Figure 2.
Plakophilin 2 interacts with RPC155. (a) Coimmunoselection of RPC155 with plakophilin 2. Extracts from cultured A-431 cells were subjected to immunoselection with plakophilin 2 antiserum (lane 1; lane 2, control showing the proteins of the same antiserum bound to protein A-Sepharose), and the proteins selected were analyzed by SDS/PAGE and stained with Coomassie brilliant blue (the sizes of reference proteins are indicated). The 155-kDa polypeptide (upper arrow) coselected with plakophilin 2 (lower arrow) was excised from the gel and identified by matrix-assisted laser desorption ionization analysis as the largest subunit of RNA polymerase III (RPC155). (b) Peptide sequences determined in the 155-kDa binding partner of plakophilin 2 by matrix-assisted laser desorption ionization analysis. In addition, myosin heavy chain and major vault protein (42) were identified as apparently nonspecifically bound coadsorbed proteins (cf. ref. 7). (c) Blot-overlay assay of polypeptides of a chromatographic fraction enriched in pol III, separated by SDS/PAGE, and visualized with Coomassie brilliant blue (lane 1) or transferred to nitrocellulose membranes (lanes 2 and 3). After incubation with in vitro translated 35S-labeled plakophilin 2, the binding partners of plakophilin 2 within the pol III fraction were detected by autoradiography (lane 2). A 155-kDa protein, which was identified subsequently as the largest subunit of pol III by immunoblot analysis using RPC155-specific antibodies (lane 3), binds plakophilin 2 selectively. The position of reference proteins is indicated on the left.