Progress 10/01/08 to 09/30/09
Outputs
OUTPUTS: Conducted and analyzed experiments designed to further the aims of our research into the control of gene expression in mitochondria of yeast. These included genetic analysis to identify mutations affecting respiratory growth of yeast, and biochemical studies to examine the interaction of feedback regulatory proteins with the products of gene expression that they regulate. The project has resulted in publications for (see below), and presentations to basic scientists. Verbal reports were given at invited seminars (Pennsylvania State University - April 2009; Cold Spring Harbor Laboratory - July 2009), and interantional conferences (FASEB Summer Research Conference on Mitochondrial Assembly and Dynamics - Carefree, AZ July 2009; 1st International FOR 967 Symposium on Mechanisms of gene expression: assembly, transport and degradation of polypeptides. Homburg-Saar, Germany - October 2009). PARTICIPANTS: Dr. Thomas D. Fox, Principle investigator; Dr. Heather L. Fiumera, Postdoctoral research associate; Lindsay Burwell, Postdoctoral researcher; Christine A. Butler, Research support specialist; Zachary Via, graduate student; Leah Elliott, graduate student; Sue Lee, undergraduate researcher; Hannah Chen, undergraduate researcher. Collaborators: Dr. Xochitl Perez-Martinez, Assistant Professor UNAM, Mexico City; Dr. Nathalie Bonnefoy, Staff scientist, CNRS, Gif-sur-Yvette, France. Dr. Bernard Trumpower, Professor, Dartmouth College. Training: This project affords opportunities for undergraduate research training, Ph.D. graduate education, and postdoctoral training. TARGET AUDIENCES: The primary target audience for this research is the basic biomedical research community, focussing on geneticists, cell biologists, and microbiologists. The lab provides teaching opportunities that enrich the educations of Cornell undergraduate students. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Expression of a limited number of genes within the mitochondria of eukaryotic cells is vitally important for the assembly of the respiratory chain. The last enzyme in this pathway, cytochrome c oxidase, During the past year we have continued to study the mechanisms employed to control mitochondrial gene expression in the model organism yeast and to target mitochondrially coded proteins to their proper destination within the organelle. We have focussed on the mechanisms by which synthesis of the largest subunit (Cox1), encoded in mitochondrial DNA and made within the organelle, is regulated in response to the the ability of the cell to assemble new complexes. This assembly-feedback control is mediated through the protein coded by the nuclear gene MSS51. Mss51 is an unusual mitochondrial mRNA-specific translational activator that has targets of action mapping genetically in the COX1 coding sequence as well as in the 5 prime UTR of the COX1 mRNA. Furthermore, Mss51 co-immune precipitates with newly synthesized Cox1. Since Mss51 is present at near-rate-limiting levels in mitochondria, these dual functions of Mss51 would allow it to link COX1 mRNA translation and assembly of cytochrome oxidase. We are dissecting these Mss51 functions genetically. We have found that a substantial fraction of cellular Mss51 is indeed tied up in complexes with unassembled Cox1, and thus presumably unavailable to promote further synthesis of Cox1. We have continued to obtain more MSS51 mutations that allow activation of translation through the COX1 mRNA 5'UTR but not assembly of cytochrome oxidase. A second project involves the study of membrane translocases required to insert Cox2 into the mitochondrial inner membrane. Two related proteins Oxa1 and Cox18 are necessary to translocate the N-terminal and C-terminal intermembrane spaces domains, respectively, through the inner membrane. We have identified several distinct mutations affecting the same amino acid residue of Oxa1 that allow overproduced Oxa1 to compensate for the lack of Cox18.
Publications
- Bonnefoy, N., H.L. Fiumera, G. Dujardin and T.D. Fox. 2009. Roles of Oxa1-related inner membrane translocases in assembly of respiratory chain complexes. Biochim. Biophys. Acta 1793(1):60-70.
- Ding, M.G., C.A. Butler, S.A. Saracco, T.D. Fox, F. Godard, J.P. di Rago, and B.L. Trumpower, (2009). An improved method for introducing point mutations into the mitochondrial cytochrome b gene to facilitate studying the role of cytochrome b in the formation of reactive oxygen species. Meth. Enzymol. 456: 491-506.
- Fiumera, H.L., M.J. Dunham, S.A. Saracco, C.A. Butler, J.A. Kelly, and T.D. Fox. 2009. Translocation and assembly of mitochondrially coded Saccharomyces cerevisiae cytochrome c oxidase subunit Cox2 by Oxa1 and Yme1, in the absence of Cox18. Genetics 182: 519-528.
