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T W McMullin

Publications and source records attributed to T W McMullin.

9 recordsLinked to original sources
PubMed1993

COX3 mRNA-specific translational activator proteins are associated with the inner mitochondrial membrane in Saccharomyces cerevisiae.

Three yeast proteins coded by nuclear genes, PET54, PET122, and PET494, are specifically required to activate translation of the mitochondrially coded COX3 mRNA, and one of them, PET122, has been shown to interact functionally with both the mitochondrial ribosomal small subunit and the COX3 mRNA leader. To determine the location of these proteins within mitochondria, we have used antisera directed against them to assay for their presence in submitochondrial fractions. Approximately half of the PET54 protein present in mitochondria from wild-type cells pelleted with membranes, while half was released from disrupted mitochondria in soluble form. The membrane-bound PET54 floated with the inner membrane during buoyant density gradient centrifugation, but was efficiently solubilized by extraction with alkaline carbonate. The PET122 and PET494 proteins could only be detected in cells overproducing these proteins. PET122 was completely membrane-bound and could not be extracted with alkaline carbonate. Most of the PET494 protein pelleted with membranes and very little could be solubilized by alkaline carbonate. Both PET122 and PET494 floated with membranes during buoyant density gradient centrifugation. These data suggest the possibility that synthesis of the highly hydrophobic subunit III of cytochrome c oxidase is activated by PET54, PET122, and PET494 at the inner mitochondrial membrane. Amino-terminal sequencing of proteins isolated from mitochondria revealed that PET54 is not processed during import to mitochondria while the PET122 precursor is cleaved between the 8th and 9th residues.

Amino Acid Sequence↗
PubMed1991

Functional interactions among two yeast mitochondrial ribosomal proteins and an mRNA-specific translational activator.

Expression of the Saccharomyces cerevisiae mitochondrial gene coding cytochrome c oxidase subunit III is specifically activated at the level of translation by at least three nuclear genes, PET122, PET494 and PET54. We have shown previously that carboxy-terminal deletions of PET122 are allele-specifically suppressed by mutations in an unlinked nuclear gene, termed PET123, that encodes a small subunit ribosomal protein. Here we describe additional pet122 suppressors generated by mutations in a second gene which we show to be the previously identified nuclear gene MRP1. Like PET123, MRP1 encodes a component of the small subunit of mitochondrial ribosomes. Our mrp1 mutations are allele-specific suppressors of carboxyl-terminal truncations of the PET122 protein and do not bypass the requirement for residual function of PET122. None of our mrp1 mutations has an intrinsic phenotype in an otherwise wild-type background. However, some of the mrp1 mutations cause a non-conditional respiratory-defective phenotype in combination with certain pet123 alleles. This synthetic defective phenotype suggests that the ribosomal proteins PET123 and MRP1 interact functionally with each other. The fact that they can both mutate to suppress certain alleles of the mRNA-specific translational activator PET122 strongly suggests that the PET122 protein promotes translation of the coxIII mRNA via an interaction with the small subunit of mitochondrial ribosomes.

DNA, Mitochondrial↗
PubMed1990

A genetic link between an mRNA-specific translational activator and the translation system in yeast mitochondria.

Translation of the Saccharomyces cerevisiae mitochondrial mRNA encoding cytochrome c oxidase subunit III (coxIII) specifically requires the products of at least three nuclear genes, PET122, PET494 and PET54. pet122 mutations that remove 24-67 amino acid residues from the carboxyterminus of the gene product were found to be suppressed by unlinked nuclear mutations. These unlinked suppressors fail to suppress both a pet122 missense mutation and a complete pet122 deletion. One of the suppressor mutations causes a heat-sensitive nonrespiratory growth phenotype in an otherwise wild-type strain and reduces translation of all mitochondrial gene products in cells grown at high temperature. This suppressor maps to a newly identified gene on chromosome XV termed PET123. The sequence of a DNA fragment carrying PET123 contains one major open reading frame encoding a basic protein of 318 amino acids. Inactivation of the chromosomal copy of PET123 by interruption of this open reading frame causes cells to become rho- (sustain large deletions in their mtDNA). This phenotype is characteristic for null alleles of genes whose products are essential for general mitochondrial protein synthesis. Thus our data strongly suggest that the PET123 protein is a component of the mitochondrial translation apparatus that interacts directly with the coxIII-mRNA-specific translational activator PET122.

Alleles↗
PubMed1990

A novel small-subunit ribosomal protein of yeast mitochondria that interacts functionally with an mRNA-specific translational activator.

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 coxIII mRNA.

Electron Transport Complex IV↗
PubMed1988

Plasmids can stably transform yeast mitochondria lacking endogenous mtDNA.

The mitochondrial gene oxi1, carried on a bacterial plasmid, has been used to transform the mitochondria of a yeast strain lacking mtDNA (rho0). 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 oxi1 sequences in the transformants were surrounded by restriction sites derived from the plasmid that were not present in wild-type mtDNA. oxi1 genetic information in these "synthetic rho-" strains could be expressed in diploids either after "marker rescue" by recombination with rho+ mtDNA carrying an appropriate oxi1 point mutation or in trans during the growth of diploids heteroplasmic for both the plasmid-derived oxi1 sequences and rho+ mtDNA with oxi1 deleted. The ability to generate such "synthetic rho-" strains by transformation will allow transfer of mutations generated in vitro to wild-type rho+ mtDNA as well as examination of the function of altered genes in trans.

DNA, Mitochondrial↗
PubMed1988

A highly evolutionarily conserved mitochondrial protein is structurally related to the protein encoded by the Escherichia coli groEL gene.

We recently reported that a Tetrahymena thermophila 58-kilodalton (kDa) mitochondrial protein (hsp58) was selectively synthesized during heat shock. In this study, we show that hsp58 displayed antigenic similarity with mitochondrially associated proteins from Saccharomyces cerevisiae (64 kDa), Xenopus laevis (60 kDa), Zea mays (62 kDa), and human cells (59 kDa). Furthermore, a 58-kDa protein from Escherichia coli also exhibited antigenic cross-reactivity to an antiserum directed against the T. thermophila mitochondrial protein. The proteins from S. cerevisiae and E. coli antigenically related to hsp58 were studied in detail and found to share several other characteristics with hsp58, including heat inducibility and the property of associating into distinct oligomeric complexes. The T. thermophila, S. cerevisiae, and E. coli macromolecular complexes containing these related proteins had similar sedimentation characteristics and virtually identical morphologies as seen with the electron microscope. The distinctive properties of the E. coli homolog to T. thermophila hsp58 indicate that it is most likely the product of the groEL gene.

Animals↗
PubMed1987

A normal mitochondrial protein is selectively synthesized and accumulated during heat shock in Tetrahymena thermophila.

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.

Animals↗
PubMed1986

Effect of heat shock on ribosome structure: appearance of a new ribosome-associated protein.

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.

Electrophoresis, Polyacrylamide Gel↗