Abstract

Animals, plants, fungi, and other eukaryotes assemble their respiratory apparatus from protein subunits made in the cytoplasm and protein subunits made inside mitochondria. We are trying to understand the basic mechanisms that allow this coordinated process to occur, since defects in this process affect the health and vitality of both plants and animals, including humans.

Issue

Oxygen is a very dangerous chemical. Cells that live in the presence of oxygen, and use it to burn their food must handle it very carefully. Interestingly, the apparatus that ultimately consumes oxygen in organelles called mitochondria is composed of proteins encoded by nuclear genes that are synthesized in the cytoplasm, and proteins encoded by genes in mitochondrial DNA that are synthesized by a separate genetic system inside the organelles. Defects in the complex interplay between these genetic systems in humans lead to disease and appear to play a significant role in the aging process, while variation in mitochondrial gene expression in maize and other plants influences male fertility and pathogen sensitivity.

Response

We study the mechanisms employed to control mitochondrial gene expression in Baker's yeast (the model organism), and to target mitochondrially coded proteins to their proper destination within the organelle. We have found that dramatically elevated levels of the COX2 mitochondrial mRNA-specific translational activator protein Pet111p interfere with respiratory growth and cytochrome c oxidase accumulation. The respiratory phenotype appears to be caused primarily by inhibition of the COX1 mitochondrial mRNA translation, a finding confirmed by lack of cox1deletion::ARG8m reporter mRNA translation. Respiratory growth is partially restored by a chimeric COX1 mRNA bearing the untranslated regions of the COX2 mRNA, and by overproduction of the COX1 mRNA-specific activators, Pet309p and Mss51p. These results suggest that excess Pet111p interacts unproductively with factors required for normal COX1 mRNA translation. Certain missense mutations in PET111 alleviate the interference with COX1 mRNA translation but do not completely restore normal respiratory growth in strains overproducing Pet111p, suggesting that elevated Pet111p also perturbs assembly of newly synthesized subunits into active cytochrome c oxidase. Thus, this imbalance in translational activator levels causes multiple problems in mitochondrial gene expression reflecting the dual role of balanced translational activators in cooperatively regulating both the levels and locations of organellar translation.

Impact

Our detailed examination of mitochondrial gene expression in yeast has revealed that organellar gene regulation is often at the level of protein synthesis instead of mRNA synthesis. And it has revealed that this regulatory mechanism plays an important role in targeting the newly synthesized mitochondrial gene products to sites where they can be efficiently assembled into respiratory complexes. These studies have taken advantage of the remarkably powerful genetic approaches that can be used to study yeast, principally replacement of genes in both nuclear and mitochondrial chromosomes, but not in more complex organisms such as plants and animals. Thus our exploratory work has provided a trailblazing look at the questions that should be studied in these more complex organisms, as well as helping to elucidate basic mechanisms and potential drug targets in fungi.

Funding Sources

• Other Federal non-USDA (e.g., NSF, NIH, DOA, DOD)

Topic Description

• Basic biological research on cellular mechanisms

Collaborators

• Dr. Thomas L. Mason, University of Massachusetts, Amherst, MA
• Dr. Dieter Söll, Yale University, New Haven, CT
• Dr. Nathalie Bonnefoy, CNRS, Gif-sur-Yvette, France

Key Personnel

• Dr. Heather Lumppio Fiumera, MBG, Cornell University
• Dr. Alessandro Fiori, MBG, Cornell University
• Dr. Tim Ellis, MBG, Cornell University