-diosgenin; Opa1, optic atrophy 1; SLC25A46, solute carrier family 25 member A46.

Article Snippet: Genes for SLC25A46, Mfn2, Opa1(253-960), and Opa1(MGD) constructs were synthesized (GenScript) and cloned into pPICZ A for P. pastoris expression.

Techniques: Sequencing, Expressing, Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: Identification of SLC25A46 interaction interfaces with mitochondrial membrane fusogens Opa1 and Mfn2

doi: 10.1016/j.jbc.2024.107740

Figure Lengend Snippet: SLC25A46 mutations in the binding interface diminish binding to Opa1. A , SLC25A46-Opa1 interface observed in AlphaFold 2 model. Top view: adjacent to the salt bridges is a cluster of hydrophobic residues comprising of I229, I230, I349, Y357, V359, and L360 of SLC25A46 and T576, F572, and A569 of Opa1. Bottom view: predicted salt-bridge interactions between R257 of SLC25A46 and E561 of Opa1, and R347 of SLC25A46 and D565 of Opa1. Interface residues highlighted. Mutated residues indicated in red . B , purified SLC25A46-His WT, I349D mutant or R257A/R347A double mutant were incubated with 2 μM of Strep-Opa1 and subjected to StrepTactin binding and elution experiments. Input and elution analyzed by Western blotting. Opa1, optic atrophy 1; SLC25A46, solute carrier family 25 member A46.

Article Snippet: Genes for SLC25A46, Mfn2, Opa1(253-960), and Opa1(MGD) constructs were synthesized (GenScript) and cloned into pPICZ A for P. pastoris expression.

Techniques: Binding Assay, Purification, Mutagenesis, Incubation, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Identification of SLC25A46 interaction interfaces with mitochondrial membrane fusogens Opa1 and Mfn2

doi: 10.1016/j.jbc.2024.107740

Figure Lengend Snippet: SLC25A46 interactions with Mfn2. A , domain diagrams of full length and truncated constructs of SLC25A46 and Mfn2 are depicted. B , SLC25A46-His coelutes upon elution of bound Mfn2-Strep. GDN extracts prepared from Pichia pastoris expressing Mfn2-Strep or coexpressing SLC25A46-His and Mfn2-Strep were subjected to StrepTactin column binding and elution. C , circular map of SLC25A46 and Mfn2 with the identified lysine-lysine crosslinks ( black arcs ). Crosslinker used is indicated. Cross-links were identified between the cytosol-exposed N-terminal K60 of SLC25A46 and the HR1 lysines K416 and K420 of Mfn2. D , model of the SLC25A46-Mfn2 interaction. The identified cross-links are depicted as dotted lines in the AlphaFold 2 generated models of SLC25A46 and Mfn2. Helical bundle 1 (HB1) indicated. The N-terminal region of SLC25A46 comprising residues 1 to 83 is predicted to be disordered and is displayed as a hand-drawn cartoon. The transmembrane helices of Mfn2 were poorly modeled and are shown as transparent cylinders, to indicate uncertainty. E , (Δ2-83)SLC25A46-His coelutes upon elution of bound Mfn2-Strep. GDN extracts prepared from P. pastoris expressing Mfn2-Strep and coexpressing SLC25A46-His or (Δ2-83)SLC25A46-His were subjected to StrepTactin column binding and elution. F , residues 1 to 88 of SLC25A46 fused to MBP and His tag at the C terminus (SLC25A46 (1-88)-MBP-His) does not coelute with immobilized Mfn2-Strep. G , a Mfn2 lacking the two transmembrane segments (Mfn2 (ΔTM)-Strep) coelutes with SLC25A46-His. H , a minimal Mfn2 construct comprising helical bundle 1 (HB1) and the GTPase domain coelutes with SLC25A46-His. GDN, glyco-diosgenin; HR1, heptad repeat 1; SLC25A46, solute carrier family 25 member A46; TM, transmembrane; Mfn, mitofusin 2.

