Study of the interaction between the TatE and TatA subunits

As mentioned previously, in WT E. coliMC4100 cells TatA and TatE are expressed constitutively along with the other Tat components (Jack et al., 2001). Moreover these two proteins have overlapping functions in protein transport by the Tat pathway (Sargent et al., 1998). In chapter 3 it has been shown that overexpressed TatE, in the absence of TatA, was able to form homo-oligomeric complexes in the membrane. It is thus reasonable to suggest that TatE may interact with TatA during the translocation process forming TatAE mixed complexes. Nevertheless the formation of these complexes has not been reported so far.

In order to investigate the association between TatE and TatA proteins, tatE was cloned with a C-terminal Strep-IITM tag into the IPTG-inducible vector pEXT22 forming the plasmid pEXT-E-Strep. Next the vectors pEXT-E-Strep and pBAD-A, which expresses TatA, were used to transformE. coli ΔtatAEcells. These vectors are compatible which means they can both be maintained within the same E. coli cells. However, induction of both promoters by IPTG and arabinose respectively cannot be achieved simultaneously because the two substances appear to inhibit each other (unpublished data). Therefore expression from each plasmid must be separated by time.

First of all the TMAO reductase (TorA) activity assay shown in chapter 3 was used to test whether the simultaneous overexpression of TatE and TatA can complementE.

coli ΔtatAE cells by restoring export activity. The pBAD-A plasmid was induced

first for ~ 2 hours under low oxygen conditions. After the removal of arabinose from the growth medium, the pEXT-E-Strep was induced by IPTG for ~ 2 hours under low oxygen conditions as well. The cells were then fractionated and periplasmic (P), membrane (M), and cytoplasmic (C) fractions prepared. Samples of each were run on a native-PAGE gel which was analyzed for the presence of TorA activity.

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

contribute to the formation of an active translocation system. As shown previously

the E. coli ΔtatAE double mutant, no periplasmic activity

export is blocked (ΔtatAE

Figure 5.2.4 Traslocation of TMAO reductase by simultaneous overexpression of TatE and TatA

The figure shows native polyacrylamide gel

activity. Periplasm, membrane and cytoplasm samples ( were prepared and analyzed from

TatA (TatE + TatA (ΔtatAE TorA is indicated. TorA* indicates

Next, to investigate the ability of TatE and TatA to associate with each other form hetero-oligomeric complexes in the membranes, affinity chromatography was used. 500 ml ofE. coli

and TatA from the plasmid pBAD IPTG separately. The cells described in chapter 2 (DDM) and applied to

If the TatA subunit ha

Strep-IITM tag, TatA should have been detected in the Streptactin

fractions co-eluting with TatE PAGE gels and subsequently

tag on TatE or against TatA. Figure 5.2.5

protein and TatA protein in the elution fractions from this column

Study of the TatE subunit interactions with the other Tat components

contribute to the formation of an active translocation system. As shown previously double mutant, no periplasmic activity was

ΔtatAEpanel).

Traslocation of TMAO reductase by simultaneous overexpression

native polyacrylamide gels stained for TMAO reductase ( activity. Periplasm, membrane and cytoplasm samples (P, M, and C respectively were prepared and analyzed fromE. coli ΔtatAEcells co-expressing

TatA (TatE + TatA (ΔtatAE)) and fromE. coli ΔtatAE cells. The mobility of active TorA* indicates a faster migrating form of TorA

to investigate the ability of TatE and TatA to associate with each other oligomeric complexes in the membranes, affinity chromatography was

E. coliΔtatAE cells expressing TatE from the plasmid pEXT

and TatA from the plasmid pBAD-A were induced first with arabinose

IPTG separately. The cells were then fractionated and membranes were isolated described in chapter 2. Total membranes were solubilized in 2% dodecyl mal (DDM) and applied to a 2 ml StreptactinTM-affinity column as described previously

If the TatA subunit had interacted with TatE which is tagged with a C , TatA should have been detected in the Streptactin

eluting with TatE. Column elution fractions were PAGE gels and subsequently immunoblotted using antibodies either

or against TatA. Figure 5.2.5 shows the presence of Strep protein and TatA protein in the elution fractions from this column

Study of the TatE subunit interactions with the other Tat components

contribute to the formation of an active translocation system. As shown previously in detected since Tat

Traslocation of TMAO reductase by simultaneous overexpression

stained for TMAO reductase (TorA) P, M, and C respectively) expressingE. coliTatE and The mobility of active TorA.

to investigate the ability of TatE and TatA to associate with each other and oligomeric complexes in the membranes, affinity chromatography was ssing TatE from the plasmid pEXT-E-Strep A were induced first with arabinose and then with and membranes were isolated as Total membranes were solubilized in 2% dodecyl maltoside as described previously.

E which is tagged with a C-terminal , TatA should have been detected in the StreptactinTM column elution elution fractions were resolved on SDS- either to theStrep-IITM he presence of Strep-tagged TatE protein and TatA protein in the elution fractions from this column. TatE was eluted

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

across elution fractions almost all the Streptactin

detected in the wash fraction before protein was eluted was bound in the case of TatE and

presence of TatA in the earlier elution fractions may interactions between the protein and

purification experiments using TatA alone quite transient and the binding constant quite we during the elution steps and that is why it may come off Another explanation could be that

detergent used during the purification.

In conclusion this result suggests that TatE and TatA may be associated in the formation of TatAE mixed complexes

unequivocally by the co

Figure 5.2.5 Co-purification of TatE and TatA by affinity chromatography

Membranes were prepared from a C-terminal Strep-II

Study of the TatE subunit interactions with the other Tat components

across elution fractions E4-E8 (Figure 5.2.5A), whereas TatA

all the StreptactinTM elution fractions (Figure 5.2.5B). No TatE or TatA was in the wash fraction before protein was eluted which suggests

ase of TatE and partially co-eluted in the case of TatA presence of TatA in the earlier elution fractions may be due to

between the protein and the column. This hypothesis could be tested by experiments using TatA alone. Moreover if the TatAE

quite transient and the binding constant quite weak then perhaps TatA became ring the elution steps and that is why it may come off the column earlier than TatE Another explanation could be that the TatAE complexes might be weakened by the

during the purification.

result suggests that TatE and TatA may be associated in the formation of TatAE mixed complexes, even though this cannot be demonstra

the co-purification experiment.

purification of TatE and TatA by affinity chromatography

Membranes were prepared from E. coliΔtatAEcells co-expressing

IITM tag andE. coli TatA. Membranes were solubilised in Study of the TatE subunit interactions with the other Tat components

TatA was detectable in o TatE or TatA was which suggests the protein e case of TatA. The be due to non specific . This hypothesis could be tested by TatAE complex was ak then perhaps TatA became free the column earlier than TatE. he TatAE complexes might be weakened by the

result suggests that TatE and TatA may be associated in the this cannot be demonstrated

purification of TatE and TatA by affinity chromatography

expressingE. coliTatE with solubilised in DDM

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

5.2.5 Blue-native polyacrylamide gel electrophoresis (BN-PAGE) of