Fig. 5. (A) ClpXP degrades a disulfide-bonded heterodimer in which just one subunit contains a ssrA tag. Three disulfide-bonded species were present in the experiment, i.e. untagged homodimers, singly tagged heterodimers and tagged homodimers, but only the untagged subunits were 35S-labeled and thus the tagged homodimer was not detected following SDS–PAGE and autoradiography. Lane 4 shows SDS–PAGE of the protein mixture used in the degradation experiment, stained for total protein with SYPRO Orange. (B) Quantitation of the autoradiogram in (A). (C) Specific trapping of the singly tagged heterodimer in ClpPDFP as measured by gel filtration chromatography The top panel shows the absorbance profile at 214 nm of samples eluting from the column. The elution positions of molecular weight standards are shown above the chromatogram. Fractions 10–35 were analyzed by SDS–PAGE and quantified by PhosphorImager analysis. The ClpP14 and ClpX peaks were identified by staining for total protein with SYPRO Orange. The intensities of the heterodimer band (middle panel) and untagged homodimer band (bottom panel) are shown.