- Xochitl Perez-Martinez, X., C.A. Butler, M. Shingu-Vazquez and T.D. Fox. 2009. Dual functions of Mss51 couple synthesis of Cox1 to assembly of cytochrome c oxidase in Saccharomyces cerevisiae mitochondria. Mol. Biol. Cell. 20: 4371-4380
|
Progress 10/01/07 to 09/30/08
Outputs
OUTPUTS: Conducted and analyzed experiments designed to further the aims of our research into the control of gene expression in mitochondria of yeast. These included genetic analysis to identify mutations affecting respiratory growth of yeast, and biochemical studies to examine the interaction of feedback regulatory proteins with the products of gene expression that they regulate. Delivered a research seminar at University of Wyoming, Laramie WY - October 2007 Invited speaker at 33rd FEBS Congress, Biochemistry of Cell Regulation - Athens, Greece June/July 2008. Invited speaker at Gordon Research Conference on Mitochondria and Chloroplasts, Biddeford, ME - August 2008. PARTICIPANTS: Dr. Thomas D. Fox, Principle investigator; Dr. Heather L. Fiumera, Postdoctoral research associate; Lindsay Burwell, Postdoctoral researcher; Christine A. Butler, Research support specialist; Zachary Via, graduate student; Leah Elliott, graduate student; Sue Lee, undergraduate researcher; Hannah Chen, undergraduate researcher. Collaborators: Dr. Xochitl Perez-Martinez, Assistant Professor UNAM, Mexico City; Dr. Nathalie Bonnefoy, Staff scientist, CNRS, Gif-sur-Yvette, France. Dr. Bernard Trumpower, Professor, Dartmouth College. Training: This project affords opportunities for undergraduate research training, Ph.D. graduate education, and postdoctoral training. TARGET AUDIENCES: The primary target audience for this research is the basic biomedical research community, focussing on geneticists, cell biologists, and microbiologists. The lab provides teaching opportunities that enrich the educations of Cornell undergraduate students. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Expression of a limited number of genes within the mitochondria of eukaryotic cells is vitally important for the assembly of the respiratory chain. The last enzyme in this pathway, cytochrome c oxidase, During the past year we have continued to study the mechanisms employed to control mitochondrial gene expression in the model organism yeast and to target mitochondrially coded proteins to their proper destination within the organelle. We have focussed on the mechanisms by which synthesis of the largest subunit (Cox1), encoded in mitochondrial DNA and made within the organelle, is regulated in response to the the ability of the cell to assemble new complexes. This assembly-feedback control is mediated through the protein coded by the nuclear gene MSS51. Mss51 is an unusual mitochondrial mRNA-specific translational activator that has targets of action mapping genetically in the COX1 coding sequence as well as in the 5 prime UTR of the COX1 mRNA. Furthermore, Mss51 co-immune precipitates with newly synthesized Cox1. Since Mss51 is present at near-rate-limiting levels in mitochondria, these dual functions of Mss51 would allow it to link COX1 mRNA translation and assembly of cytochrome oxidase. We are dissecting these Mss51 functions genetically. We have found that a substantial fraction of cellular Mss51 is indeed tied up in complexes with unassembled Cox1, and thus presumably unavailable to promote further synthesis of Cox1. We have continued to obtain more MSS51 mutations that allow activation of translation through the COX1 mRNA 5'UTR but not assembly of cytochrome oxidase. A second project involves the study of membrane translocases required to insert Cox2 into the mitochondrial inner membrane. Two related proteins Oxa1 and Cox18 are necessary to translocate the N-terminal and C-terminal intermembrane spaces domains, respectively, through the inner membrane. We have identified nuclear mutations that allow overproduced Oxa1 to compensate for the lack of Cox18. In collaboration with M. Dunham of University of Washington, we have mapped thesemutations to the genes MGR1 and MGR3, which encode subunits of the Yme1 membrane protease. Respiratory growth in these strains depends upon active Yme1, which we believe is operating as a chaperone under these conditions.