Article Snippet: Genes for SLC25A46, Mfn2, Opa1(253-960), and Opa1(MGD) constructs were synthesized (GenScript) and cloned into pPICZ A for P. pastoris expression.

Techniques: Construct, Expressing, Binding Assay, Generated

Journal: The Journal of Biological Chemistry

Article Title: Identification of SLC25A46 interaction interfaces with mitochondrial membrane fusogens Opa1 and Mfn2

doi: 10.1016/j.jbc.2024.107740

Figure Lengend Snippet: Mutations disrupting Opa1 interaction alter mitochondrial morphology. A , identified SLC25A46 disease mutations mapped on AlphaFold 2 model of SLC25A46-Opa1 complex. Box regions enlarged on the right to display the mutations. B , graph bar representing the relative proportion of scored mitochondrial morphologies in HCT116 WT, SLC25A46-KO, or SLC25A46-KO cells expressing SLC25A46 variants. Mitochondrial morphology for 300 cells were scored for each condition and evaluated (see Materials and Methods). Significance of difference is tested relative to WT using unpaired t test with Welch’s correction. Blue ∗∗∗∗: p < 0.0001 when compared to scored WT cells showing fragmented mitochondria. Green ∗: p < 0.05 when compared to scored WT cells showing interconnected mitochondrial network. C , Western blot of expression levels for Opa1, Mfn2, SLC25A46, or MTCO1 in HCT116 WT, SLC25A46-KO, or SLC25A46-KO cells expressing SLC25A46 variants. (Δ2-83)SLC25A46 not detected as commercially available antibodies detect the N-terminal epitope of SLC25A46. MTCO1 is used as loading control. Mfn, mitofusin 2; Opa1, optic atrophy 1; SLC25A46, solute carrier family 25 member A46.

Article Snippet: Genes for SLC25A46, Mfn2, Opa1(253-960), and Opa1(MGD) constructs were synthesized (GenScript) and cloned into pPICZ A for P. pastoris expression.

Techniques: Expressing, Western Blot, Control

Journal: bioRxiv

Article Title: SLC25A46 is in contact with lysosomes and plays a role in mitochondrial cholesterol homeostasis

doi: 10.1101/2024.04.01.587618

Figure Lengend Snippet: SLC25A46 is required for starvation-induced mitochondrial hyperfusion . The mitochondrial morphology of control fibroblasts and the SLC25A46 knock-out cell line was analyzed by immunofluorescence after treatment with cycloheximide (CHX) for 2 hours, with HBSS for 4 hours, with the mTOR inhibitor Ink128 for 24 h or CCCP for 1 hour. (a) Representative images of fibroblasts decorated with anti-PRDX3 (mitochondria) in white and DAPI (nuclei) in blue. A zoomed image of the indicated area is shown on the right. Scale bars: 10 µm. Images were analyzed using MitoMAPR to describe number of mitochondrial networks (b) and the average length of mitochondrial networks (c) (N=3, n=10) per condition shown as violin plots. (d) SDS-PAGE analysis of steady-state levels of indicated proteins in control fibroblasts and SLC25A46 knock-out cells after the indicated treatments.

Article Snippet: Antibodies directed against the following proteins were used in this study: SLC25A46 (Santa Cruz; G2-SC-515823 and Proteintech; 12277-1-AP), MFN2 (Cell Signaling; 11925S), MFN1 (Cell Signaling; 14739S), OPA1 (BD bioscience, 612607) for WB, VDAC1 (Abcam; ab14734), 4E-BP (Cell Signaling; 9644S), Cav1 (BD bioscience; 610060).

