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. 2017 May 9;8(3):e00574-17. doi: 10.1128/mBio.00574-17

FIG 4 .

FIG 4 

Complementation of the endogenous TcMCU and reconstitution of Ca2+ transport in yeast. (A) Ca2+ uptake reconstitution in digitonin-permeabilized TcMCU-KO epimastigotes transfected with pTREX-h and pTREX-h-TcMCU-PAM-HA (TcMCU). Experimental conditions were as in Fig. 1I. (B) Quantification of data in panel A. Relative Ca2+ uptake at 600 s compared with WT. (C) Western blot analysis of total protein extracts of wild-type (WT), TcMCU-KO plus pTREX-h empty vector (KO + EV), and TcMCU-KO plus TcMCU-PAM-HA (KO + MCU) epimastigotes, using anti-HA antibodies. Anti-α-tubulin antibodies were used as a loading control. (D) Ca2+ uptake reconstitution in digitonin-permeabilized TcMCU-KO epimastigotes transfected with TcMCUR214W,D219V (MCU-KO + R214W,D219V), TcMCUD223N,E226Q (MCU-KO + D223N,E226Q), or TcMCUb (MCU-KO + TcMCUb). Experimental conditions were as in Fig. 1I. (E) Quantification of data in panel D. Relative Ca2+ uptake at 600 s compared with WT (TcMCUR214W,D219V [shown as R214W,D219V] and TcMCUD223N,E226Q [shown as D223N,E226Q]). (F) Western blot analysis of total protein extracts of wild-type (WT), TcMCU-KO (MCU-KO), TcMCU-KO plus pTREX-h empty vector (KO + EV), TcMCUD223N,E226Q (D223N,E226Q), TcMCUR214W,D219V (R214W,D219V), and TcMCUb (KO + MCUb) epimastigotes, using anti-c-Myc antibodies. Anti-α-tubulin antibodies were used as a loading control. (G) Ca2+ uptake reconstitution in digitonin-permeabilized TcMCU-KO epimastigotes transfected with HsMCU or pTREX-p. Experimental conditions were as in Fig. 1I. (H) Quantification of data in panel G. Relative Ca2+ uptake at 600 s compared with WT. (I) Western blot analysis of total protein extracts of WT, TcMCU-KO (MCU-KO), TcMCU-KO plus pTREX-p empty vector (KO + EV), and TcMCU-KO plus HsMCU (KO + HsMCU) epimastigotes, using anti-c-Myc antibodies. Anti-α-tubulin antibodies were used as a loading control. (J) Ca2+ uptake by spheroplasts from yeast transformed with pACT2 empty vector, TcMCU, ScMTSTcMCU, or DdMCU. Experimental conditions were as described in Materials and Methods. (K) Quantification of data in panel J. Relative Ca2+ uptake at 350 s compared with DdMCU. (L) Western blot analysis of lysates and mitochondrial fractions of yeast complemented with empty vector, TcMCU-HA (TcMCU), or ScMTSTcMCU-HA (ScMTSTcMCU) using anti-HA antibodies. Anti-cytochrome c antibodies were used as a loading control. (M) Western blot analysis of lysates and mitochondrial fractions of yeast complemented with DdMCU-Flag (DdMCU) using anti-Flag antibodies. Anti-cytochrome c antibodies were used as a loading control. Values in panels B, E, H, and K are means ± SD (n = 3). *, P < 0.05; ***, P < 0.001.