Publications
- Ding, M.G., C.A. Butler, S.A. Saracco, T.D. Fox, F. Godard, J.-P. diRago and B.L. Trumpower, 2008. Introduction of cytochrome b mutations in Saccharomyces cerevisiae by a method that allows selection for both functional and nonfunctional cytochrome b proteins. Biochim. Biophys. Acta 1777:1147-1156
|
Progress 10/01/06 to 09/30/07
Outputs
OUTPUTS: This project entailed conducting laboratory experiments on the mechanisms by which Saccharomyces cerevisiae (bakers yeast) cells assemble the mitochondrial respiratory chain/ oxidative phosphrylation apparatus. Yeast has proven to be an ideal simple organism for studying cellular processes that occur in all eucaryotic cells, including those of plants and humans. Differences in mitochondrial gene expression play a role in determining such traits as cytoplasmic male sterility and pathogen sensitivity in maize. Thus, a detailed understanding of mitochondrial regulatory mechanisms is important for agronomical research. Mitochondrial dysfunction in humans is associated with many disease states and with the aging process. Our development of tools in yeast with which to study nucleo-mitochondrial interactions demonstrates potential pathways to achieving a robust understanding of these processes in plants and animals, including humans. The project has resulted in publications for
(see below), and presentations to basic scientists. Verbal reports were given at invited seminars (University of Miami, April 2007; University of Wyoming, October 2007), and three interantional conferences (FEBS Advanced Lecture Series: Mitochondria in Life, Death, and Disease - Aussois, France April 2007; FASEB Summer Research Conference on Assembly of the Mitochondrial Respiratory Chain - Tucson, AZ August 2007; American Society for Cell Biology annual meeting - Washington DC, December 2007).
PARTICIPANTS: Individuals: Dr. Thomas D. Fox, Principle investigator; Dr. Heather L. Fiumera, Postdoctoral research associate; Christine A. Butler, Research support specialist; Zachary Via, graduate student; Jessica Kelly, Temporary research technician. Collaborators: Dr. Xochitl Perez-Martinez, Assitant Professor UNAM, Mexico City; Dr. Nathalie Bonnefoy, Staff scientist, CNRS, Gif-sur-Yvette, France. Training: This project affords opportunities for undergraduate research training, Ph.D. graduate education, and postdoctoral training.
TARGET AUDIENCES: The primary target audience for this research is the basic biomedical research community, focussing on geneticists, cell biologists, and microbiologists. The lab provides teaching opportunities that enrich the educations of Cornell undergraduate students.
Impacts
Expression of a limited number of genes within the mitochondria of eukaryotic cells is vitally important for the assembly of the respiratory chain. The last enzyme in this pathway, cytochrome c oxidase, During the past year we have continued to study the mechanisms employed to control mitochondrial gene expression in the model organism yeast and to target mitochondrially coded proteins to their proper destination within the organelle. We have focussed on the mechanisms by which synthesis of the largest subunit (Cox1), encoded in mitochondrial DNA and made within the organelle, is regulated in response to the the ability of the cell to assemble new complexes. This assembly-feedback control is mediated through the protein coded by the nuclear gene MSS51. Mss51 is an unusual mitochondrial mRNA-specific translational activator that has targets of action mapping genetically in the COX1 coding sequence as well as in the 5 prime UTR of the COX1 mRNA. Furthermore, Mss51
co-immune precipitates with newly synthesized Cox1. Since Mss51 is present at near-rate-limiting levels in mitochondria, these dual functions of Mss51 would allow it to link COX1 mRNA translation and assembly of cytochrome oxidase. We are dissecting these Mss51 functions genetically. An mss51 mutation that allows expression of a chimeric mRNA with the COX1 UTRs flanking the reporter ARG8m, but not a chimeric mRNA with the COX2 UTRs flanking the COX1 coding sequence, suggests that these functions may correlate with distinct Mss51 domains. Recessive nuclear cox14 mutations disrupt both cytochrome oxidase assembly and the assembly-feedback control of COX1 mRNA translation (Barrientos, et al. EMBO J. 23, 3472-3482, 2004). We have found that the interaction between Mss51 and newly synthesized Cox1 is dependent upon the presence of Cox14. This result suggests that Cox14 may act prior to, or together with, Mss51 in the pathway leading to assembly of Cox1 into cytochrome oxidase. The fact
that Cox14 is required for assembly-feedback control suggests the possibility that Cox14 could be a negative regulator of completed Cox1 translation that is antagonized by Mss51. The fact that Cox14 is required for assembly of cytochrome oxidase and the interaction between newly synthesized Cox1 and Mss51, suggests that the Cox1-Mss51 interaction may also be necessary for subsequent cytochrome oxidase assembly. A second project involves the study of membrane translocases required to insert Cox2 into the mitochondrial inner membrane. Two related proteins Oxa1 and Cox18 are necessary to translocate the N-terminal and C-terminal intermembrane spaces domains, respectively, through the inner membrane. We have found that overproduced Oxa1 will not compensate for the lack of Cox18 in normal strains. However, we have isolated recessive mutations in at least two genes that do allow overproduced Oxa1 to support respiratory growth of cox18 deficient mutants. In collaboration with M. Dunham of
Princeton University we have mapped one of these mutations to the gene FMP24, which encodes a mitochondrial protein of as yet unknown function.
