Figure 1. Natural resistance-associated macrophage protein (NRAMP) transporters link Mn²⁺-import to rapamycin resistance.

(A) Schematical outline of yeast Mn²⁺ transporters and their intracellular localization. PM, plasma membrane; PVE, pre-vacuolar endosomes; ER, endoplasmic reticulum. Note that Bsd2 is a specific adaptor protein for Rsp5-mediated Smf1 and Smf2 ubiquitination in response to Mn²⁺ overload. The Golgi Mn²⁺ transporter Gdt1 is omitted for clarity. (B–D) Growth on MnCl₂ and/or rapamycin-containing medium (RAP). Ten-fold dilutions of exponentially growing cells are shown. Strains and compound concentrations are indicated. Note that the medium used in (D) was supplemented with 10 mM CaCl₂. Data obtained in a medium without CaCl₂ are shown in Figure 1—figure supplement 3. (E) Southern blot analysis of telomere length. Genomic DNA was derived from cells grown in a medium supplemented or not with CaCl₂ and cleaved by XhoI before agarose gel electrophoresis. The 1.3 kb average length of telomeres from WT cells (dashed white line, black arrow) and size marker (M) are shown. (F) Growth of pmr1∆ smf2∆ double mutants transformed with plasmids expressing yeast Smf2, Mus musculus SLC11A1, or SLC11A2 on rapamycin-containing medium. Complementary data showing that pmr1∆ rapamycin resistance is not linked to Gap1 or Tor1 localization is provided in Figure 1—figure supplement 1. Rapamycin sensitivity of bsd2∆ cells overexpressing the Vcx1-M1 transporter are shown in Figure 1—figure supplement 2.

Figure 1—figure supplement 1. pmr1∆ rapamycin resistance is not linked to Gap1 or Tor1 localization.

(A) Sensitivity of indicated strains to rapamycin (RAP). Ten-fold dilutions of exponentially growing cells are shown. (B) Spinning disk microscopic analysis of GFP-Tor1 localization WT and pmr1∆ cells upon treatment with 200 ng/ml rapamycin (+RAP) for 30 min. Scale bar represents 5 µm. (C) Exponentially growing cells expressing GFP-Tor1 from the endogenous locus were treated for up to 2 hr with 200 ng/ml rapamycin (RAP). GFP-Tor1 protein levels were analyzed by immunoblotting with an anti-GFP antibody. G-6-PDH levels were used as a loading control.

Figure 1—figure supplement 2. Growth of bsd2∆ mutants expressing Vcx1-M1 from plasmid pVCX1-M1 on MnCl₂ and/or rapamycin (RAP) containing medium.

Ten-fold dilutions of exponentially growing cells are shown. Compound concentrations are indicated.

Figure 1—figure supplement 3. Natural resistance-associated macrophage protein (NRAMP) transporters mediate rapamycin resistance of pmr1∆ mutants.

Sensitivity of the indicated strains to rapamycin (RAP). Ten-fold dilution of exponentially growing cells are shown. Data obtained in medium supplemented with CaCl₂ is shown in Figure 1D. Note that growth of the pmr1∆ smf1∆ double mutant is strongly impaired without CaCl₂ addition.