Skip to main content
. Author manuscript; available in PMC: 2024 Jun 15.
Published in final edited form as: Mol Cell. 2023 Jun 15;83(12):2059–2076.e6. doi: 10.1016/j.molcel.2023.05.031

Figure 3. DELE1 is stabilized on mitochondria by an iron deficiency-dependent mitochondrial import arrest.

Figure 3

(A) Tet-on DELE-HA stable HeLa cells were treated with DFO or CCCP for 16 hours. The subcellular localization of DELE1-HA was determined by immunocytochemistry (ICC). Scale bars; 10 μm. Line profiles for the indicated fluorescent intensities determined along the white lines are shown to the right. (B) Crude mitochondrial fractions were isolated from Endogenous DELE1-HA HEK293T cells that were treated with CHX for 30 min with or without the pre-treatment with DFO for 16 hours, and were subjected to in vitro proteinase K protection assay at the indicated proteinase K concentration in the indicated buffer conditions. Sensitivities of endogenous DELE1-HA and other mitochondrial marker proteins to proteinase K were determined by immunoblotting (IB).

(C) Schematic representation for the TEV cleavage assay. Suppression of the proteases-mediated cleavage of DELE1 by import arrest (upper panel). Insertion of the TEV cleavage sequence after the predicted MPP cleavage site of DELE1 [DELE1(TEV30)] (lower panel).

(D) DELE1 KO cells were transfected with the TEV sequence-inserted DELE1 (DELE1(TEV30)-HA) with or without the matrix-targeted TEV protease (Su9-TEV protease-HA). After 24 hours, cells were treated with DFO for the indicated time periods. Cell lysates were separated in an SDS-PAGE 7.5% polyacrylamide Midi gel and analyzed by IB.

(E) IB for lysates of endogenous DELE1-HA HEK293T cells treated with CHX for 30 min with or without pre-treatment of DFO for 16 hours (lane 1–4), or transfected with the indicated siRNAs for 72 hours, and subsequently treated with CHX for 30 min (lane 5–10).

(F) Knockdown efficiency of indicated proteins in (E) determined by IB.

(G to I) HeLa cells transiently transfected with tag (−) PINK1 (G), Tet-on Su9-DHFR-3xFlag stable HeLa cells (H), or Tet-on DELE1-HA stable HeLa cells (I), were treated with DFO, DFP or CCCP for 16 hours. Doxycycline was added 8 hours prior to DFO treatment to induce the expression of Su9-DHFR-3xFlag (H) or DELE1-HA (I). Cell lysates were analyzed by IB. *; non-specific bands.

See also Figure S2.