Techniques: Knock-Out, Immunofluorescence, SDS Page

Journal: bioRxiv

Article Title: SLC25A46 is in contact with lysosomes and plays a role in mitochondrial cholesterol homeostasis

doi: 10.1101/2024.04.01.587618

Figure Lengend Snippet: SLC25A46 knock-out cells have differential mRNA expression for genes encoding lysosomal proteins. Transcriptomic data was analysed for control and SLC25A46 knock-out cell lines in fibroblasts and HeLa cells. (a) Principle component analysis of gene expression quantified by RNA-seq in control and SLC25A46 knock-out fibroblast cells. (b) Only transcripts that were differently expressed in both cell lines (adjusted p-value < 0.1, Log2FC > |0.3|) were chosen to be analyzed (n = 1,790) (SLC25A46-responsive subset). (c) List of Gene Ontology (GO) terms with their p- value and the number of proteins of the group.

Article Snippet: Antibodies directed against the following proteins were used in this study: SLC25A46 (Santa Cruz; G2-SC-515823 and Proteintech; 12277-1-AP), MFN2 (Cell Signaling; 11925S), MFN1 (Cell Signaling; 14739S), OPA1 (BD bioscience, 612607) for WB, VDAC1 (Abcam; ab14734), 4E-BP (Cell Signaling; 9644S), Cav1 (BD bioscience; 610060).

Techniques: Knock-Out, Expressing, RNA Sequencing Assay

Journal: bioRxiv

Article Title: SLC25A46 is in contact with lysosomes and plays a role in mitochondrial cholesterol homeostasis

doi: 10.1101/2024.04.01.587618

Figure Lengend Snippet: (a) Immunofluorescence analysis of SLC25A46 contacts with lysosomes in fibroblasts stably overexpressing SLC25A46-GFP (green). Mitochondria were stained with MitoTracker Deep Red (white) and lysosomes were stained with LysoTracker Red (magenta). Images were captured every 1 s for a period of 1 min. Time-lapse imaging of mitochondria-lysosome contact. Orange arrows indicate SLC25A46-GFP signal that contacts a lysosome. Scale bar: 10 μm. (b) Quantification of mitochondria-lysosome contacts positive for SLC25A46. Five cells were analyzed with a total of 94 mitochondria- lysosome contacts. (c) Quantification of the percentage of lysosomes in contact with mitochondria in control fibroblasts and in SLC25A46 knock-out cell line. Seven cells per condition were analyzed with a total of 230 lysosomes in control cells and 188 lysosomes in knock-out cells. (d) Quantification of mitochondrial fission sites with a lysosome present (lysosome pos) and positive for SLC25A46 (SLC25A46-GFP pos). Seven cells were analysed with 22 fission events. Data shown as mean+SEM.

Article Snippet: Antibodies directed against the following proteins were used in this study: SLC25A46 (Santa Cruz; G2-SC-515823 and Proteintech; 12277-1-AP), MFN2 (Cell Signaling; 11925S), MFN1 (Cell Signaling; 14739S), OPA1 (BD bioscience, 612607) for WB, VDAC1 (Abcam; ab14734), 4E-BP (Cell Signaling; 9644S), Cav1 (BD bioscience; 610060).

Techniques: Immunofluorescence, Stable Transfection, Staining, Imaging, Knock-Out

Journal: bioRxiv

Article Title: SLC25A46 is in contact with lysosomes and plays a role in mitochondrial cholesterol homeostasis

doi: 10.1101/2024.04.01.587618

Figure Lengend Snippet: SLC25A46 influences mitochondrial cholesterol homeostasis. Heatmap of differently expressed (adjusted p-value < 0.1, Log2FC > |0.3|) SLC25A46- responsive transcripts in control and SLC25A46 knock-out fibroblasts coding for proteins involved in fatty acid biosynthesis (GO:0006633) (a) and cholesterol binding (GO:0015485) (b). Log2 fold changes are centered and represented as z-scores. Total cholesterol and non- esterified (“free”) cholesterol in whole cells (c) and in sucrose-gradient isolated mitochondria (d) from control fibroblasts, SLC25A46 knock-out cells (KO) and SLC25A46 knock-out cells expressing the wildtype SLC25A46 protein (Rescue) was measured with a Cholesterol/Cholesteryl Ester Assay Kit (Abcam, ab65359).

