Figure 4. Four gap1 mutants altered in the N-terminal tail resist ammonium-induced down-regulation.

(A) Strain EK008 (gap1Δ ura3) transformed with the pJOD10 (YCpGAL-GAP1-GFP) plasmid expressing the native permease (Gap1) or with an equivalent plasmid expressing no Gap1 protein (−), the Gap1^K9,16R form resistant to ubiquitylation, or one of the indicated Gap1 mutants was tested for growth on solid medium containing the indicated nitrogenous compound(s). For each growth condition, the seven strains have been grown on the same plate. (B) Strain EK008 (gap1Δ ura3) transformed with the pJOD10 plasmid (Gap1) or with an equivalent plasmid encoding the Gap1^K9,16R or Gap1-112 mutant was grown on galactose-proline medium and examined by fluorescence microscopy before and two hours after addition of ammonium (20 mM). (C) Western blot analysis of Gap1-GFP constructs in total cell extract prepared before (t0) and several times after addition of ammonium. Strains and growth conditions were as in (B).