Gln3 phosphorylation levels in glutamine- or proline-grown wild type, sit4Δ single and sit4Δ,ure2Δ double-mutant cells in the presence (+Rap) or absence of rapamycin treatment. Wild type (TB123), sit4Δ (TB136-2a), ure2Δ (TB138-1a), and ure2Δ,sit4Δ (FV071) strains were grown to an A600nm ∼0.5 in YNB-glutamine (A–C) or YNB-proline (D and E). (B and E) Untreated cultures were sampled without addition of rapamycin or following 20 min incubation with 200 ng/ml rapamycin (+Rap). (C) Wild type (JK9-3da) or ure2Δ mutant (RR215) were transformed with wild-type (pRR536) or gln3 NES mutant (pRR752) plasmids and grown as described in (A). All samples were processed for western blot analysis as described in Materials and Methods.