Article Snippet: Antibodies directed against the following proteins were used in this study: SLC25A46 (Santa Cruz; G2-SC-515823 and Proteintech; 12277-1-AP), MFN2 (Cell Signaling; 11925S), MFN1 (Cell Signaling; 14739S), OPA1 (BD bioscience, 612607) for WB, VDAC1 (Abcam; ab14734), 4E-BP (Cell Signaling; 9644S), Cav1 (BD bioscience; 610060).

Techniques: Knock-Out, Binding Assay, Isolation, Expressing

Journal: bioRxiv

Article Title: SLC25A46 is in contact with lysosomes and plays a role in mitochondrial cholesterol homeostasis

doi: 10.1101/2024.04.01.587618

Figure Lengend Snippet: Immunofluorescence analysis of cholesterol trafficking in control human fibroblasts and SLC25A46 knock-out cells. Cells were pulsed for 2 min with the fluorescent cholesterol probe TopFluor® (in magenta) and fixed after the indicated time to track the probe. PRDX3 was used as a mitochondrial marker (green). A zoomed image of the boxed area is shown on the right. Scale bars: 10 μm. (b) Quantification of colocalizing TopFluor signal with mitochondria divided by the total TopFluor signal in the cell at indicated time points. n=10 per condition.

Article Snippet: Antibodies directed against the following proteins were used in this study: SLC25A46 (Santa Cruz; G2-SC-515823 and Proteintech; 12277-1-AP), MFN2 (Cell Signaling; 11925S), MFN1 (Cell Signaling; 14739S), OPA1 (BD bioscience, 612607) for WB, VDAC1 (Abcam; ab14734), 4E-BP (Cell Signaling; 9644S), Cav1 (BD bioscience; 610060).

Techniques: Immunofluorescence, Knock-Out, Marker

Journal: bioRxiv

Article Title: SLC25A46 is in contact with lysosomes and plays a role in mitochondrial cholesterol homeostasis

doi: 10.1101/2024.04.01.587618

Figure Lengend Snippet: Cholesterol rescues the mitochondrial fragmentation phenotype of the SLC25A46 knock-out cell line and interaction of SLC25A46 with Cav1. The mitochondrial morphology of control and SLC25A46 human fibroblasts was analyzed after treating the cells with 20 μM cholesterol for 4h. Representative immunofluorescent images with the mitochondrial marker PRDX3 (white) (a). Scale bars: 10 μm. Images were analyzed using MitoMAPR across max projections keeping all parameters constant (N=3, n=30) to describe number of mitochondrial networks (b) and the average length of mitochondrial networks (c) per condition shown as violin plots. SDS-PAGE analysis of control and SLC25A46 knock-out cell lines (d). VDAC was used as a loading control. (e) Venn diagram of proteins co- immunoprecipitated with SLC25A46. Two different antibodies (G2: Santa Cruz; G2-SC-515823 and Proteintech: 12277-1-AP) against the endogenous SLC25A46 protein we used for immunoprecipitation of crude mitochondria isolated from control fibroblasts. The co- immunoprecipitated proteins were identified by mass spectrometry.

Article Snippet: Antibodies directed against the following proteins were used in this study: SLC25A46 (Santa Cruz; G2-SC-515823 and Proteintech; 12277-1-AP), MFN2 (Cell Signaling; 11925S), MFN1 (Cell Signaling; 14739S), OPA1 (BD bioscience, 612607) for WB, VDAC1 (Abcam; ab14734), 4E-BP (Cell Signaling; 9644S), Cav1 (BD bioscience; 610060).

