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The mitochondrial gene oxil, carried on a bacterial plasmid, has been used to transform the mitochondria of a yeast strain lacking mtDNA (rhoo). The plasmid DNA behaved in a manner entirely consistent with the known properties of normal yeast rho-mtDNA after its introduction by high-velocity microprojectile bombardment. Like the mtDNA sequences retained in natural rho-strains, the plasmid DNA in the transformants was reiterated into concatemers whose size was indistinguishable from that of wild-type mtDNA. The oxil sequences in the transformants were surrounded by restriction sites derived from the plasmid that were not present in wild-type mtDNA. oxil genetic information in these "synthetic rho"-strains could be expressed in diploids either after "marker rescue" by recombination with rho+ mtDNA carrying an appropriate oxil point mutation or in trans during the growth of diploids heteroplasmic for both the plasmid-derived oxil sequences and rho' mtDNA with oxil deleted. The ability to generate such "synthetic rho-" strains by transformation will allow transfer of mutations generated in (7), where it became incorporated into preexisting organellar DNA (rho+ in the case ofyeast) by recombination.We report here that yeast mitochondria lacking mtDNA can be transformed by bombardment of a rho' strain with microprojectiles carrying a mitochondrial gene, oxil (8-10), on a plasmid. Genetic and molecular analysis of the transformants demonstrates that they exhibit all the expected properties outlined above for a rho-strain carrying reiterated copies of the plasmid. The ability to generate such "synthetic rho-" strains by transformation will now allow transfer of mutations generated in vitro to wild-type rho+ mtDNA as well as examination of the function of altered genes in trans. MATERIALS AND METHODSMedia, Yeast Strains, and Genetic Methods. Minimal medium (SD), complete medium containing glucose (YPD), complete medium containing the nonfermentable carbon sources ethanol and glycerol (YPEG), and standard genetic manipulations were as described (8,11). Key strains and their genotypes are listed in Table 1. TD28rho°was generated by treatment of TD28 (12) with ethidium bromide (13). TF138 and TF140 were progeny of crosses between DAU2 (MATa, ade2, ura3, [rho+]) and the oxil mutants M8-171 and M11-30, respectively (8,14,15). TF145 was similarly derived from a spontaneous oxil deletion isolated in this laboratory. All other strains have been described previously (8,16,17).Plasmids and Transformations. The plasmid pMT36 (18) carries the wild-type oxil gene inserted at the Pst I site of pBR322 (19) after GC-tailing such that the mtDNA insert could be cleaved out by using either the Pst I sites derived from the plasmid or the Msp I (Hpa II) sites derived from mtDNA (8) (see Fig. 1). pCGE137 carries the wild-type URA3 gene and a 1.56-kb fragment containing the origin of replication of the 2-,um plasmid of yeast in pBR322 and has been used previously (20). Equal amounts of these plasmid DNAs were mixed and precipitated o...

We have identified and purified a 58-kilodalton protein of Tetrahymena thermophila whose synthesis during heat shock parallels that of the major heat shock proteins. This protein, hsp58, was found in both non-heat-shocked as well as heat-shocked cells; however, its concentration in the cell increased approximately two-to threefold during heat shock. The majority of hsp58 in both non-heat-shocked and heat-shocked cells was found by both cell fractionation studies and immunocytochemical techniques to be mitochondrially associated. During heat shock, the additional hsp58 was found to selectively accumulate in mitochondria. Nondenatured hsp58 released from mitochondria of non-heat-shocked or heat-shocked cells sedimented in sucrose gradients as a 20S to 25S complex. We suggest that this protein may play a role in mitochondria analogous to the role the major heat shock proteins play in the nucleus and cytosol.Virtually every organism tested to date responds to hyperthermal stress by transiently inducing the synthesis and accumulation of a specific array of polypeptides commonly referred to as heat shock proteins (hsps) or stress proteins (summarized in references 4, 32, and 48). The major hsps of most organisms fall into size classes of approximately 80 to 90, 68 to 75, and 15 to 30 kilodaltons (kDa). Evolutionary conservation of DNA and amino acid sequences exhibited by the groups of major hsps, especially the hsp7O group (6, 10, 24-26, 32, 47), suggest that they must play fundamental metabolic roles in the cell. In many organisms, a variable array of minor hsps are also synthesized during heat shock (17,33,38,58,60). Collectively, these hsps are thought to protect the cell from the adverse effects of heat shock as well as other physiological stresses (summarized in references 4, 10, 32, and 48).While mutations in specific heat shock genes have been shown to affect the viability of the cells harboring those mutations (11,35,62), the exact functions of individual hsps are still not well understood. One approach to ascertaining the functions of individual hsps has been to determine the subcellular locations of those proteins. These studies have revealed that during heat shock certain hsps, such as some members of the 70-kDa group, and several of the small hsps tend to accumulate in the nucleus, while others, such as members of the 80-to 90-kDa group, remain cytosolic (3,17,35,51,56,59). Additionally, several of the small hsps appear to form cytoplasmic aggregates (2,40,49). In plants, several hsps were found to associate with chloroplasts (27, 57), ribosomes (31), and mitochondria (31,50). Although these studies clearly show differences in the localizations of the hsps, particular functions have not yet been ascribed to these proteins.In Tetrahymena thermophila, the heat shock response is typical and can be induced by shifting the organism from 30 to 41°C (16, 19, 20, 63). This induces the immediate and transient synthesis of the major hsps (73 kDa, 80 kDa, and 29 to 35 kDa) as well as several minor hsps (20,37). * Co...

