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. 2015 Jan 30;10(1):e0116750. doi: 10.1371/journal.pone.0116750

Figure 1. Topology of YuaG.

Figure 1

(A) A cartoon of the two putative topologies of SNAP-YuaG is shown. YuaG has either a hairpin loop tethering it to the membrane or a trans-membrane helix as predicted by bioinformatics tools. SNAP dye TMR-Star is membrane permeable and hence sufficient to label in- and outside the cell, labelling with SNAP dye BG-488 (SNAP-surface 488), which is membrane impermeable, is only possible with an extracellular SNAP tag. (B) Cells expressing SNAP-YuaG or free SNAP were labelled with the cell impermeable SNAP dye BG-488 and the cell permeable SNAP dye TMR-Star. SNAP dyes are all false coloured in green. Zoomed in regions are indicated with a red frame. Scale bar 2 µm. (C) A representative Proteinase K (PK) sensitivity assay is shown for protoplasted B. subtilis cells. Control experiments using wild type cells incubated with TMR-Star and unlabeled cells expressing SNAP-YuaG confirmed that TMR-Star does not unspecifically label B. subtilis proteins. As a positive control protoplasts were resuspended in water instead of MSMNB and TritonX-100 was added to a final concentration of 1%. (D) The in gel-fluorescence of the PK assay was quantified. Fluorescence of 0 minutes PK was always defined as 100%. Standard deviation is shown; n = 4. (E) Alignment of the hydrophobic helix of B. subtilis YuaG with different flotillins from various bacteria. Note the conserved glycine residue, highlighted in red. The alignment was performed using Kalign with default settings [43].