Techniques: Knock-Out, Marker, SDS Page, Immunoprecipitation, Isolation, Mass Spectrometry

Journal: bioRxiv

Article Title: SLC25A46 is in contact with lysosomes and plays a role in mitochondrial cholesterol homeostasis

doi: 10.1101/2024.04.01.587618

Figure Lengend Snippet: An altered cellular response during nutrient starvation in the SLC25A46 knock- out cells. (a) Kernel density estimation adjusted p-value distributions for differentially expression genes in control and SLC25A46 KO cells upon treatment with HBSS, as well as genes where the differential expression upon HBSS treatment was dependent on SLC25A46 expression. A higher density of low adjusted p-values is indicative of enrichment in changes in gene expression. Heatmaps of changes in levels of transcripts of lysosomal proteins (GO:0005764) (b), proteins involved in fatty acid biosynthesis (GO:0006633) (c) and in cholesterol binding (GO:0015485) (d) in control and SLC25A46 knock-out fibroblasts in normal conditions and during nutrient starvation. Genes displayed are those where HBSS-induced changes in expression were dependent on SLC25A46 in the DESeq2 model with the design “expression ∼ genotype + condition + genotype x condition” (adjusted p-value < 0.1 and Log2FC > |0.3| for the “genotype x condition” term). Log2 fold changes are centered and represented as z-scores. (e) List of Gene Ontology (GO) terms with their p-value and the number of proteins in the group. (f) Representative images of the mitochondrial morphology of control and SLC25A46 human fibroblasts after 4 hours of incubation in normal medium (DMEM) or under nutrient starvation (HBSS), either without supplementation (no treatment) or with supplementation of 20 μM cholesterol for 4h. Mitochondrial marker PRDX3 (white). Scale bars: 10 μm. (g) Images were analyzed using MitoMAPR on ROIs keeping all parameters constant to quantify number of junction per mitochondrial networks (N=3, n=30).

Article Snippet: Antibodies directed against the following proteins were used in this study: SLC25A46 (Santa Cruz; G2-SC-515823 and Proteintech; 12277-1-AP), MFN2 (Cell Signaling; 11925S), MFN1 (Cell Signaling; 14739S), OPA1 (BD bioscience, 612607) for WB, VDAC1 (Abcam; ab14734), 4E-BP (Cell Signaling; 9644S), Cav1 (BD bioscience; 610060).

Techniques: Knock-Out, Expressing, Binding Assay, Incubation, Marker

Journal: bioRxiv

Article Title: SLC25A46 is in contact with lysosomes and plays a role in mitochondrial cholesterol homeostasis

doi: 10.1101/2024.04.01.587618

Figure Lengend Snippet: SLC25A46 is required for starvation-induced mitochondrial hyperfusion . The mitochondrial morphology of control fibroblasts and the SLC25A46 knock-out cell line was analyzed by immunofluorescence after treatment with cycloheximide (CHX) for 2 hours, with HBSS for 4 hours, with the mTOR inhibitor Ink128 for 24 h or CCCP for 1 hour. (a) Representative images of fibroblasts decorated with anti-PRDX3 (mitochondria) in white and DAPI (nuclei) in blue. A zoomed image of the indicated area is shown on the right. Scale bars: 10 µm. Images were analyzed using MitoMAPR to describe number of mitochondrial networks (b) and the average length of mitochondrial networks (c) (N=3, n=10) per condition shown as violin plots. (d) SDS-PAGE analysis of steady-state levels of indicated proteins in control fibroblasts and SLC25A46 knock-out cells after the indicated treatments.

Article Snippet: In these two experiments we used two different antibodies (G2 and Proteintech), both indicating an interaction of SLC25A46 with Cav1 ( , Supplementary Table 2).

Techniques: Control, Knock-Out, Immunofluorescence, SDS Page

Journal: bioRxiv

Article Title: SLC25A46 is in contact with lysosomes and plays a role in mitochondrial cholesterol homeostasis

doi: 10.1101/2024.04.01.587618

Figure Lengend Snippet: SLC25A46 knock-out cells have differential mRNA expression for genes encoding lysosomal proteins. Transcriptomic data was analysed for control and SLC25A46 knock-out cell lines in fibroblasts and HeLa cells. (a) Principle component analysis of gene expression quantified by RNA-seq in control and SLC25A46 knock-out fibroblast cells. (b) Only transcripts that were differently expressed in both cell lines (adjusted p-value < 0.1, Log2FC > |0.3|) were chosen to be analyzed (n = 1,790) (SLC25A46-responsive subset). (c) List of Gene Ontology (GO) terms with their p- value and the number of proteins of the group.

