(A) The growth defects of the strains lacking Gtr1GDP-Gtr2 or the Ragulator components Lam1 ~4 were partially complemented by supplementing the YES medium with 5 mg/ml NH4Cl. The indicated strains were grown in liquid EMM and their serial dilutions were spotted onto the indicated agar media at 30 ˚C. TORC1 activity was monitored by immunoblotting as in Figure 6 after wild-type cells growing in YES at 30°C were shifted to the same medium supplemented with NH4Cl or 200 ng/ml rapamycin (Rap) in order to confirm that NH4Cl does not significantly affect TORC1 activity in YES. (B–C) Those mutants also showed severe growth defects on minimal media containing amino acid (20 mM Pro or Arg) as sole nitrogen source, while the phenotype was ameliorated by adding 5 mg/ml NH4Cl (B). In comparison to wild-type cells, the mutants were more resistant to canavanine (‘Can’, 60 µg/ml) and ethionine (‘Eth’, 30 µg/ml), toxic analogs of arginine and methionine, respectively (C). (D–E) gtr1∆ and iml1∆ mutant strains with amino-acid auxotrophy show severe growth defects even in the presence of amino acids supplemented to the growth medium. The indicated prototrophic, arginine-auxotrophic (arg3-D4) and leucine-auxotrophic (leu1-32) strains were grown in liquid YES supplemented with 200 ng/ml rapamycin, washed with EMM liquid, and their serial dilutions were spotted onto EMM agar media with 225 mg/L arginine (D) or 250 mg/L leucine (E) at 30 ˚C. (F) Unlike the gtr1∆ and iml1∆ mutants, the growth defects of the tor2-13 and tor2-287 are not suppressed by NH4Cl in the growth medium. Growth of the indicated strains was tested as in (A). (G) In contrast to the tor2 hypomorphic mutants, cells lacking Gtr1GDP-Gtr2 or Ragulator are not smaller than wild-type cells. The indicated strains were grown in EMM at 30 ˚C, and a particle analyzer was used to measure their cell volume, which is presented as equivalent spherical diameter in the bar graph (means ±SD, n = 3).