Fig. 5: uSCL35A4 is a mitochondrial outer membrane protein and regulates cell proliferation and mitochondrial membrane potential. | Nature Communications

Fig. 5: uSCL35A4 is a mitochondrial outer membrane protein and regulates cell proliferation and mitochondrial membrane potential.

From: Widespread stable noncanonical peptides identified by integrated analyses of ribosome profiling and ORF features

Fig. 5

a, b uSLC35A4 protein interacts with mitochondrial outer membrane proteins. Immunoblotting analysis of uSLC35A4 co-IP lysates (a). Co-immunostaining analysis of uSLC35A4 protein using uSLC35A4_Flag stably expressing MCF-7 cells (b). VDAC1/3 and TOMM20 are mitochondrial outer membrane (OM) markers, AIF is the inner membrane space (IMS) marker, and OXCT1 is the matrix marker. Pseudocolored confocal images are shown on the left. Scale bar, 10 μm. The intensity profiles of Flag (red) and indicated mitochondrial marker (green) along the white arrows in the merged images are shown on the right. c Overexpression of wild-type but not start codon-mutated uSLC35A4 impairs MCF-7 Cell growth. Data are shown as mean values ± SD of four replicates. (***P < 0.001, P = 0.0009; ns, not significant, P = 0.2452; two-tailed t-test). d Heatmap showing differentially expressed genes in uSLC35A4_Flag stably expressing MCF-7 cells. The cells expressing start codon-mutated (AAA instead of AUG) ORF sequences were used as the control. Blue: down-regulated; Red: up-regulated. OE, overexpression. e Gene ontology analyses of differentially expressed genes in (d). f, g The mitochondrial membrane potential of MCF-7 cells measured by TMRE (tetramethylrhodamine, ethyl ester) staining. CCCP (a mitochondrial oxidative phosphorylation uncoupler) was used as system control. Representative confocal images are shown in (f). Scale bar, 25 μm. The statistic results are shown as mean values ± SD of five replicates in (g). Compared with control, wide-type uSLC35A4 overexpression (OE_WT) decreased MMP (***P < 0.001, P = 0.0003), which was rescued by uSLC35A4 knock out (KO) (**P < 0.01, P = 0.0023). Cell growth was unaffected by overexpressing mutated uSLC35A4 (OE_AAA) or a combination with KO (ns not significant, two-tailed t-test). Experiments (a–c, and f–g) were performed three times with similar results. Source data are provided as a Source Data file.

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