Article Snippet: In these two experiments we used two different antibodies (G2 and Proteintech), both indicating an interaction of SLC25A46 with Cav1 ( , Supplementary Table 2).

Techniques: Knock-Out, Expressing, Control, Gene Expression, RNA Sequencing

Journal: bioRxiv

Article Title: SLC25A46 is in contact with lysosomes and plays a role in mitochondrial cholesterol homeostasis

doi: 10.1101/2024.04.01.587618

Figure Lengend Snippet: (a) Immunofluorescence analysis of SLC25A46 contacts with lysosomes in fibroblasts stably overexpressing SLC25A46-GFP (green). Mitochondria were stained with MitoTracker Deep Red (white) and lysosomes were stained with LysoTracker Red (magenta). Images were captured every 1 s for a period of 1 min. Time-lapse imaging of mitochondria-lysosome contact. Orange arrows indicate SLC25A46-GFP signal that contacts a lysosome. Scale bar: 10 μm. (b) Quantification of mitochondria-lysosome contacts positive for SLC25A46. Five cells were analyzed with a total of 94 mitochondria- lysosome contacts. (c) Quantification of the percentage of lysosomes in contact with mitochondria in control fibroblasts and in SLC25A46 knock-out cell line. Seven cells per condition were analyzed with a total of 230 lysosomes in control cells and 188 lysosomes in knock-out cells. (d) Quantification of mitochondrial fission sites with a lysosome present (lysosome pos) and positive for SLC25A46 (SLC25A46-GFP pos). Seven cells were analysed with 22 fission events. Data shown as mean+SEM.

Article Snippet: In these two experiments we used two different antibodies (G2 and Proteintech), both indicating an interaction of SLC25A46 with Cav1 ( , Supplementary Table 2).

Techniques: Immunofluorescence, Stable Transfection, Staining, Imaging, Control, Knock-Out

Journal: bioRxiv

Article Title: SLC25A46 is in contact with lysosomes and plays a role in mitochondrial cholesterol homeostasis

doi: 10.1101/2024.04.01.587618

Figure Lengend Snippet: SLC25A46 influences mitochondrial cholesterol homeostasis. Heatmap of differently expressed (adjusted p-value < 0.1, Log2FC > |0.3|) SLC25A46- responsive transcripts in control and SLC25A46 knock-out fibroblasts coding for proteins involved in fatty acid biosynthesis (GO:0006633) (a) and cholesterol binding (GO:0015485) (b). Log2 fold changes are centered and represented as z-scores. Total cholesterol and non- esterified (“free”) cholesterol in whole cells (c) and in sucrose-gradient isolated mitochondria (d) from control fibroblasts, SLC25A46 knock-out cells (KO) and SLC25A46 knock-out cells expressing the wildtype SLC25A46 protein (Rescue) was measured with a Cholesterol/Cholesteryl Ester Assay Kit (Abcam, ab65359).

Article Snippet: In these two experiments we used two different antibodies (G2 and Proteintech), both indicating an interaction of SLC25A46 with Cav1 ( , Supplementary Table 2).

Techniques: Control, Knock-Out, Binding Assay, Isolation, Expressing

Journal: bioRxiv

Article Title: SLC25A46 is in contact with lysosomes and plays a role in mitochondrial cholesterol homeostasis

doi: 10.1101/2024.04.01.587618

Figure Lengend Snippet: Immunofluorescence analysis of cholesterol trafficking in control human fibroblasts and SLC25A46 knock-out cells. Cells were pulsed for 2 min with the fluorescent cholesterol probe TopFluor® (in magenta) and fixed after the indicated time to track the probe. PRDX3 was used as a mitochondrial marker (green). A zoomed image of the boxed area is shown on the right. Scale bars: 10 μm. (b) Quantification of colocalizing TopFluor signal with mitochondria divided by the total TopFluor signal in the cell at indicated time points. n=10 per condition.

