Fig 1.
Protein sequences are provided in S1 Fig and plasmid sequences are available from Addgene. A short linker (L) is indicated in gray. The TEV protease cleaves within the TEV recognition sequence (ENLYFQG) between Q and G.
Fig 2.
Oligomerization state of TorD and spTorA-mCherry.
(A) Oligomerization state of Ni-NTA purified TorD under native gel conditions. TorD is largely monomeric (~17 μM load), although some higher order oligomers (starred bands) are eliminated by βME (143 mM) and hence are disulfide-linked. (B) Oligomerization state of TorD analyzed by size-exclusion chromatography. Ni-NTA purified TorD contains higher order oligomers at high concentration (top, ~170 μM total load), yet the monomeric form in the FPLC-purified fractions was stable for at least a month at -80°C when preserved in the presence of 50% glycerol and 5 mM DTT at a lower concentration (bottom, ~7.0 μM load). (C) Ni-NTA purified spTorA-mCherry. The fully denatured unfolded form of mCherry (boiled sample) runs slower on SDS-PAGE and is non-fluorescent. The starred (*) band is a heat-dependent cleavage product of mCherry [60]. (D) Oligomerization state of spTorA-mCherry analyzed by size-exclusion chromatography. The Ni-NTA purified spTorA-mCherry is largely monomeric (top, ~290 μM total load), and remains stably monomeric upon re-chromatographing (bottom, ~7.1 μM total load). The molecular weight axis on the top of the size-exclusion chromatograms was generated by a standard curve from the peak elution positions of conalbumin (75 kDa), carbonic anhydrase (29 kDa), RNase (13.7 kDa), and aproptinin (6.5 kDa). The ordinates are milli-absorbance units (mAU).
Table 1.
Plasmids encoding overproduced proteins.
Fig 3.
TorD and spTorA-mCherry form a 1:1 complex.
(A & B) The TorD/spTorA-mCherry complex. The spTorA-mCherry protein (~7 μM) was mixed with TorD in a 1:1 (A) or 1:2 (B) ratio and analyzed by size-exclusion chromatography (bottom chromatograms). For the 1:1 mixture, the peaks corresponding to the individual spTorA-mCherry (top chromatogram) and TorD (middle chromatogram) proteins are virtually absent, and the new peak at ~45 kDa reflects the TorD/spTorA-mCherry complex. For the 1:2 mixture, approximately half of the TorD was recovered uncomplexed with spTorA-mCherry. Integrated signal intensities from Western blots of the elution fractions from the spTorA-mCherry + TorD mixtures confirm a 1:1 stoichiometry of the spTorA-mCherry and TorD proteins in the high molecular weight peak (dashed boxes). The lower molecular weight band for purified spTorA-mCherry (*) is a known product of heat-dependent self-cleavage (see text). The spTorA-mCherry and TorD load in the standard lanes was 2 pmol. (C & D) Non-binding controls. TorD does not form a complex with either mCherry, which has no signal peptide, or pre-SufI, which has a non-cognate signal peptide.
Fig 4.
Dissociation of the TorD/spTorA-mCherry complex.
Fractions 19–21 of the 1:1 TorD/spTorA-mCherry complex in Fig 3A were pooled, centrifuged to remove aggregates (4°C, 32,000 g for 10 minutes), and immediately re-run on the size-exclusion column. Approximately half of the TorD dissociated from spTorA-mCherry (see text).
Fig 5.
In vitro transport efficiencies of Tat substrates.
(A) Purified spTorA-GFP(Alexa532) after TEV protease cleavage of H6-spTorA-GFP(Alexa532). (B & C) Transport assays analyzed by in-gel fluorescence (B) or Western blotting (C). Transport assays were conducted with 50 nM precursor proteins and Tat++ IMVs (A280 = 5) for 10 min at 37°C. Transport efficiencies are indicated as the amount of protease-protected (571 μg/mL proteinase K treatment for 20 min) mature-length protein as the percent of total added precursor from at least 4 independent assays. These data suggest that the transport of spTorA-GFP is reduced by ~5- and 3-fold with a 6xHis-tag at the N- or C-terminus, respectively (but see Fig 6), and that the transport efficiency of spTorA-GFP is ~80% of that observed for pre-SufI. Transport was not observed in the absence of NADH (control).
Fig 6.
Western blots underestimate Tat precursor concentrations in the presence of Tat++ IMVs.
Different amounts of the indicated Tat precursors were electrophoresed in the absence or presence of Tat++ IMVs (A280 = 5). In the graph at the top, the intensity dataset for each gel is normalized to the intensity for the 0.18 pmol load in the absence of IMVs: solid curves, no IMVs; dashed curves, +IMVs. The amount of spTorA-GFP(Alexa532) detected by in-gel fluorescence in the absence or presence of Tat++ IMVs is linear with the load, and similar under the two conditions. In contrast, Western blots of H6-spTorA-GFP and spTorA-GFP-H6C using anti-6xHis antibodies substantially underestimate the presence of these proteins when electrophoresed with Tat++ IMVs. The starred (*) band indicates a partially cleaved protein product, which is not the mature-length protein.
Fig 7.
TorD reduces the binding of spTorA-GFP to Tat++ IMVs.
The spTorA-GFP(Alexa532) protein (50 nM) was pre-incubated with various concentrations of TorD (10 min, 37°C), incubated with Tat++ IMVs (A280 = 5; 10 min at 37°C), and centrifuged to remove unbound protein (32,000 g, 4°C for 10 min). The pellets were analyzed by SDS-PAGE, and the amount of IMV-bound spTorA-GFP(Alexa532) was quantified by in-gel fluorescence imaging using the standard lanes for calibration (% of 50 nM). Data are normalized to the 0 μM TorD sample (100%; N = 3). Assuming that the TorD interaction with spTorA-GFP prevents the precursor from binding to the IMVs, three binding regimes are apparent: first phase (~30%), linear fit; second phase (~40%), single site Langmuir binding isotherm (KD = 1.3±0.3 μM) [46]; third phase (~30%), no apparent binding to TorD. The first two phases (independent fits in black) are also simultaneously fit to a single site binding model that accounts for the precursor concentration, but not the IMV binding sites (red curve) [31].
Fig 8.
TorD has similar affinities for ΔTat and Tat++ IMVs.
Different concentrations of TorD(Alexa532) were incubated with ΔTat and Tat++ IMVs (A280 = 5; 10 min at 37°C). IMV pellets were recovered and analyzed for the amount of bound TorD using the approach described for Fig 7. Data were fit using a single site Langmuir binding isotherm model [46], yielding KD values of 100±33 nM and 112±43 nM as well as [TorD]bound(max) values of 28±3 nM and 29±4 nM for ΔTat and Tat++ IMVs, respectively.
Fig 9.
spTorA-GFP transport is inhibited by TorD.
Tat-dependent transport of spTorA-GFP(Alexa532) (50 nM) was measured in the presence of increasing concentrations of TorD (conditions of Fig 5). Markers were not used for this experiment since all lanes were used for the assay. The standard lanes provide a suitable reference (N = 3).