Publications
- Zambrano, A., Fontanesi, F., Solans, A., de Oliveira, R.L., Fox, T.D. Tzagoloff, A., and Barrientos, A. 2007. Aberrant translation of cytochrome c oxidase subunit 1 mRNA species in the absence of Mss51p in the yeast Saccharomyces cerevisiae. Mol. Biol. Cell 18:523-535.
- Bonnefoy, N. and Fox, T.D. . 2007. Directed alteration of Saccharomyces cerevisiae mitochondrial DNA by biolistic transformation and homologous recombination. In: Methods in Molecular Biology, Mitochondria Practical Protocols. D. Leister and J.M. Herrmann, eds., (Humana Press Inc.), pp. 153-166.
- Bonnefoy, N, Remacle, C. and Fox, T. D. 2007. Genetic transformation of Saccharomyces cerevisiae and Chlamydomonas reinhardtii mitochondria. Meth. Cell Biol. 80: 525-548.
- Williams, E. H., Butler, C. A., Bonnefoy, N., and Fox, T. D. 2007 Translation initiation in Saccharomyces cerevisiae mitochondria: Functional interactions among mitochondrial ribosomal protein Rsm28p, initiation factor 2, methionyl-tRNA-formyltransferase, and novel protein Rmd9p. Genetics 175: 1117-1126.
- Fiumera, H. L., Broadley, S. A. and Fox, T. D. 2007 Translocation of mitochondrially synthesized Cox2p domains from the matrix to the intermembrane space. Mol. Cell. Biol. 27: 4664-4673.
|
Progress 01/01/06 to 12/31/06
Outputs
Expression of a limited number of genes within the mitochondria of eukaryotic cells is vitally important. During the past year we have continued to study the mechanisms employed to control mitochondrial gene expression in the model organism yeast and to target mitochondrially coded proteins to their proper destination within the organelle. We have focussed on the mechanisms by which the nuclear gene MSS51 participates in assembly-feedback-control of translation of the mitochondriall coded mRNA for cytochrome c oxidase subunit I (Cox1). We have found previously that the Mss51 protein has a target of action in the Cox1 mRNA 5'-leader through which it participates in translational activation. It also has a target within the Cox1 coding sequence itself. Furthermore, Mss51interacts with newly synthesized Cox1, and this interaction appears to be necessary for assembly of newly synthesized Cox1 in to the enzyme complex. We have now established that the interaction between
Mss51 and Cox1 depends upon the presence of the assembly factor Cox14, which appears to act upstream of Mss51 in the pathway. We are currently isolating mutations in MSS51 that selectively affect its ability to assemble Cox1 without destroying its translational activation function. A second project involves the study of membrane translocases required to insert Cox2 into the mitochondrial inner membrane. Two related proteins Oxa1 and Cox18 are necessary to translocate the N-terminal and C-terminal intermembrane spaces domains, respectively, through the inner membrane. We have found that overproduced Oxa1 will not compensate for the lack of Cox18 in normal strains. However, we have isolated recessive mutations in at least two genes that do allow overproduced Oxa1 to support respiratory growth of cox18 deficient mutants. These genes are very likely to be involved in the membrane insertion process and we are actively seeking their identity. Efforts to clone these genes directly have not
succeeded. However, we have collaborated with M. Dunham of Princeton University to use microarray assisted bulk segregation analysis to map one of these mutations to a limited region of chromosome XIII of yeast.
Impacts
This work represents a basic investigation into cellular processes using genetic analysis in the model organisms yeast. Yeast has proven to be an ideal simple organism for studying cellular processes that occur in all eucaryotic cells, including those of plants and humans. Differences in mitochondrial gene expression play a role in determining such traits as cytoplasmic male sterility and pathogen sensitivity in maize. Thus, a detailed understanding of mitochondrial regulatory mechanisms is important for agronomical research. Mitochondrial dysfunction in humans is associated with many disease states and with the aging process. Our development of tools in yeast with which to study nucleo-mitochondrial interactions demonstrates potential pathways to achieving a robust understanding of these processes in plants and animals, including humans.
