Fig. 4

Metabolic stress regulates CHTM1-AIF1 interactions. (a) Representative fluorescent photomicrographs showing cellular localization of HA-tagged CHTM1 (Rhodamine stained, red) and endogenous AIF1 (FITC stained, green) in HeLa cells. Scale bar, 10 μM. (b, left panel) Western blot analyses of S-tag pull-down to demonstrate the interaction between endogenous AIF1 and exogenous HA-tagged CHTM1. (Middle panel) Western blot analyses of immunoprecipitation to demonstrate the interaction of endogenous AIF1 with endogenous CHTM1. (Right panel) Metabolic stress disrupts CHTM1-AIF1 interaction. S-tag pull-down assay was performed on A549 cells transiently transfected with HA-S-tagged CHTM1 or vector-only, and cultured in the presence or absence of glucose/glutamine for 4 h. (c, left upper panel) Schematic of deletion strategy in CHTM1 and Western blot analyses showing interactions between AIF1 and wild type (WT, full-length) or the deletion variants of CHTM1. (c, left lower panel) Western blot analyses showing interactions between AIF1 and wild type or the deletion variants of CHTM1. (c, right upper panel) Schematic of mutation strategy in CHTM1. (c, right lower panel, Western blot analyses showing increased AIF1 interaction with CHTM1 mutant, Mut3 harboring replacement of cysteine with alanine at positions 58, 68 and 89 (Lane 11). Decreased AIF1 interaction with CHTM1 mutant, Mut4 harboring replacement of cysteine with alanine at positions 58, 68, 79, 89 (Lane 12), indicating the contribution of residue C79 in CHTM1-AIF1 interaction