FIGURE 4.
TorA cleavage between amino acid positions 49 and 50. A, TorA and FLAG-LULL1 were transiently expressed in ExpiTM 293 cells and purified via FLAG tag. The purified complex was reconstituted in liposomes and incubated with DTT in the presence of ATP. TorA was analyzed by SDS-PAGE/immunoblotting (IB). B, the schematic view of TorA N-terminal processing position identified by Edman degradation. Bold letters, amino acid residues identified by Edman degradation. Black arrow, signal peptidase cleavage site. Red arrow, deduced cleavage site from Edman degradation. SS, signal sequence. Hy, hydrophobic region (underlined). SPase, signal peptidase. C, design of HA-tagged TorA Δ49 construct using the signal sequence of MHC-1 (MHC-1 SS; dark red). D, 293T cells transfected with C-terminal HA-tagged TorA or TorA processed product (TorA Δ49-HA) were treated with DTT and analyzed by immunoblotting following deglycosylation. E, phase separation comparison of TorA and TorA Δ49. 293T cells were transfected as above and lysed in Triton X-114. The aqueous and hydrophobic phases of the lysates were separated by varying the temperature (see “Experimental Procedures”) and analyzed by immunoblotting. Total, lysates without phase separation treatment. aq., aqueous phase. det., detergent phase. PDI serves as a control.