Article Snippet: In these two experiments we used two different antibodies (G2 and Proteintech), both indicating an interaction of SLC25A46 with Cav1 ( , Supplementary Table 2).

Techniques: Immunofluorescence, Control, Knock-Out, Marker

Journal: bioRxiv

Article Title: SLC25A46 is in contact with lysosomes and plays a role in mitochondrial cholesterol homeostasis

doi: 10.1101/2024.04.01.587618

Figure Lengend Snippet: Cholesterol rescues the mitochondrial fragmentation phenotype of the SLC25A46 knock-out cell line and interaction of SLC25A46 with Cav1. The mitochondrial morphology of control and SLC25A46 human fibroblasts was analyzed after treating the cells with 20 μM cholesterol for 4h. Representative immunofluorescent images with the mitochondrial marker PRDX3 (white) (a). Scale bars: 10 μm. Images were analyzed using MitoMAPR across max projections keeping all parameters constant (N=3, n=30) to describe number of mitochondrial networks (b) and the average length of mitochondrial networks (c) per condition shown as violin plots. SDS-PAGE analysis of control and SLC25A46 knock-out cell lines (d). VDAC was used as a loading control. (e) Venn diagram of proteins co- immunoprecipitated with SLC25A46. Two different antibodies (G2: Santa Cruz; G2-SC-515823 and Proteintech: 12277-1-AP) against the endogenous SLC25A46 protein we used for immunoprecipitation of crude mitochondria isolated from control fibroblasts. The co- immunoprecipitated proteins were identified by mass spectrometry.

Article Snippet: In these two experiments we used two different antibodies (G2 and Proteintech), both indicating an interaction of SLC25A46 with Cav1 ( , Supplementary Table 2).

Techniques: Knock-Out, Control, Marker, SDS Page, Immunoprecipitation, Isolation, Mass Spectrometry

Journal: bioRxiv

Article Title: SLC25A46 is in contact with lysosomes and plays a role in mitochondrial cholesterol homeostasis

doi: 10.1101/2024.04.01.587618

Figure Lengend Snippet: An altered cellular response during nutrient starvation in the SLC25A46 knock- out cells. (a) Kernel density estimation adjusted p-value distributions for differentially expression genes in control and SLC25A46 KO cells upon treatment with HBSS, as well as genes where the differential expression upon HBSS treatment was dependent on SLC25A46 expression. A higher density of low adjusted p-values is indicative of enrichment in changes in gene expression. Heatmaps of changes in levels of transcripts of lysosomal proteins (GO:0005764) (b), proteins involved in fatty acid biosynthesis (GO:0006633) (c) and in cholesterol binding (GO:0015485) (d) in control and SLC25A46 knock-out fibroblasts in normal conditions and during nutrient starvation. Genes displayed are those where HBSS-induced changes in expression were dependent on SLC25A46 in the DESeq2 model with the design “expression ∼ genotype + condition + genotype x condition” (adjusted p-value < 0.1 and Log2FC > |0.3| for the “genotype x condition” term). Log2 fold changes are centered and represented as z-scores. (e) List of Gene Ontology (GO) terms with their p-value and the number of proteins in the group. (f) Representative images of the mitochondrial morphology of control and SLC25A46 human fibroblasts after 4 hours of incubation in normal medium (DMEM) or under nutrient starvation (HBSS), either without supplementation (no treatment) or with supplementation of 20 μM cholesterol for 4h. Mitochondrial marker PRDX3 (white). Scale bars: 10 μm. (g) Images were analyzed using MitoMAPR on ROIs keeping all parameters constant to quantify number of junction per mitochondrial networks (N=3, n=30).

Article Snippet: In these two experiments we used two different antibodies (G2 and Proteintech), both indicating an interaction of SLC25A46 with Cav1 ( , Supplementary Table 2).

Techniques: Knock-Out, Expressing, Control, Quantitative Proteomics, Gene Expression, Binding Assay, Incubation, Marker