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Characterization of the Essential Gene

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During the summer after herfreshman year, Reshmi discov-ered the opportunity forresearch that led her to work atUCI’s Cumsky Laboratory.According to Reshmi, partici-patinginresearchhas enhancedher education in more than afew ways, and has given her afoundation for pursuing hergoal of attending medicalschool. “I have been madeaware of the limitless scope ofresearch and the potentialimpact it can have on everydaylife,” she says. She is an activemember of UCI’s FlyingSamaritans, enjoys playing thepiano and collecting first edi-tion books.When Reshmi began work on the MIA1 gene, our lab group knewlittle about it. Her work, and that of her primary collaborator Dr.Virginia Bilanchone, established that MIA1 encodes a protein criti-cal for mitochondrial biogenesis. Furthermore, this work deter-mined the scope and direction of subsequent studies that have con-firmed Reshmi’s findings. These studies have shown that the MIA1protein interacts with proteins of the mitochondrial import motor,and therefore may be a component of this molecular machine. In addition, it wasfound that a Drosophila homolog of MIA1 causes early embryogenic death in fruitflies when defective. Reshmi’s project is a wonderful example of excellence in under-graduate research. It also underscores the value of this experience for both studentand laboratory. Reshmi’s work is a significant contribution to both our laboratory’sresearch and mitochondrial research in general. Key TermswEssential GenewMIA1wMitochondriawRespirationwSaccharomyces cerevisiaeCharacterization of theEssential Gene MIA1inSaccharomyces cerevisiaeReshmi SinhaAnthropology and Biological Sciences Michael CumskySchool of Biological SciencesThe biogenesis of functional mitochondria is essential to the survival of alleukaryotic cells. Mitochondria are organelles whose chief function is to supplya cell with energy in the form of adenosine triphosphate (ATP). The final step inATP production requires cytochrome c oxidase, a multi-subunit enzyme complex.One of the essential subunits of cytochrome c oxidase is Va. Ongoing studies seekto identify the mitochondrial proteins responsible for the import and sorting of sub-unit Va to the mitochondrial inner membrane. Past research in the Cumsky labora-tory has identified a previously uncharacterized gene named MIA1. This gene isencoded by nuclear DNA, encodes a 16.2 kDa gene product localized in the mito-chondrial inner membrane, and is essential for the viability of yeast cells. The goalof this project was to investigate the role of the Mia1 protein (Mia1p) in mitochon-drial biogenesis. It was found that cells depleted of Mia1p show loss of respiratorycomplexes, lose mitochondrial DNA, and display altered mitochondrial morpholo-gies. MIA1 has a human homolog whose product, CG1-136 protein, has 65% simi-larity in amino acid sequence. Therefore, it is probable that defects in the MIA1homolog in humans will result in severe disease. Further understanding of proteinimport and localization in mitochondria may help with the development of therapiesthat will address such problems.55The UCI Undergraduate Research JournalAuthorAbstractFaculty MentorIntroductionAll eukaryotic cells require the biogenesis of functionalmitochondria for viability. Mitochondria are organelles thatoperate in the metabolism of heme proteins and iron(Beinert et al., 1997) and provide much of the biochemicalenergy required to sustain normal biological functions.Adenosine triphosphate (ATP) stores this energy and is pro-duced by the interaction of different proteins within themitochondria. The final step in ATP production requirescytochrome c oxidase (CCO), a multi-subunit complex. Ineukaryotes, CCO is a heteroligomer containing up to 13 dif-ferent polypeptide subunits that are products of both thenuclear and mitochondrial genomes. The three largest sub-units (I, II and III) are the products of mitochondrial DNAand are transcribed and translated within the organelle. Theremaining subunits are nuclear gene products and areimported into the mitochondria after translation in the cyto-plasm. One of the essential subunits of CCO, Va, containsthe protein COX5a that is encoded by nuclear DNA andsynthesized outside the mitochondria in the cytoplasm.Mitochondrial gene products of nuclear origin, such asCOX5a, must be targeted to the mitochondria and thensorted to one of the four intra-mitochondrial compart-ments. These mitochondrial proteins must not only beimported from the cytoplasm, but they must also be prop-erly localized within the organelle (Lill et al., 1996).Therefore, many genes involved in mitochondrial importand sorting have proven to be essential for the survival ofeukaryotic cells (Voos et al., 1999).Previous investigations in the Cumsky laboratory have uti-lized a high-copy DNA library to identify the componentsinside yeast mitochondria that are responsible for importand sorting of COX5a into the mitochondrial inner mem-brane. The approach is based on the phenotype of cellscontaining the mutant protein COX5a109R. The incorrectsorting or localization of this mutant protein results in adefect in cytochrome c oxidase assembly and loss of CCOactivity (manuscript in preparation). The genetic screenselects for wild-type genes, which, when present in highcopy, suppress the growth defect of cells containing themutant protein. Through this screen, the Cumsky laborato-ry has thus far identified several genes involved in theimport and/or assembly of CCO. One of these genes,MIA1, represents an open reading frame that has not previ-ously been characterized. Through previous experiments ithas been found that MIA1 is encoded by nuclear DNA,localized in the mitochondrial inner membrane, and speci-fies a 16.2 kDa gene product that is essential for the survivalof yeast cells (unpublished). The current study sought to investigate the role of the pro-tein encoded by MIA1 in the cell. Because MIA1 is essen-tial, yeast cells will die without the gene product and so asystem of conditional expression needed to be established.This was accomplished by constructing a plasmid,pgalMIA1, in which the native promoter of MIA1 wasreplaced with the GAL1 promoter. Expression of genespositioned adjacent to this promoter can be controlled atthe level of transcription and induced or repressed by grow-ing cells in media containing galactose and glucose respec-tively.This system of conditional expression was used to examinethe effects


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