Extended Data Fig. 3: Related to relative HK1-OMM binding varies with nutrient conditions and HK detachment from the OMM promotes cell growth.
(a) Immunoblots for expression of HK2 in whole-cell lysates (WC) and mitochondria isolated (IP:HA) from K562 cells that expressed 3xMyc-eGFP-OMP25 (Control-MITO) or 3xHA-eGFP-OMP25 (HA-MITO). COXIV served as a mitochondrial loading control for HK1 (immunoblot displayed in Fig. 3a) and HK2 (immunoblot displayed in this panel) from the same samples. Immunoblot for expression of HK2 is identical to that displayed in Fig. 3a. (b) HK signal normalized by COXIV signal in IP:HA versus WC from the same respective cells. nd, not detected. Comparison of normalized signals between HK1 and HK2 is derived by using the same experimental samples run across different blots and processed in parallel. (c, i, m) Immunoblots for expression of HK1 and HK2 (c) or Flag (i, m) in dounce homogenates (DH) and corresponding cytosolic (Cyt) and organellar (Org) fractions isolated from HK2-knockout cells. GAPDH served as cytosolic control marker. COXIV served as a mitochondrial control marker. (d, j) HK (d) or Flag (j) signal normalized by COXIV signal in Org versus DH from the same respective cells. Comparisons of normalized signal between HK1 and HK2 are derived using the same experimental samples run across different blots and processed in parallel (d). Comparisons of normalized signal are derived from samples run on the same blot (j). (e, f, k, l) Immunoblots for expression of Flag (e, g), HK1 (e), or HK2 (f, k, l) in HK2-knockout and control cells. GAPDH served as the loading control in each case. (g) Immunoblot for expression of Flag in WC and IP:HA from HK2-knockout cells that expressed HA-MITO. COXIV served as a mitochondrial control marker. S6K served as a non-mitochondrial control marker. (h) HK1 (mean ± s.d., n = 3) or Flag signal normalized by COXIV signal in IP:HA versus WC from the same respective cells. P value, Two-tailed Welch’s t-test between the indicated bars identical to that displayed in Fig. 3c. For comparisons involving normalized Flag signal, MBD-deficient HK1 and MBD-deficient HK2 samples were run on the same blot. Wild-type HK1 samples were run on a different blot and were processed on a separate day. (n) Pseudocolor Coomassie-stained gel imaged using a LI-COR Odyssey FC. 1: TOM20-HK2-3xFLAG; 2: M.W. standards. HK1 signal normalized by COXIV signal in IP:HA versus WC from the same respective cells (mean ± s.e.m., n = 3 biologically independent samples). (o) G6P levels measured from reactions of recombinant TOM20-HK2 with glucose and ATP (mean ± s.d., n = 3 independent reactions). (p) Immunoblots for expression of HK1 and HK2 from HK2-knockout and control cells used for lysate-based HK activity assays. GAPDH served as the loading control in both cases. (q) Relative growth of HK2-knockout versus control cells (mean ± s.d., n = 3 biologically independent samples). Two-tailed Welch’s t-test comparing the respective mean ± s.d. (bar) versus mean ± s.d. (control cells) between bars. In a and c, L.B., low brightness; H.B., high brightness.
