Fig. 5.

Cysteine cross-linking inhibits peptide binding or translocation. Membranes (500 μg of protein) containing variants of TAP1 in combination with TAP2(E602C) were incubated in the presence or absence of CuPhe for 1 min at 4 °C. (A) Samples (20 μg of protein per lane) were analyzed by nonreducing 6% SDS/PAGE and immunoblotting. (B) Peptide binding to TAP treated with (black bars) or without (gray bars) CuPhe. TAP-containing membranes (35 μg of protein) were incubated with radio-labeled RRYQKSTEL (1 μM) at 4 °C for 15 min. Unspecific binding was determined in the presence of an excess of RRYQKSTEL (200 μM). Peptide binding to TAP in the absence of CuPhe was normalized to 100%. (C) Peptide transport via TAP treated with (black bars) or without (gray bars) CuPhe. TAP-containing membranes (150 μg of total protein) were incubated with fluorescein-labeled RRYQNSTC^(F)L (1 μM) for 5 min at 32 °C in the presence or absence of 3 mM ATP. To show reversibility, cross-linked samples were subsequently reduced with 100 mM 2-ME for 5 min at 4 °C (dark gray). ATP-dependent TAP transport activity in the absence of CuPhe was normalized to 100%. Data were collected from 3 independent experiments. The error bars represent the standard deviation.