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. 2022 Apr 27;11:e72822. doi: 10.7554/eLife.72822

Figure 2. In vitro translocation assay for reconstitution of Slam-dependent SLP translocation.

(a) Model of the proposed in vitro proteoliposomes translocation assay for TbpB secreted directly from E. coli spheroplast. As E. coli cell expressing TbpB, the cells were converted into spheroplast that has an intact inner membrane. Purified LolA was added to release mature TbpB (processed by Lgt, Lsp, and Lnt) from the LolCDE complex in the inner membrane. The secreted TbpB was incubated with proteoliposomes for translocation, followed by PK digestion to quantify the amount of TbpB that had been translocated inside the liposomes. (b) Representative α-flag western blots obtained for the in vitro translocation assay. Slam1 proteoliposomes were incubated either with spheroplasts expressing TbpB (spheroplast-dependent translocation, upper panel) or supernatant of spheroplasts that have been induced for TbpB production (spheroplast-independent translocation, lower panel). Empty liposomes and Bam proteoliposomes were used as controls. Proteoliposomes containing Bam + Slam1 were used to test if the Bam complex plays an accessory role to Slam in TbpB translocation. For each proteoliposome, no proteinase K treatment (−PK), proteinase K treatment (+PK) and proteinase K + Triton X-100 treatment (+PK + T) samples are shown. The % TbpB protection shown was calculated by dividing the intensity of the mature TbpB band (~75 kDa) for each sample by the −PK sample. (*) Partial TbpB fragment which is only seen in the presence of Slam1 proteoliposomes. (c) Quantification of TbpB protection in proteoliposomes through densitometry analysis. The plot represents data obtained from at least three biological replicates for both spheroplast-dependent translocation and spheroplast-independent assay. Individual data points were included on the graph. (d) Representative α-flag western blot of spheroplast-independent TbpB translocation into Bam and Slam1 proteoliposomes over time. Spheroplast-secreted TbpB was incubated with proteoliposomes in 1:1 ratio at room temperature. Samples were collected every 5 or 10 min and left on ice before proteinase K treatment. (e) Quantification of spheroplast-secreted TbpB translocation into Bam proteoliposomes and Slam1 proteoliposomes over the course of 60 min.

Figure 2—source data 1. Quantification of in vitro translocation of spheroplast-secreted TbpB.
Figure 2—source data 2. Quantification of time-dependent translocation of spheroplast-secreted TbpB.

Figure 2.

Figure 2—figure supplement 1. Purification and functional characterization of LolA from E. coli.

Figure 2—figure supplement 1.

(a) Model of the release of SLPs from spheroplasts upon addition of purified E. coli LolA. SLPs are synthesized in the cytoplasm and transported to the periplasm via the Sec translocon. After the addition of the lipid anchor, SLPs are transferred to the LolCDE complex and released into the periplasm by LolA. In spheroplasts, SLPs are released into the supernatant upon LolA addition. (b) Uncut 6x-His tagged E. coli LolA (~22 kDa) after Ni-NTA affinity chromatography purification. (c) Release of Mcat TbpB in the presence of purified LolA over a time course of 60 min. E. coli cell pellets were converted into spheroplasts and induced for expression of TbpB in the presence or absence of 10 μM LolA. Samples were collected every 15 min, spun down at 13,500 rpm for 5 min and the supernatants were loaded on sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE). The amount of TbpB (~75 kDa) that was released into the supernatant in the presence and absence of LolA was accessed using a α-flag antibody western blot. Lower bands are degradation products of TbpB which also increase over time.
Figure 2—figure supplement 2. Characterization of spheroplast-secreted TbpB.

Figure 2—figure supplement 2.

(a) Western blot accessing the protection of secreted TbpB in the absence of liposomes. Similar background protection was observed in no liposomes and liposome negative control (8.5% and 9.3%, respectively). Note: The input (−PK/−T) has been diluted by 1:10 to enhance the signal of the background protection for quantification. The protection percentage was calculated using the densitometry method and accounted for the dilution. Protection for TbpB in Slam1 proteoliposomes is still four times higher than the background protection (40.7%). (b) Functionality test of TbpB-containing proteoliposomes after translocation. After inhibiting proteinase K using PMSF, protected TbpB-proteoliposomes samples were treated with 0.1% Triton X and further incubated with human transferrin-conjugated beads to pulldown functional TbpB (flag-tagged). The lower band in Slam1-elution represents partially degraded TbpB. Note: The input of TbpB from Bam proteoliposomes and Slam1 proteoliposomes are different as they are outputs of the PK digestion assay (5% vs. 45% protected TbpB as the input for Bam and Slam1 proteoliposomes respectively).