FIGURE 5.

Functional properties of mutant Can1 proteins. A, subcellular locations of the mutant Can1 proteins. Strain 22Δ8AA was transformed with the pKG036 (YCpCAN1-GFP) plasmid expressing the native Can1 protein coupled to GFP or with an equivalent plasmid encoding a mutant Can1 protein. Cells were grown on glucose-ammonium medium and examined by fluorescence microscopy. B, strain 22Δ8AA transformed with the empty vector YCpFL38, the pKG036 (YCp-CAN1) plasmid expressing the native permease (Can1), or one of five derived plasmids encoding Can1 mutants were grown on solid minimal medium with arginine (Arg) or ammonium (Am) as the sole nitrogen source and with canavanine (Can) when indicated. Cells were incubated at 29 °C for 5 days. C, strain 22Δ8AA transformed with pKG036 (CAN1-GFP) plasmid or pKG065 (CAN1-S176N-GFP), pKG046 (CAN1-T456S-GFP), or pKG066 (CAN1-S176N/T456S-GFP) plasmid was grown on glucose-ammonium medium. Crude cell extracts were prepared and immunoblotted with anti-GFP or with anti-Pma1 antibodies.