Figure 3. Expression and activity of E327Q LmrP mutants with a reinserted carboxyl residue.
(A) Immunodetection of total membrane proteins in plasma membrane vesicles (10 μg of protein/lane) containing WT LmrP or mutants LmrP, or no LmrP protein (Ctrl). Proteins were transferred from SDS-PAGE gel to HybondP membrane by electroblotting and probed with anti-His-tag and secondary antibodies. For clarity of presentation, blot was cropped in Adobe Illustrator CC 2015.3; full-length blot is available at http://dx.doi.org/10.17863/CAM.6219. (B) Cells expressing the T331E/E327Q double mutant LmrP were diluted in KPi buffer (pH 7.0) supplemented with 5 mM MgSO4 and pre-equilibrated with 2 μM propidium. (C–G) Transport experiments as in (B) for A355E/E327Q (C), N123D/E327Q (D), T324E/E327Q (E), T33E/E327Q (F), and A53E/E327Q (G). See legend to Fig. 2 for further details. Data represent observations in 3 or more independent experiments with independently prepared batches of membrane vesicles or cells. Values in histograms are expressed as mean ± s.e.m. (one-way analysis of variance; ****P < 0.0001).