Fig 1. Npr1 is required for Mep1 and Mep3 inherent transport activity.

(a) Immunodetection of Mep1 and Mep3 from membrane-enriched cell extracts. Wild-type (23344c) and npr1Δ (30788a) cells were grown in the presence of proline (0.1%) as nitrogen source. The plasma membrane proton ATPase Pma1 was detected as a loading control. (b) Immunodetection of Mep1 and Mep3 from membrane-enriched extracts treated (+) or not (-) with alkaline phosphatase (ALP). Wild-type (23344c) and npr1Δ (30788a) cells were grown in the presence of proline (0.1%) as nitrogen source. (c) Mep1-GFP and Mep3-GFP localization was observed by fluorescence microscopy in triple-mepΔ (31019b) and triple-mepΔ npr1-1 (31052c) cells transformed with the pGAL1Mep1-GFP or pGAL1Mep3-GFP low-copy-number vectors and grown in the presence of proline (0.1%), galactose (3%) and glucose (0.3%). (d) [¹⁴C]-methylammonium (0.5 mM) accumulation in proline-grown triple-mepΔ (31019b,│,□) and triple-mepΔ npr1 ^ts (MB063,▲,Δ) cells transformed with YCpMep1 after transfer in a similar medium preheated at 29°C (│,▲) or 37°C (□,Δ). (e) Immunodetection of Mep1 from membrane-enriched extracts of cells collected after the temperature shift as described in Fig 1d.