Extended Data Fig. 3: TANGO2 proteins regulate haem homeostasis in yeast and mammalian cells.
From: HRG-9 homologues regulate haem trafficking from haem-enriched compartments
a, Growth curve of wild type, hem1Δ, and tango2Δ yeast cells treated with or without 5-aminolevulinic acid (ALA). OD, optical density at 600 nm. n = 2 technical replicates. b, The tango2Δ yeast cells exhibit increased expression (middle) and activity (top) of Ctt1p. GAPDH is used as a control. c,d, Dynamics of haem trafficking to mitochondria (c) and the cytosol (d) in wild type and tango2Δ cells. Labile haem levels in mitochondria and the cytosol were monitored by measuring fractional saturation of the haem sensors specifically targeted to these two locations, respectively, following initiation of haem synthesis. e, Flag-tagged human TANGO2 colocalizes with the mitochondrial marker COX IV in HEK293 cells. Scale bars, 5 μm. f, Western analysis of TANGO2 in whole lysates as well as the mitochondrial and non-mitochondrial fractions of HEK293 cells. The mitochondrial protein COX IV and the cytosolic protein GAPDH were used as controls. g, Strategy to knock out TANGO2 in HEK293 cells and verification of the knockout by genotyping. The sgRNA targeting sites and the positions of genotyping primers are indicated. h, Immunoblotting analysis validating the knockout of TANGO2 in HEK293 cells. i, Total haem content of the wild type and ΔTANGO2 HEK293 cells after treatment with succinylacetone (SA) for 24 h. j, Strategy to knock out Tango2 in MEL cells and verification by PCR genotyping. The sgRNA targeting sites and the positions of genotyping primers are indicated. k, Immunoblotting analysis validating the knockout of Tango2 in MEL cells. l, o-dianisidine staining of haemoglobin in wild type and Tango2-knockout MEL cells. Scale bar, 20 μm. m,n, Basal oxygen consumption rates (OCR, m) and maximal OCR (n) of wild type and TANGO2-knockout HEK293 and MEL cells. o, Representative images of TMRE staining in wild type and TANGO2-knockout HEK293 and MEL cells. Scale bars, 20 μm. n = 3 independent experiments for c,d,i,m,n. Data in a,c,d,i,m,n are presented as mean ± s.e.m. P values were determined by one-way ANOVA followed by Tukey’s test.
