What's so interesting about the cell biology of mitochondria?
The oxygen we breath 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 genomes 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 is 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
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 codedmRNAs is activated mRNA-specifically by nuclearly encoded proteinswhich recognize sites in the mRNA 5'-untranslated leaders. Thetranslational activators for the three mitochondrial mRNAs encodingsubunits of cytochrome c oxidase interact with each other on the innersurface of the inner membrane and thus appear to co-localize synthesisof these proteins. We believe that this is an adaptation allowingthe efficient assembly of the core of cytochrome c oxidase within themembrane 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.
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 theMitochondrial Inner Membrane of Saccharomyces cerevisiae
Overexpression of the COX2 translational activator, Pet111p, preventstranslation 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 untranslatedportions of their mRNAs
Highly Diverged Homologs of Saccharomyces cerevisiae MitochondrialmRNA-Specific Translational Activators Have Orthologous Functions inOther Budding Yeasts
2) The role of ribosomes and other general factors in controlling mitochondrial translation initiation
Studies of genetic interactions involving mitochondrial mRNAs and theirmRNA-specific translational activators have revealed that mitochondrialribosomal proteins play important roles in controllingtranslation. Only some of these mitochondrial ribosomal proteinsare 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 WithAdditional 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 thatinteracts 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 factorsnecessary for the export of the N- and C- terminal hydrophilic domainsof Cox2p (cytochrome c oxidase subunit II), as well as the features ofCox2p that are recognized as export signals. Interestingly, themechanisms of translocation for these two domains of Cox2p are distinct.
Translocation and assembly of mitochondrially coded Saccharomyces cerevisiae cytochrome c oxidase subunit Cox2 by Oxa1 and Yme1 in the absence of Cox18
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, Requiredfor 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 proteinfrom the matrix
Membrane translocation of mitochondrially coded Cox2p: distinctrequirements for export of N and C termini and dependence on theconserved 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.