Publications
- No publications reported this period
|
Progress 01/01/05 to 12/31/05
Outputs
Expression of a limited number of genes within the mitochondria of eukaryotic cells is vitally important. During the past year we have continued to study the mechanisms employed to control mitochondrial gene expression in the model organism yeast and to target mitochondrially coded proteins to their proper destination within the organelle, focusing on a dispensable small subunit ribosomal protein, Rsm28p, which was identified by mutations that suppressed a mitochondrially inherited defect in translation of the COX2 mRNA. To explore further the function of Rsm28, we looked for mutations in other genes that would cause a synthetic respiratory defective phenotype together with an rsm28 deletion. We identified mutations in two nuclear genes known to function in translation initiation: IFM1, which encodes mitochondrial translation initiation factor 2 (IF2), and FMT1, which encodes the methionyl-tRNA formyltransferase (generates fMet-tRNA). These results support the notion
that Rsm28 has a role in mitochondrial translation initiation. In addition, we isolated mutations in RMD9, a gene with no known function. Bacterial IF2 is a GTP/GDP-binding protein that interacts with initiator fMet-tRNA and positions it in the ribosomal P site. IF2 binds to the assembled ribosome at the interface between large and small subunits, and is thought to bind first to the small subunit prior to monosome assembly. While ifm1 deletion mutations fail to grow by respiration, they have residual protein synthesis and can maintain rho+ mtDNA. It appears that Ifm1p and Rsm28p may both promote initiator tRNA binding and/or ribosomal subunit joining. Surprisingly, formylation of initiator Met-tRNA is dispensable in yeast mitochondria. However, in the absence of Rsm28, fMet-tRNA becomes critical for translation efficiencies needed to support respiratory growth. Rmd9p is located in mitochondria, and roughly half is bound to membranes. We ran sucrose gradients of solubilized
mitochondria to ask whether Rmd9-TAP was bound to ribosomes. In standard high salt conditions most of the protein remained at the top of the gradient, while a small amount trailed in evenly as though it were falling off a larger structure. In lower salt concentration most of the protein was again at the top, but there was a distinct peak of Rmd9-TAP in the gradient at the position where both large subunit and small subunit control ribosomal proteins peaked together. Thus, Rmd9p is not a ribosomal protein per se, but rather a factor associated most tightly with assembled monosomes. rmd9 deletion mutants are rho-, indicating that it is essential for mitochondrial protein synthesis. Although RMD9 is essential for respiratory growth, genes encoding homologous proteins have been found only in the genomes of other budding yeasts, including Candida albicans. This contrasts with the genes IFM1 and FMT1 whose widely conserved functions are apparently less critical for mitochondrial translation
in S. cerevisiae.
Impacts
This work represents a basic investigation into cellular processes using genetic analysis in the model organisms yeast. Yeast has proven to be an ideal simple organism for studying cellular processes that occur in all eucaryotic cells, including those of plants and humans. Differences in mitochondrial gene expression play a role in determining such traits as cytoplasmic male sterility and pathogen sensitivity in maize. Thus, a detailed understanding of mitochondrial regulatory mechanisms is important for agronomical research. Mitochondrial dysfunction in humans is associated with many disease states and with the aging process. Our development of tools in yeast with which to study nucleo-mitochondrial interactions demonstrates potential pathways to achieving a robust understanding of these processes in plants and animals, including humans.
Publications
- Williams, E.H., Bsat, N., Bonnefoy, N., Butler C.A. and Fox, T.D. 2005. Alteration of a novel dispensable mitochondrial ribosomal small subunit protein, Rsm28p, allows translation of defective COX2 mRNAs. Eukaryot. Cell 4: 337-345.
- Fiori, A., Perez-Martinez, X. and Fox, T.D.. 2005. Overexpression of the COX2 translational activator, Pet111p, prevents translation of COX1 mRNA and cytochrome c oxidase assembly in mitochondria of Saccharomyces cerevisiae. Mol. Microbiol. 56: 1689-1704.
|
Progress 01/01/04 to 12/31/04
Outputs
Expression of a limited number of genes within the mitochondria of eukaryotic cells is vitally important. During the past year we have continued to study the mechanisms employed to control mitochondrial gene expression in the model organism yeast and to target mitochondrially coded proteins to their proper destination within the organelle. and to regulate their synthesis. 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. Interference with Cox1p synthesis depends to a limited extent upon increased translation of the COX2 mRNA, but is largely independent of it. 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 severe imbalance in translational activator levels appears to cause 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.
