Extended Data Fig. 4 |. Characterization of TMEM175–AKT interaction and its role in TMEM175 activation.

a, Association between heterologously expressed TMEM175 and AKT. HEK293T cells were transfected with GFP-tagged wild-type or mutant TMEM175 and HA-tagged human AKT1 (HA–AKT), as indicated. Lysates were immunoprecipitated with anti-GFP and blotted with anti-GFP or with anti-AKT. Whole-cell lysate was also blotted for input control. b–d, Experiments demonstrating that S241 is not essential for TMEM175 activation. b, Sequence alignment of the region around S241 of TMEM175. GenBank access numbers are in parentheses. c, Lysosomal I_K recorded from HEK293T cells transfected with S241A/T338A or S241A substitutions. Averaged I_K sizes (at 100 mV) are shown in the bar graphs. Data are mean ± s.e.m. Numbers of lysosomes recorded are in parentheses. d, Association between wild-type or S241A-mutant TMEM175 and AKT. HA-tagged AKT was transfected alone (lane 1) in HEK293T cells or together with GFP-tagged wild-type or S241A-mutant human TMEM175 (lanes 2 and 3). Whole-cell lysates were blotted with anti-GFP (bottom) or anti-HA (middle), or immunoprecipitated with anti-GFP followed by immunoblotting with anti-HA (top). In c, arrows are used to indicate curves that overlap and are not easily distinguished. Black, recorded from bath containing K⁺; blue, recorded from bath containing K⁺ and SC79. e, Mapping the AKT-interacting region to the second repeat of TMEM175. A diagram illustrating the GFP-tagged TMEM175 fragments used in the experiments is shown. Lysates from HEK293T cells transfected with HA-tagged AKT1 (HA–AKT) alone (lane 1) or together with GFP-tagged TMEM175 fragments (lanes 2–6) were blotted with anti-GFP (bottom) or anti-HA (middle) or immunoprecipitated with anti-GFP followed by blotting with anti-HA (top). f, Phosphorylation status of T308 and S473 of TMEM175-associated AKT. HEK293T cells were transfected with GFP-tagged TMEM175 (with nontransfected cells as control). Endogenous AKT associated with TMEM175 was pulled down with immunoprecipitation using anti-GFP and was probed with antibodies against total AKT (pan-AKT), AKT phosphorylated at T308 (pAKT(T308)) or at S473 (pAKT(S473)). Total cell lysates (input) before immunoprecipitation were also loaded for comparison. g, Association between TMEM175(M393T) and TMEM175(Q65P) and AKT. Lysates from HEK293T cells transfected with GFP-tagged wild-type or mutant TMEM175 (with nontransfected cells as control) were blotted with anti-GFP, anti-AKT or immunoprecipitated with anti-GFP followed by blotting with anti-GFP or anti-AKT. For gel source data, see Supplementary Fig. 1.