PMC

SmpA copurifies with the YaeT complex. (A) Silver-stained SDS/polyacrylamide gel of samples from cell lysates of wild-type (WT), wt/pSmpA-His strains immunoprecipitated with anti-His tag antibody. (B) Silver-stained SDS/polyacrylamide gel of samples from cell lysates of WT, yfgL⁻/pYfgL-His, yfiO::kan/pYfiO-His, and nlpB⁻/pNlpB-His strains immunoprecipitated with anti-His tag antibody. MW, molecular weight.

Affinity purification of the YaeT complex in lipoprotein mutants. (A–D) Affinity purification was performed in WT and lipoprotein mutant strains that contain pYaeT-His (A and B) and pSmpA-His (C and D), respectively. (A and C) Protein levels in the total membrane fraction isolated from cells before any purification. (B and D) Samples after they have passed through an Ni-affinity column. Samples were blotted against antibodies that recognize YaeT, YfgL, and YfiO and the His tag.

smpA genetic interactions. Western blot analysis shows the levels of DegP, LamB, MBP, YfgL, and YfiO proteins in strains carrying mutations that affect different YaeT complex members and in smpA double mutants during midlogarithmic growth at 30°C. The smpA yfgL depletion strain was grown in the presence of l-arabinose (A) to induce expression of yfgL or in the presence of d-fucose (F) to prevent its expression. Below the Western blots is a chart that shows the quantification of LamB (gray bars) and DegP (black bars). The values of the y axis represent the areas of the bands as quantified by using ImageJ image-processing software.

SmpA is not essential for growth in E. coli. (A) Growth curves for wild-type cells (♦) or smpA cells (▫) at 37°C are shown. Growth was monitored by measuring the optical density at 600 nm every hour for 8 h. The results of a representative experiment are shown above. (B) Levels of envelope proteins in an smpA mutant during midlogarithmic growth at 37°C and 30°C. Western blot analysis shows a very small reduction in the levels of OmpA and LamB in an smpA mutant strain when compared with a wild-type (WT) strain. Levels of the periplasmic chaperone/protease DegP are elevated 1.5- to 2-fold in an smpA mutant. (C) Unfolded LamB accumulates in an smpA degP double mutant. Cells were grown to midlogarithmic growth at 37°C and 30°C and harvested by centrifugation. These samples were gently lysed to preserve the native conformation of OMPs. Western blot analysis by using LamB antiserum was used to visualize unfolded LamB (U-LamB). Levels of MBP are shown as a loading control.