Fig. 2.
Interaction of mutant forms of Tim50 with Tim23. (a) Radiolabeled Tim501-361 and mutant forms were imported into yeast mitochondria carrying protein A-tagged Tim23 or into wild-type (WT) mitochondria. After lysis with digitonin and affinity-purification, mature-sized (m) Tim501-361 was analyzed by SDS-PAGE and autoradiography (see Supplementary Material). Control proteins, Tim17 and Atp4, were detected by Western blotting. Load, 10%; elution, 100%. (b) Quantification of co-purification of wild-type (WT) and mutant Tim501-361 forms with Tim23, normalized to input/load. The yield of co-purification of imported WT Tim501-361 was set to 100%. Shown are averages ± SEM (n=4) (range with n=2 for the R214A K217N double mutant form). (c) In vitro crosslinking of purified Tim50164-361 forms and Tim231-96. Crosslinking reactions with glutaraldehyde were carried out at room temperature for 20 min. 10 μM Tim231-96 were incubated with equimolar amounts of Tim50164-361 or mutant forms in 20 mM HEPES, 0.1 M NaCl, pH 8 buffer. 0.005% glutaraldehyde was added. The crosslinking reaction was quenched by adding SDS-PAGE sample buffer, and products were analyzed by 12% SDS polyacrylamide gel electrophoresis.