FIGURE 5:

Expression of Rsf, but not of Rsf-M, in the cytosol reduces cellular fitness. (A) Rsf-HA (corresponding to residues 1–278 of Atp25) was expressed in yeast cells. A cell extract was loaded onto a linear 10–34% sucrose gradient. The gradient was harvested by pumping it through a photometer recording the distribution of ribosomes at 260 nm. Proteins from 14 fractions were precipitated and analyzed by Western blotting. (B) Tenfold serial dilutions were dropped either on glucose (noninducing) or galactose (inducing) plates. The plates were incubated at 35°C. Expression of Rsf but not of Rsf-M interfered with cell growth. (C) Growth curves of the same strains as in B in glucose- and galactose-containing media. Expression of Rsf retarded cell growth (arrowhead). Mean values of four replicates. SDs are plotted as gray bars. (D) Extracts from the stains used in B were loaded onto linear 10–34% sucrose gradients. The distribution of ribosomes was analyzed spectroscopically. Relative area volumes of the monosome peaks. (E, F) Yeast cells expressing Rsf-M, Rsf, or M in the cytosol were grown to log phase, and [³⁵S]methionine was added for the times indicated. Cells were harvested and lysed. Proteins were resolved either by SDS–PAGE or dot blot analysis and visualized by autoradiography. (G) A C-terminally hexahistidine-tagged version of Rsf (residues 43–292) was expressed in E. coli, purified from inclusion bodies, refolded, and added to reticulocyte lysate (Promega) for an in vitro translation reaction. Bovine serum albumin (BSA) was used for control. Addition of Rsf prevented protein synthesis of this cytosolic translation system. (H) Model for the import and processing of Atp25. In mitochondria, Rsf associates with the mitochondrial ribosome in the matrix. Our observations suggest that Rsf can interfere with the cytosolic translation machinery, which is prevented by its synthesis as a composite Atp25 precursor protein.