Fig. 2.
Nramp2 protein expression in
smf1/smf2 mutant yeast and functional complementation of growth at alkaline pH. (A) Crude membrane fractions fromsmf1/smf2 yeast cells (pVT) expressing either wild-type (Nramp2) or individual mutant variants of Nramp2 (indicated on top) were separated by SDS-polyacrylamide gel electrophoresis. Immunoblotting was performed using an affinity-purified rabbit antimouse polyclonal anti-Nramp2 antibody. Apparent electrophoretic mobility of the immunoreactive species is consistent with a molecular mass of 60 to 65 kDa. (B) Functional complementation of the growth defect of the smf1/smf2 mutant was tested on solid YPD agar adjusted at alkaline pH (pH 7.9). Serial 10-fold dilutions of cultures corresponding to individual Nramp2 mutants (identified) were spotted (from top to bottom) on YPD agar plates (pH 7.9), followed by incubation for 48 hours at 30°C and photography.