RESEARCH ARTICLE Udo Krause-Buchholz Æ Kathrin Barth Cora Dombrowski Æ Gerhard Ro¨del Saccharomyces cerevisiae translational activator Cbs2p is associated with mitochondrial ribosomes Received: 9 January 2004 / Revised: 22 March 2004 / Accepted: 27 March 2004 / Published online: 4 May 2004 Ó Springer-Verlag 2004 Abstract A characteristic feature of the mitochondrial expression system in Saccharomyces cerevisiae is the requirement for gene-specific translational activator proteins. Translation of mitochondrial apocytochrome b mRNA requires the nucleus-encoded proteins Cbs1p and Cbs2p. These proteins are thought to tether cyto- chrome b mRNA to the mitochondrial inner membrane via binding to the 5¢ untranslated mRNA leader. Here, we demonstrate by the use of affinity chromatography and coimmunoprecipitation that Cbs2p interacts with the mitoribosomes. We further provide evidence that the C-terminus of Cbs2p is important for ribosome associ- ation, while the N-terminal portion is essential for the formation of homomeric structures. Keywords Saccharomyces cerevisiae Æ Translational activator proteins Æ Cbs2p Æ Mitochondrial ribosomes Introduction Most mitochondrial (mt) proteins are encoded by nu- clear genes and are imported into the organelle after translation on cytosolic ribosomes. A few key compo- nents, however, are products of the mtDNA. With the exception of Var1p, a protein of the small mt ribosomal (mitoribosomal) subunit, all these proteins are subunits of the oxidative phosphorylation (OXPHOS) system. The mtDNA-encoded proteins are synthesized on mi- toribosomes, which are preferentially detected at the matrix side of the inner mt membrane (Watson 1972). In the yeast Saccharomyces cerevisiae, genetic data led to the identification of specific translational activator pro- teins which are essential for the translation of individual mt transcripts. In the absence of a functional activator the protein is not synthesized, despite the presence of its target mRNA. When translation of intron-encoded RNA-maturases is necessary for RNA maturation [as in the case of apocytochrome b (COB) RNA], a lack of translational activator proteins results in the accumula- tion of unspliced precursor RNAs. Analysis of mt sup- pressor mutations suggested that the 5¢ untranslated RNA leader sequences (5¢UTL) are the target sites of the translational activator proteins (for a review, see Fox 1996). For example, translation of Cox3p is no longer dependent on its activator Pet494p, when the COX3 gene is fused to the 5¢UTL of COB mRNA due to a rearrangement of mtDNA (Ro¨del and Fox 1987). Translation of this novel chimeric gene requires the COB-specific activators Cbs1p and Cbs2p (Ro¨del 1997). Similarly, mutations affecting Cbs1p or Cbs2p can be overcome by mt mutations which replace the COB 5¢UTL by the ATP9 5¢UTL (Ro¨del 1986). The idea that 5¢UTLs directly interact with activator proteins is strongly supported by the finding that alterations or overproduction of genes encoding translational activa- tors were able to bypass COX2 and COX3 5¢UTL mutations, respectively (Mulero and Fox 1993; Wie- senberger et al. 1995). All translational activator proteins studied so far are firmly bound or at least associated with the inner mt membrane (Michaelis et al. 1991; McMullin and Fox 1993). Because of this association, it was proposed that the activators act by tethering their target RNA to specific sites at the inner membrane to promote trans- lation and concomitant assembly of the nascent proteins into the OXPHOS complexes (Michaelis et al. 1991). Strong experimental evidence for this view was obtained by Sanchirico et al. (1998). Those authors showed that Communicated by M. Brunner U. Krause-Buchholz (&) Æ K. Barth Æ C. Dombrowski Æ G. Ro¨del Institut fu¨r Genetik, Technische Universita¨t Dresden, 01062 Dresden, Germany E-mail: ukrause@rcs.urz.tu-dresden.de Tel.: +49-351-46335960 Fax: +49-351-46337725 Present address: K. Barth Institut fu¨r Anatomie, Medizinische Fakulta¨t Carl Gustav Carus, 01307 Dresden, Germany Curr Genet (2004) 46: 20–28 DOI 10.1007/s00294-004-0503-y