Mitochondrial translation of the mRNA encoding cytochrome c oxidase subunit III (coxIII) specifically requires the action of three position activator proteins encoded in the nucleus of Saccharomyces cerevisiae. Some mutations affecting one of these activators, PET122, can be suppressed by mutations in an unlinked nuclear gene termed PET123. PET123 function was previously demonstrated to be required for translation of all mitochondrial gene products. We have now generated an antibody against the PET123 protein and have used it to demonstrate that PET123 is a mitochondrial ribosomal protein of the small subunit. PET123 appears to be present at levels comparable to those of other mitochondrial ribosomal proteins, and its accumulation is dependent on the presence of the 15S rRNA gene in mitochondria. Taken together with the previous genetic data, these results strongly support a model in which the mRNA-specific translational activator PET122 works by directly interacting with the small ribosomal subunit to promote translation initiation on the coxll mRNA.Protein synthesis in mitochondria of Saccharomyces cerevisiae depends on nuclear genes that encode components of the general translation machinery such as mitochondrial ribosomal proteins, translation factors, and tRNA synthetases (1, 21; M. C. Costanzo and T. D. Fox, Annu. Rev. Genet., in press). In addition to these general components, other proteins are required specifically to promote the translation of particular mitochondrial mRNA species (1, 18, 21; Costanzo and Fox, in press). For example, the proteins coded by the nuclear genes PET122, PET54, and PET494 have been shown to be required specifically for mitochondrial translation of cytochrome c oxidase subunit III (coxIII) (2,5,8,10,26,27,31). All three of these coxIII-specific translational activators have been shown to accumulate in mitochondria (5, 9, 35; unpublished results), and their site (or sites) of action has been mapped genetically to the 5'-untranslated leader of the coxIII mRNA (6). A possible explanation for these findings is that one or more of the three translational activator proteins interact directly both with the coxIII 5' leader and with some component of the general mitochondrial translation apparatus to activate coxIII translation.To understand better the mechanism of action of the coxIII-specific translational activators, we have been isolating and analyzing allele-specific nuclear suppressors of mutations in PET122, PET54, and PET494. Such allele-specific suppressors can frequently identify genes encoding proteins that directly interact (23,25

After a nonlethal but heat shock protein-inducing hyperthermic treatment, ribosomes isolated from Tetrahymena thermophila contained an additional 22-kilodalton protein (p22). When maximally ribosome associated, this protein was found to be on the small subunit in a 1:1 stoichiometric ratio with other ribosomal proteins. Using an antiserum directed against the purified 22-kilodalton protein, we found that non-heat-shocked and heat-shocked cells contain identical amounts of this protein, the only difference being that in the stressed cells p22 is entirely ribosome bound, whereas in the unstressed cells p22 has little or no detectable ribosome association. Because the two-dimensional electrophoretic properties of p22 showed no alterations after heat shock, this change in state of ribosome-p22 interaction does not appear to be caused by a chemical modification of p22. When not strongly ribosome associated, p22 is not found free in the cytoplasm. During that time in heat shock when p22 is first becoming ribosome associated, it is found preferentially on polysomal ribosomes. Subsequently, all ribosomes, whether polysome bound or not, obtain a bound p22. The functional significance of this association is discussed.