FIG. 4.
Role of RIC45p and RIC21p in import. (A) Inhibition by anti-RIC45p antibody. Mitoplasts were incubated with the indicated antiserum (1:50), washed, and assayed for import (a) or binding (b) of the indicated 32P-labeled tRNA substrate (S) in the presence or absence of the indicated effector (E). Ab, antibody; n, nonimmune serum; α, anti-RIC 45p serum. (B) Immunoprecipitation of cross-linked tRNA. Left, 5-BrU-, 32P-labeled tRNATyr (20 fmol) was incubated with mitoplasts in the presence (lane 6) or absence (lanes 1 to 5 and 7) of low-specific-activity tRNAIle (2 fmol) under binding conditions. In lanes 1 to 3, the mitoplasts were solubilized and directly loaded; lanes 4 to 7 show immunoprecipitates. RNA-protein complexes were UV cross-linked (except lane 1, a no-UV control) and immunoprecipitated with anti-RIC45p (lanes 5 and 6), anti-RIC21p (lane 7), or nonimmune serum (lane 4). Right, photo-cross-linking of 5-BrU-labeled D arm oligonucleotide (Fig. 3). Lane 10, input RNA. Lane 8, cross-linked product. Lane 9, anti-RIC45p immunoprecipitate. (C) Inhibition by anti-RIC21p antibody. Mitoplasts were incubated with nonimmune or anti-RIC21p antibody and assayed for import (a) or binding (b) of the indicated substrate in the absence or presence of effector. (D) Cross-linking of RIC21p to intact tRNAIle (left) or tRNAIle [42-66] (right). Mitoplasts were incubated with the indicated substrates and effectors before UV irradiation. Lane 4, input tRNAIle. Lane 5, cross-linked product. Lanes 1 to 3, 6, and 7, immunoprecipitates with anti-RIC21p (lanes 1 and 2), nonimmune serum (lanes 3 and 7), or anti-45p antibody (lane 6). Right, tRNAIle [42-66] was incubated with mitoplasts in the absence (lane 8) or presence (lanes 9 and 10) of tRNATyr effector and then irradiated. Lane 10, anti-21p immunoprecipitate.