Impacts
This work represents a basic investigation into cellular processes using genetic analysis in the model organisms yeast. Yeast has proven to be an ideal simple organism for studying cellular processes that occur in all eucaryotic cells, including those of plants and humans. Differences in mitochondrial gene expression play a role in determing such traits as cytoplasmic male sterility and pathogen sensitivity in maize. Thus, a detailed understanding of mitochondrial regulatory mechanisms is important for agronimical research. Mitochondrial dysfunction in humans is associated with many disease states and with the aging process. Our development of tools in yeast with which to study nucleo-mitochondrial interactions demonstrates potential pathways to achieving a robust understanding of these processes in plants and animals, including humans.
Publications
- Williams, E.H., Perez-Martinez, X. and Fox, T.D.. 2004. MrpL36p, a highly diverged L31 ribosomal protein homolog with additional functional domains in Saccharomyces cerevisiae mitochondria. Genetics 167: 65-75.
|
Progress 01/01/03 to 12/31/03
Outputs
Expression of a limited number of genes within the mitochondria of eukaryotic cells is vitally important. During the past year we have continued to study the mechanisms employed to control mitochondrial gene expression in the model organism yeast and to target mitochondrially coded proteins to their proper destination within the organelle. and to regulate their synthesis. The activator proteins specific for cytochrome oxidase subunit mRNAs interact with each other, as detected by two-hybrid analysis and by co-immune precipitation. Thus it appears that interacting translational activators may co-localize translation of the three largest cytochrome oxidase subunits, facilitating their assembly into active enzyme complexes. Furthermore, one of these, Mss51p, also interacts with newly synthesized cytochrome oxidase subunit I, and thus may play a role in assembly feedback control of Cox1p synthesis.
Impacts
This work represents a basic investigation into cellular processes using genetic analysis in the model organisms yeast. Yeast has proven to be an ideal simple organism for studying cellular processes that occur in all eucaryotic cells, including those of humans. Mitochondrial dysfunction in humans is associated with many disease state and possibly aging. Our development of tools in yeast with which to study nucleo-mitochondrial interactions demonstrates potential pathways to achieving the same level of understanding of these processes in humans.
Publications
- Mireau, H., N. Arnal and T.D. Fox. 2003. Expression of Barstar as a selectable marker within yeast mitochondria. Mol. Gen. Genet. 270:1-8
- Naithani, S., Saracco, S.A., Butler, C.A. and T.D. Fox. 2003. Interactions among COX1, COX2 and COX3 mRNA-specific translational activator proteins on the inner surface of the mitochondrial inner membrane of Saccharomyces cerevisiae. Mol. Biol. Cell 14: 324-333.
- Williams, E.H. and T.D. Fox. 2003. Antagonistic Signals within the COX2 mRNA Coding Sequence Control Its Translation in Saccharomyces cerevisiae Mitochondria. RNA 9: 419-431. Fiori, A., T.L. Mason and T.D. Fox. 2003. Evidence that synthesis of the Saccharomyces cerevisiae mitochondrially-encoded ribosomal protein Var1p may be membrane localized. Eukaryot. Cell 2: 651-653.
- Demlow, C.M. and T.D. Fox. 2003. Activity of mitochondrially synthesized reporter proteins is lower than imported proteins, and is increased by lowering cAMP in glucose-grown Saccharomyces cerevisiae cells. Genetics 165: 961-974.
- Perez-Martinez, X., S.A. Broadley and T.D. Fox. 2003. Mss51p promotes mitochondrial Cox1p synthesis and interacts with newly synthesized Cox1p. EMBO J. 22: 5951-5961.
|
Progress 01/01/02 to 12/31/02
Outputs
Expression of a limited number of genes within the mitochondria of eukaryotic cells is vitally important. During the past year we have continued to study the mechanisms employed to control mitochondrial gene expression in the model organism yeast and to target mitochondrially coded proteins to their proper destination within the organelle. We have found that mRNA specific translational activator proteins, encoded by nuclear genes, are present on the inner surface of the mitochondrial inner membrane. Their interactions with the 5'-untranslated leaders of mitochondrially coded mRNAs play a role in targeting the products of mitochondrial protein synthesis to sites where cytochrome oxidase is assembled. The activator proteins specific for cytochrome oxidase subunit mRNAs interact with each other, as detected by two-hybrid analysis and by co-immune precipitation. Thus it appears that interacting translational activators may co-localize translation of the three largest
cytochrome oxidase subunits, facilitating their assembly into active enzyme complexes.
