Fig. 4.
Cysteine cross-linking between the CLs of TAP1 and the X-loop of TAP2. (A) Oxidative cysteine cross-linking of TAP1(A381C)/TAP2(E602). TAP-containing membranes (500 μg of protein) were incubated in the presence or absence of 1 mM CuPhe for 1 min at 4 °C. Samples were subjected to nonreducing 6% SDS/PAGE (20 μg of protein per lane) followed by immunoblotting against TAP1 and TAP2. The positions of the cross-linked TAP1–TAP2 (highlighted in bold), homodimeric (TAP1)2 and (TAP2)2, as well as monomeric TAP1 and TAP2 are indicated. (B and C) The cross-linking of the single-cysteine mutants of CL1 (B) and CL2 (C) in TAP1 with E602C of TAP2. The conditions are identical as described above. The efficiency of TAP1–TAP2 cross-linking was derived from TAP2 immunoblots.