Fox Lab

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

Mitochondria and Nuclear
Mictochondial (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.

Aspects of mitochondrial biology we are studying, with links to some 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.

See:

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.
See:

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.
See:

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. 

General contact info:

Email:  tdf1@cornell.edu

Phone:  +1.607.254.4835

 

Mailing address:

Department of Molecular Biology and Genetics

Biotechnology Building

Cornell University

Ithaca, NY 14853-2703

USA