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. 2022 Jul 29;11:e80497. doi: 10.7554/eLife.80497

Figure 4. TORC1 regulates natural resistance-associated macrophage protein (NRAMP) transporter levels.

Figure 4.

(A) GFP-Smf1 expressing cells were cultivated for 5–6 hr in a synthetic medium devoid of manganese sulfate before being treated with 200 ng/ml rapamycin (RAP), 25 µg/ml cycloheximide (CHX), or both compounds (RAP + CHX) for the indicated times. GFP-Smf1 and cleaved GFP (*GFP) protein levels were analyzed by immunoblotting using an anti-GFP antibody. Alcohol dehydrogenase (Adh1) protein levels, probed with anti-Adh1 antibodies, served as a loading control. (B) Quantification of GFP-Smf1. Percentages of GFP-Smf1 relative to the total GFP signal (GFP-Smf1 + GFP) are plotted. Data represent means ± SEM of independent experiments (n=3). Statistical analysis: unpaired two-tailed t-test. *p<0.05; **p<0.01, ***p<0.001, ****p<0.0001. (C) Microscopic analysis of GFP-Smf1 localization. Cells co-expressing GFP-Smf1 and the vacuolar marker Vph1-mCherry were grown exponentially in a manganese-free medium for 5–6 hr, then treated with 200 ng/ml rapamycin for 2 hr. Scale bar represents 5 µm. (D–E) Cells expressing Smf2-GFP from its endogenous locus were grown, treated, and processed as in (A). (F) Microscopic analysis of Smf2-GFP localization. Cells co-expressing Smf2-GFP and the vacuolar marker Vph1-mCherry were cultivated, treated, and examined as in (C).

Figure 4—source data 1. Quantification of blots for graphs shown in Figure 4B–E.
Figure 4—source data 2. Uncropped blots shown in Figure 4A–D and quantified in Figure 4B–E.
Figure 4—source data 3. Raw blots shown in Figure 4A–D and quantified in Figure 4B–E.