TatA
E4
6.5 kDa
16.5 kDa
25 kDa
32.5 kDa
42.5 kDa
63.0 kDa
Figure 3.2.16 Circular Dichroism spectra of purified E. coli TatABCs and B. subtilisTatAdCds
Purified E. coli TatABCs and B. subtilis TatAdCds was used to obtain CD spectra over 200-260 nm using a Jasco J-815 spectrophotometer as described in Chapter 2 (2.11). The spectra shown is an average taken from 8 consecutive scans.
-2 -1.5 -1 -0.5 0 0.5 1 200 210 220 230 240 250 260 -10 -8 -6 -4 -2 0 2 4 200 210 220 230 240 250 260
TatAdCd
TatABC
Wavelength (nm)
(nm)
Wavelength (nm)
(nm)
Mean residue
ellipticity
(litre mol
-1cm
-1)
Mean residue
ellipticity
(litre mol
-1cm
-1)
3.3 Discussion
In this Chapter the Tat complexes of the B. subtilis TatAdCd pathway have been studied in detail. The absence of a TatB component in Gram-positive bacteria suggested that the Tat systems of these bacteria differ significantly from the Tat systems of Gram-negative bacteria.
InE. colia 370 kDa membrane localised TatABC complex has been isolated (Oateset al., 2005) that is thought to act as the initial receptor for Tat substrates (Alami et al., 2003). Cross-linking studies indicate that the TatC and TatB components play a critical role in this substrate binding (Alamiet al., 2003; Holzapfelet al., 2007). Whilst the TatA protein appears to only be loosely associated with TatBC in the TatABC complex, TatB and TatC form a tight association. A translational fusion of TatB-TatC ofE. colihas been found to be active (Bolhuiset al., 2001).
Most of the TatA protein found within the membrane forms separate homo-oligomeric complexes of variable mass (100 kDa -500 kDa) (Oates et al., 2005). Data from the thylakoidal Tat pathway point to a model that upon interaction of substrate proteins with the TatABC complexes, free TatA complexes are recruited (Dabney-Smith et al., 2006). Electron microscopy data show the E. coli TatA complexes form pore-like structures with central cavities of variable diameter (Gohlke et al., 2005) and taken together these data point to TatA as being the most likely candidate for the formation of a channel through which substrates can cross the membrane.
A major aim of this work was to identify any differences, particularly differences in translocation mechanism between the Tat pathways of Gram-negative and Gram- positive bacteria using the TatAdCd system as a model. The TatAdCd pathway was found to support translocation of the E. coli Tat substrate TorA when the tatAdCd
genes were expressed in an E. colibackground. Furthermore, TorA was not the only substrate that the TatAdCd pathway could translocate. Efficient translocation of a fusion of the TorA signal peptide and GFP was observed in addition to the tatAdCd
genes ability to complement E. coli Δtat cells for their filamentous phenotype, indicating translocation of AmiA and AmiC. This clearly demonstrates that the Tat
and shows that there are no factors that are essential for translocation inB. subtilisthat are absent in E. coli.This focuses attention on the TatAd and TatCd proteins as being the critical components of this pathway.
The absence of a TatB component in B. subtilisled to the suggestion that the TatAd protein may be bifunctional and perform the roles of both E. coli TatA and TatB (Jongbloedet al., 2006). The fact the TatAdCd pathway can translocate theE. coli Tat substrate TorA for which TatA and TatB have been shown to have distinct and essential roles, strongly suggests that the TatAd protein is indeed bifunctional. The bifunctional nature of the TatAd protein is studied in more detail in chapter 6.
The Tat complexes ofB. subtilisshow both similarities to and differences from theE. coli Tat complexes. Expression of tatAdCd in E. coli and subsequent affinity chromatography of the TatCd protein allowed the isolation of both a TatAdCd containing complex and TatAd complex. This organisation into two types of complex is common to the Tat pathways of a number of Gram-negative bacteria studied in the same way (Oates et al., 2003), and taken together with the ability of the TatAdCd pathway to translocate different E. coli Tat substrates, points to a similar mode of action.
The TatAdCd and TatAd complexes however, also exhibit striking differences to their
E. coli counterparts. Firstly the TatAdCd complex is significantly smaller than the E. coil TatABC complex (~230 kDa for TatAdCd and ~370 kDa for TatABC). This difference might be attributed to the absence of a TatB component. Just one TatAdCd unit with equimolar stochiometry (as seen withE. coliTatBC) has a mass of ~35 kDa compared to E. coli TatABC that is 56 kDa. It may also be possible however that the TatAdCd complex is composed of a different number of TatAdCd domains and this point is currently being addressed by electron microscopy.
very broad range of fractions. It was suggested that the variation in TatA complex size is linked to its ability to translocate substrates that vary in size and shape (Oateset al., 2005; Gohlke et al., 2005). This made sense as it provided a way that the cell could move different sized proteins across the membrane without compromising its integrity. The more restricted size variation of the TatAd complex points to a mechanism involving a single defined translocon rather than a spectrum of size variants. Despite this the TatAdCd pathway can still translocate a number of substrates that vary considerably in size, from GFP (27 kDa), to PhoD (62.5 kDa), to TorA (85 kDa). Not only does the restricted size of the TatAd complex raise questions about the functional significance of the size variation of theE. coli TatA complexes, but it also raises questions about the nature of the translocation channel. The TatAd complex is fairly small. Estimated to be just 160 kDa by gel filtration chromatography, and since the detergent micelle accounts for a proportion of this estimate it is likely to be even smaller. It is difficult to see how the coalescence of a single TatAdCd (~270 kDa) and TatAd (~160 kDa) complex could result in the formation of a channel capable of accommodating the largest Tat substrates such as TorA. It is possible that the translocation mechanism is more complex than previously thought and perhaps involves the recruitment of several TatAd complexes.
In addition to identifying novel features of the TatAdCd and TatAd complexes this work has provided a solid foundation for future structural analysis.