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IDENTIFICATION OF CELL LOCALIZATION OF ADE13 IN SACCHAROMYCES CEREVISIAE VI GREEN FLUORESCENT PROTEIN TAGGING

Abstract

Saccharomyces cerevisiae has been an instrumental eukaryotic model system for a diverse array of research. The genome of 12,068 kilobases were sequenced 27 years ago, making S. cerevisiae the inaugural organism to undergo full genomic sequencing. Since this discovery, vast effort has been put forth to describe the function of each open reading frame (ORF). The genome sequencing identified 6,753 potential ORFs, however a significant portion of these ORF’s function and localization remain unknown. Interestingly, 23% of the S. serevisiae genome encompasses human orthologs, which are associated with various diseases. Therefore, there is value in exploring unknown cellular localization in less well characterized ORFs. We explored the localization of 3 such proteins, Gid10, Fsh3, and Ade13. To determine the cellular localization of these protiens, we used a C-terminal GFP tagging strategy and fluorescence microscopy. Here, we are highlighting the localization studies of Ade13, and adenyl succinate lyase, which plays a vital role in purine nucleotide synthesis. The malfunction of human orthologue of Ade13 leads to disease in humans, known as adenylosuccinate deficiency. Based on comparative analysis of Ade13 protein in several model organisms, we predicted the localization of Ade13 to be potentially mitochondrial and confirmed the mitochondrial localization with Mito tracker fluorescent stain. Visualization of GFP tagged proteins in a single cell organism such as S. cerevisiae serves as a valuable strategy to gain insight into unknown protein localization and functions that may be conserved in higher eukaryotic cells.

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