Impacts
This work represents a basic investigation into cellular processes using genetic analysis in the model organisms yeast. Yeast has proven to be an ideal simple organism for studying cellular processes that occur in all eucaryotic cells, including those of humans. Mitochondrial dysfunction in humans is associated with many disease state and possibly aging. Our development of tools in yeast with which to study nucleo-mitochondrial interactions demonstrates potential pathways to achieving the same level of understanding of these processes in humans.
Publications
- Saracco, S. A. and Fox T. D. 2002. Cox18p is required for export of the mitochondrially encoded Saccharomyces cerevisiae Cox2p C-tail, and interacts with Pnt1p and Mss2p in the inner membrane. Mol. Biol. Cell 13: 1122-1131.
|
Progress 01/01/01 to 12/31/01
Outputs
Expression of a limited number of genes within the mitochondria of eukaryotic cells is vitally important. During the past year we have continued to study the mechanisms employed to control mitochondrial gene expression in the model organism yeast and to target mitochondrially coded proteins to their proper destination within the organelle. Using genetic analysis we have begun to elucidate the mechanisms by which protein domains encoded in mitochondrial DNA are translocated across the inner membrane, focussing on cytochrome oxidase subunit II (Cox2p). This past year we have demonstrated that three membrane proteins encoded in the nucleus, Mss2p, Cox18p and Pnt1p interact to carry out the translocation of the carboxyl-terminal tail of Cox2p. Surprisingly however, they have no apparent role in the export of the amino terminal tail. Analysis of mutations affecting Cox2p itself has revealed that export of the C-tail depends upon cleavage of the leader peptide from the
exported N-tail, suggesting some kind of trans-membrane signalling. This work has also produced tantalizing hints that the C-terminal domain must be at least partially folded on the matrix side of the membrane before export.
Impacts
This work represents a basic investigation into cellular processes using genetic analysis in the model organisms yeast. Yeast has proven to be an ideal simple organism for studying cellular processes that occur in all eucaryotic cells, including those of humans. Mitochondrial dysfunction in humans is associated with many disease state and possibly aging. Our development of tools in yeast with which to study nucleo-mitochondrial interactions demonstrates potential pathways to achieving the same level of understanding of these processes in humans.
Publications
- Bonnefoy, N. and T.D. Fox. 2001. Genetic transformation of Saccharomyces cerevisiae mitochondria. Meth. Cell Biol. 65: 381-396.
- Bonnefoy, N., N. Bsat, and T. D. Fox. 2001. Mitochondrial translation of Saccharomyces cerevisiae COX2 mRNA is controlled by the nucleotide sequence specifying the pre-Cox2p leader peptide. Mol. Cell. Biol. 21: 2359-2372.
- Green-Willms, N.S., C. A. Butler, H. M. Dunstan and T. D. Fox. 2001. Pet111p, an inner membrane-bound translational activator that limits expression of the Saccharomyces cerevisiae mitochondrial gene COX2. J. Biol. Chem. 276: 6392-6397.
- Cohen, J.S., and T.D. Fox. 2001. Expression of Green Fluorescent Protein from a recoded gene inserted into Saccharomyces cerevisiae mitochondrial DNA. Mitochondrion 1: 181-189.
- Broadley, S.A., C.M. Demlow and T.D. Fox. 2001. A peripheral mitochondrial inner membrane protein, Mss2p, required for export of the mitochondrially coded Cox2p C-tail in Saccharomyces cerevisiae. Mol. Cell. Biol. 21: 7663-7672.
|
Progress 01/01/00 to 12/31/00
Outputs
Expression of a limited number of genes within the mitochondria of eukaryotic cells is vitally important. During the past year we have continued to study the mechanisms employed to control mitochondrial gene expression in the model organism yeast and to target mitochondrially coded proteins to their proper destination within the organelle. Using genetic analysis we have found that the level of mitochondrial gene expression is responsive to levels of the important signaling molecule cAMP and are continuing to try to elucidate the mechanism by which this occurs. We have also identified a novel mechanism of translational control affecting the cytochrome oxidase subunit Cox2p, that is mediated by the nucleotide sequence of the protein coding portion of the mRNA. Finally we have completed several important collaborative projects with other laboratories. On of these was to develop a system that could be used to monitor the genetic stability of mtDNA in vivo, and to study
environmental and genetic factors that affect it. In the other, we designed a system that could detect in vivo function of cytoplasmic tRNAs that were imported into yeast mitochondria.
