Characterization of Two Second-Site Mutations Preventing Wild Type Protein Aggregation Caused by a Dominant Negative PMA1 Mutant
Figure 9
Analysis of the protein complexes formed by wild type and mutant Pma1 variants.
A) Total membranes from yeast strains expressing HA-tagged wild type Pma1 and myc-tagged wild type Pma1 (lanes 1,3, 9 and 11), dominant lethal myc-Pma1-D378T (lanes 2, 4, 10, and 12), double mutant protein myc-Pma1-D378T/L151F (lanes 5, 7, 13, and 15) or myc-Pma1-D378T/ΔC409-A412 (lanes 6, 8, 14 and 16) were treated with 3% Triton X-100 (lanes 1, 2, 5, 6, 9, 10, 13 and 14) or 1% SDS (lanes (3, 4, 7, 8, 11, 12, 15 and 16) as described in Materials and Methods. The solubilized proteins were separated by BN-PAGE in duplicated gels and immunoblotted with anti-HA (lanes 1–8) or anti-myc (lanes 9–16) antibodies. The relative mobilities of molecular mass markers are indicated on the left. B) Total membranes from yeast strains expressing myc-tagged wild type Pma1 and single mutant protein HA-Pma1-L151F (lanes 1, 3, 5 and 7) or HA-Pma1-ΔC409-A412 (lanes 2, 4, 6 and 8) were treated with Triton X-100 (lanes 1, 2, 5 and 6) or 1% SDS (lanes (3, 4, 7 and 8) and the solubilized proteins separated by BN-PAGE and immunoblotted with anti-myc (lanes 1–4) or anti-HA (lanes (5–8).