Descripción de las fases del procedimiento de PML a aplicar

In order to investigate the dimensions of the cytosolic TatE and TatA complexes, they were analyzed by BN-PAGE.

E. coli ΔtatAE cells expressing TatE or TatA from the plasmid pBAD-E-Strep and pBAD-A-Strep respectively were fractionated. The cytosolic fractions were collected and loaded directly onto BN-gels in absence of any detergent. The gels were then subjected to immunoblotting with antibodies against the Strep-IITM tag on TatE and TatA. The Figure 4.2.6 (left panel) shows the presence of a band corresponding to a cytosolic TatE complex that migrated just above the 66 kDa marker. The dimension of this complex was similar of that corresponding to the uppermost band in the BN- gel of solubilised membranes expressing TatE (Figure 3.2.8, chapter 3). In contrast, as shown in the Figure 4.2.6 (right panel), cytosolic TatA ran in the BN-gel as a high molecular weight band corresponding to a complex of ~ 669 kDa. This complex resembles the high molecular weight complexes or aggregates formed by the cytosolic TatAd and TatAy inB. subtilis(Barnettet al., 2008; Barnettet al., 2009).

Chapter 4.

Figure 4.2.6 BN-PAGE

E. coli ΔtatAE cells expressing either TatE (left panel) or TatA (right panel) were

fractionated and the cytosol

immunoblotted to antibodies to the cytosolic TatE and TatA complexe markers are indicated on the left.

Chapter 4.Characterization of soluble TatE and TatA complexes

PAGE of cytosolic TatE and TatA complexes

cells expressing either TatE (left panel) or TatA (right panel) were cytosolic samples were subjected to BN-PAGE

immunoblotted to antibodies to the Strep-IITMtag on TatE or TatA.

TatE and TatA complexes are indicated on the right. Molecular mass are indicated on the left.

Characterization of soluble TatE and TatA complexes

cells expressing either TatE (left panel) or TatA (right panel) were PAGE. The gels were TatA. Mobilities of s are indicated on the right. Molecular mass

Chapter 4.Characterization of soluble TatE and TatA complexes

4.3 Discussion

The functional significance of a soluble population of TatA-type proteins is still a matter of controversy, and its role in the translocation process has not been unequivocally resolved.

InB. subtilisTatAd has been found to have a dual localization in the membrane and

in the cytoplasm. In this last compartment it has been shown to interact specifically with the prePhoD signal peptide (Popet al., 2003).

Another study has found that soluble TatAd could assemble into micelle-like structures (Westermannet al., 2006). The hydrophobic centre of the protein micelles could accommodate the hydrophobic domain of the PhoD signal peptide, whereas the negative charged C-terminal region of TatAd could interact with the twin-arginine motif (Westermann et al., 2006). A further study has shown that cytosolic TaAd could serve to target the substrates to the translocase since it binds to cytosolic loops of the membrane protein TatCd (Schreiberet al., 2006).

Interestingly in the Gram-positive bacteriumStreptomyces lividanswhich is the only Gram-positive bacterium having a TatABC type system, both TatA and TatB are present in the cytoplasm as well as in the membrane (De Keersmaekeret al., 2005).

In E. coli TatA has been found alongside its membrane localised form in the

cytoplasm too, where it forms homo-multimeric tube-like structures inside the cytoplasm (Berthelmann et al., 2008). This suggests that the Tat pathways found in Gram-positive and Gram-negative bacteria may operate by a similar mechanism.

Nevertheless another study that used in vivo single-molecule imaging showed that the TatA protein is instead localised exclusively to the plasma membrane (Leake et

Chapter 4.Characterization of soluble TatE and TatA complexes

carried out in this study support the hypothesis that the observed soluble Tat complexes are mis-localised. Still the contradictory results make a meaningful interpretation of the literature difficult and further work is needed to resolve this point.

It remains possible that the cytosolic TatE and TatA proteins, just like Tha4 in plants (Frielingsdorf et al., 2008), may form an additional pool of protein that inserts into the membrane, perhaps improving the efficiency of Tat-dependent transport. Obviously further experiments are needed to confirm this hypothesis.

The BN-PAGE data presented in this chapter show that cytosolic TatE and TatA proteins, like their membrane localised counterparts (chapter 3), are organized into complexes of different size. Cytosolic TatE forms a complex of ~ 66 kDa, similar to the largest TatE complex seen in the BN-gel of solubilised membranes expressing TatE (Figure 3.2.8, chapter 3).

In contrast cytosolic TatA, unlike membrane localized TatA (Figure 3.2.7, chapter 3), is not organized into a range of oligomeric complexes but instead it forms a distinct cytosolic complex of ~ 669 kDa which resembles in part the high molecular weight complexes or aggregates formed by the cytosolic TatAd and TatAy in B. subtilis (Barnettet al., 2009; Barnettet al., 2008).

These results show that the TatE complexes have similar features regardless of whether they have a membrane or cytosolic localisation, whilst TatA does not appear to form modular complexes in the cytoplasm. Perhaps this observation suggests that cytosolic TatA has to be present as a discrete cytosolic complex in order to execute its function in the transport mechanism. Further studies are of course needed to address whether cytosolic TatE and TatA proteins are functional or not.

Chapter 5.Study of the TatE subunit interactions with the other Tat components

Chapter 5.

Study of the TatE subunit interactions with