Impacts
This work represents a basic investigation into cellular processes using genetic analysis in the model organisms yeast. Yeast has proven to be an ideal simple organism for studying cellular processes that occur in all eucaryotic cells, including those of humans. Mitochondrial dysfunction in humans is associated with many disease state and possibly aging. Our development of tools in yeast with which to study nucleo-mitochondrial interactions and genetic instability in mitochondria demonstrates potential pathways to achieving the same level of understanding of these processes in humans.
Publications
- Bonnefoy, N. and T.D. Fox. 2000. In vivo analysis of mutated initiation codons in the mitochondrial COX2 gene of Saccharomyces cerevisiae fused to the reporter gene ARG8m reveals lack of downstream reinitiation. Mol. Gen. Genet. 262:1036-1046.
- Sia, E.A., C.A. Butler, M. Dominska, P. Greenwell, T.D. Fox and T.D. Petes. 2000. Analysis of microsatellite mutations in the mitochondrial DNA of Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. USA 97: 250-255.
- Kolesnikova, O. A., N. S. Entelis, H. Mireau, T. D. Fox, R. P. Martin, and I. A. Tarassov. 2000. Suppression of mutations in mitochondrial DNA by tRNAs imported from the cytoplasm. Science 289:1931-1933.
- Costanzo, M.C, N. Bonnefoy, E. H. Williams, G. D. Clark-Walker and T. D. Fox. 2000. Highly diverged homologs of Saccharomyces cerevisiae mitochondrial mRNA-specific translational activators have orthologous functions in other budding yeasts. Genetics 154: 999-1012.
- Souza, R.L., N.S. Green-Willms, T.D. Fox, A. Tzagoloff, and F.G. Nobrega. 2000. Cloning and characterization of COX18, a Saccharomyces cerevisiae pet gene required for the assembly of cytochrome oxidase. J. Biol. Chem. 275: 14898-14902.
- Mireau, H., A. Cosset, L. Marechal-Drouard, T. D. Fox, I. D. Small and A. Dietrich. 2000. Expression of Arabidopsis thaliana mitochondrial alanyl-tRNA synthetase is not sufficient to trigger mitochondrial import of tRNAAla in yeast. J. Biol. Chem. 275: 13291-13296.
|
Progress 01/01/99 to 12/31/99
Outputs
During the past year we have continued to identify nuclear genes encoding mitochondrial membrane proteins that participate in the export of mitochondrially coded protein domains through the inner membrane. We have also identified by mutation amino acids in mitochondrially coded Cox2p that are required for this process. These mutants were identified in a screen developed during the previous reporting period.
Impacts
This work represents a basic investigation into cellular processes using genetic analysis in the model organisms yeast. Yeast has proven to be an ideal simple organism for studying cellular processes that occur in all eucaryotic cells, including those of humans.
Publications
- He, S. and T.D. Fox. 1999. Mutations affecting a yeast mitochondrial inner membrane protein, Pnt1p, block export of a mitochondrially synthesized fusion protein from the matrix. Mol. Cell. Biol. 19:6598-6607.
|
Progress 01/01/98 to 12/31/98
Outputs
During the past year we have established that information necessary for proper targeting of mitochondrially synthesized proteins in yeast, depends on signals in the untranslated portions of their mRNAs. This information is recognized by membrane bound mRNA-specific translational activators that mediate mRNA-ribosome interactions at the surface of the inner membrane. We have also identified several nuclear genes encoding mitochondrial membrane proteins that participate in the export of mitochondrially coded protein domains through the inner membrane. These genes were identified in a screen developed during the previous reporting period. One of these genes, PNT1, encodes a protein known to be involved in the sensitivity to pentamidine, and important antifungal agent used against Pneumocystis carinii, and opportunistic pathogen.
Impacts
(N/A)
Publications
- Green-Willms, N.S., Fox, T.D. and Costanzo, M.C. 1998. Functional interactions between yeast mitochondrial ribosomes and mRNA 5'-untranslated leaders. Mol. Cell. Biol. 18:1826-1834.
- Sanchirico, M.E., Fox, T.D. and Mason, T.L. 1998. Accumulation of mitochondrially synthesized Saccharomyces cerevisiae Cox2p and Cox3p depends on targeting information in untranslated portions of their mRNAs. EMBO J. 17: 5796-5804.
|
|