Prof. Fox is no longer mentoring undergraduates, graduate students, and postdocs in laboratory research.
What have we found interesting about the cell biology of mitochondria?
The mitochondrion is arguably the most complex organelle in the budding yeast cell cytoplasm. It is essential for viability as well as respiratory growth. Its innermost aqueous compartment, the matrix is bounded by the highly structured inner membrane, which is in turn bounded by the intermembrane space and the outer membrane. Approximately 1000 proteins are present in these organelles, of which 8 major constituents are coded and synthesized in the matrix.
The oxygen we breathe and the food we eat are ultimately consumed in mitochondria by respiration, which is coupled with oxidative phosphorylation to capture energy. The enzyme complexes that carry this out are assembled in the inner mitochondrial membrane from protein subunits coded by both nuclear genes and genes in mitochondrial DNA.
While mitochondria are derived from eubacterial ancestors, the organellar genetic systems found today in animals, plants and fungi do not closely resemble those of modern eubacteria. To understand how these small but important genomes are expressed, we need to study the organelles themselves.
Our research has been aimed at understanding how expression of genes in mitochondrial DNA is controlled by nuclear genes, and how mitochondrially coded proteins are assembled with nuclearly coded proteins into the respiratory chain complexes. Budding yeast (Saccharomyces cerevisiae) is a wonderful organism in which to study these interactions, since mutations in both genetic systems can be isolated and manipulated.
Furthermore, genetic transformation and homologous recombination allow the replacement of wild-type by mutant, or novel, DNA sequences in both the nuclear and mitochondrial genomes.
Inheritance through meiosis
Mitochondrial (Left) and Nuclear (Right)
Our studies have revealed that translation of mitochondrial mRNAs within the organelle is tightly controlled, and apparently highly organized on the surface of the inner membrane. This translational regulation and organization facilitates efficient assembly of the respiratory chain complexes.
Studies in mitochondrial biology with key papers
1) mRNA-specific translational activation and homeostatic control of Cox1 translation
Translation of at least five of the seven major mitochondrially coded mRNAs is activated mRNA-specifically by nuclearly encoded proteins which recognize sites in the mRNA 5'-untranslated leaders. The translational activators for the three mitochondrial mRNAs encoding subunits of cytochrome c oxidase interact with each other on the inner surface of the inner membrane and thus appear to co-localize synthesis of these proteins. We believe that this is an adaptation allowing the efficient assembly of the core of cytochrome c oxidase within the membrane bilayer.
The COX1 mRNA activator Mss51 interacts not only with the mRNA 5'-UTL, but also with newly synthesized Cox1 protein itself. Sequestration of Mss51 with unassembled Cox1 in assembly intermediates limits the amount available for translation, thus coupling synthesis and assembly. This appears to be an adaptation to prevent overproduction of unassembled Cox1, a known pro-oxidant species.
- Inventory control: cytochrome c oxidase assembly regulates mitochondrial translation
- The carboxyl-terminal end of Cox1 is required for feedback-assembly regulation of Cox1 synthesis in Saccharomyces cerevisiae mitochondria
- Dual functions of Mss51 couple synthesis of Cox1 to assembly of cytochrome c oxidase in Saccharomyces cerevisiae mitochondria
- Interactions among COX1, COX2, and COX3 mRNA-specificTranslational Activator Proteins on the Inner Surface of the Mitochondrial Inner Membrane of Saccharomyces cerevisiae
- Overexpression of the COX2 translational activator, Pet111p, prevents translation of COX1 mRNA and cytochrome c oxidase assembly inmitochondria of Saccharomyces cerevisiae
- Accumulation of mitochondrially synthesized Saccharomyces cerevisiaeCox2p and Cox3p depends on targeting information in untranslated portions of their mRNAs
- Highly Diverged Homologs of Saccharomyces cerevisiae MitochondrialmRNA-Specific Translational Activators Have Orthologous Functions in Other Budding Yeasts
2) The role of ribosomes and other general factors in controlling mitochondrial translation initiation
Studies of genetic interactions involving mitochondrial mRNAs and their mRNA-specific translational activators have revealed that mitochondrial ribosomal proteins play important roles in controlling translation. Only some of these mitochondrial ribosomal proteins are recognizably related to bacterial ribosomal proteins.
- Translation initiation in Saccharomyces cerevisiae mitochondria: Functional interactions among mitochondrial ribosomal protein Rsm28p, initiation factor 2, methionyl-tRNA-formyltransferase, and novel protein Rmd9p
- Alteration of a Novel Dispensable Mitochondrial RibosomalSmall-Subunit Protein, Rsm28p, Allows Translation of Defective COX2mRNAs
- MrpL36p, a Highly Diverged L31 Ribosomal Protein Homolog With Additional Functional Domains in Saccharomyces cerevisiae Mitochondria
- Evidence that Synthesis of the Saccharomyces cerevisiae MitochondriallyEncoded Ribosomal Protein Var1p May Be Membrane Localized
- A novel small-subunit ribosomal protein of yeast mitochondria that interacts functionally with an mRNA-specific translational activator
- Functional Interactions between Yeast Mitochondrial Ribosomes and mRNA 5' Untranslated Leaders
3) Insertion of mitochondrially coded proteins into and through the inner membrane
Several protein domains synthesized on mitochondrial ribosomes must be inserted into, or translocated through, the inner mitochondrialmembrane. We are using genetic approaches to identify factors necessary for the export of the N- and C- terminal hydrophilic domains of Cox2p (cytochrome c oxidase subunit II), as well as the features of Cox2p that are recognized as export signals. Interestingly, the mechanisms of translocation for these two domains of Cox2p are distinct.
- Multiples roles of the Cox20 chaperone in assembly of Saccharomyces cerevisiae cytochrome c oxidase
- Roles of Oxa1-related inner-membrane translocases in assembly of respiratory chain complexes
- Translocation and assembly of mitochondrially coded Saccharomyces cerevisiae cytochrome c oxidase subunit Cox2 by Oxa1 and Yme1 in the absence of Cox18
- Translocation of mitochondrially synthesized Cox2p domains from the matrix to the intermembrane space
- Cox18p Is Required for Export of the Mitochondrially EncodedSaccharomyces cerevisiae Cox2p C-Tail and Interacts with Pnt1p andMss2p in the Inner Membrane
- Peripheral Mitochondrial Inner Membrane Protein, Mss2p, Required for Export of the Mitochondrially Coded Cox2p C Tail in Saccharomy cerevisiae
- Mutations affecting a yeast mitochondrial inner membrane protein,Pnt1p, block export of a mitochondrially synthesized fusion protein from the matrix
- Membrane translocation of mitochondrially coded Cox2p: distinct requirements for export of N and C termini and dependence on the conserved protein Oxa1p
4) A collaboration with Ophry Pines, using a synthetic mitochondrial gene, to explore the functions of cytoplasmic and mitochondrial fumarase
Fumarase (also known as fumarate hydratase) is a nuclearly encoded enzyme found in both the cytoplasm and mitochondria of yeast and other eukaryotes, including humans. By inserting a synthetic mitochondrial gene encoding the enzyme into mtDNA, we were able to generate strains specifically lacking the enzyme in the cytoplasm and nucleus. This led to the surprising discovery that the yeast cytosolic fumarase plays a key role in the protection of cells from DNA damage, particularly from DNA double-strand breaks. This finding may explain the tumor-suppressor activity of fumarase in humans.
Email: tdf1 [at] cornell.edu
Department of Molecular Biology and Genetics
Ithaca, NY 14853-2703