FIG. 2.

Tom40 forms homo-oligomers and interacts with Tom6. The indicated cross-linking reagents (see Materials and Methods) were added to intact mitochondria (A) or OMV (B). Samples were incubated for 30 min at 25°C before the cross-linkers were quenched. Proteins were analyzed by SDS-PAGE under nonreducing conditions and immunostaining with antibodies against Tom40. (C) The Tom40-containing 45-kDa band is a cross-linking adduct of Tom40 and Tom6. OMV were incubated with the cross-linker EDC or DSP for 30 min. Aliquots of each sample were analyzed by immunostaining with antibodies (Ab) against Tom40 and Tom6. Tom6 is only weakly stained due to its poor blotting efficiency. (D) Tom40 cross-linking products in yeast mitochondria. Isolated yeast mitochondria were treated with the indicated cross-linkers and analyzed by immunostaining for Tom40 as described for panel A. (E) The Tom40 cross-linking products are formed by using purified N. crassa TOM complex (21). As described for panel A, cross-linkers were added to the purified TOM complex and samples were incubated for 90 min at 0°C. Further analysis was performed as described for panel A. (F) The isoelectric point of the Tom40 cross-linking products is identical to that of the Tom40 monomer. Cross-linking with DSG was performed as described for panel B, using OMV. The sample was separated in the first dimension by isoelectric focusing and in the second dimension by SDS-PAGE (2). The pI values and the molecular masses of marker proteins are indicated.