Winter 2014 Biomedical Seminar Series
January 24, 2014
High Copy Suppressor Screen of Yeast Mating Defects
Fernando Vargas, MARC U*STAR Scholar
Summer Research Site: Department of Molecular and Quantitative & Computational Biology at Princeton University Author:
Fus2p is a pheromone-induced protein that is required for cell fusion during mating in S. cerevisiae cells. Evidence from several mutants defective for cell fusion suggests that Fus2p regulates the fusion of vesicles that localize to the zone of cell fusion and release hydrolases that break down the cell wall between mating cells. A high copy suppressor screen previously performed in the lab found that overexpression of Kel1p, a Kelch Domain-containing protein involved in cell morphogenesis and fusion, suppressed cell fusion defects in fus2 mutants to ~20% of wild-type mating efficiency. We aim to find additional suppressors of the fus2 mutant phenotype using a high copy suppressor screen, to gain insight into mechanisms of cell fusion. Wild-type FUS2, as well as two FUS2 mutants were first integrated into the genome at the FUS2 locus of a MATa strain and were then transformed with a KEL1 overexpression plasmid. A second plasmid from the suppressor screen library was then introduced into the cell. To assay for cell fusion, these strains were mated against a MAT? strain and the number of diploids were observed. During my rotation, we tested three suppressor candidates, but future experiments will include a screen of all plasmids found in the yEP24 library. The suppressors discovered will shed light on the mechanisms of cell wall removal during mating. Many of the proteins identified from the study of yeast cell fusion have homologs that are involved in higher eukaryotic cell fusion events; therefore, our research may help understand the general processes of eukaryotic cell fusion.
Characterization of the AT-Hook & ABD2 Domains on the MS1 Protein
Oscar Diaz, RISE MS to Ph.D. Scholar
Summer Research Site: MHIRT at the King’s College London
Cardiac hypertrophy, an adaptation of the heart to chronic pressure overload, is associated with the Myocyte stress-1 (MS1) protein. Operating as a mechanical stress factor, the MS1 protein utilizes F-actin binding properties to detect and activate gene regulatory factors within the nucleus. Previous studies of the murine aortic banding model, a model for left ventricular hypertrophy (LVH), has shown these transcriptional signaling pathways leading to the development of heart failure and even death. We believe that the highly conserved actin binding domain-2 (ABD2) and the AT-hook, located on the C-terminus of MS1, retain DNA binding activity of the MS1 protein. Using protein purification and systematic evolution of ligands by exponential enrichment (SELEX), our study would uncover a specific DNA sequence that binds to the MS1 construct containing only the AT-hook and ABD2 domains. Therefore, further investigation may indicate direct gene regulation by MS1 via AT-hook and ABD2 synergistic DNA binding. 1. Aravind L, Landsman, D. 1998. AT-hook motifs identified in a wide variety of DNA-binding proteins. Nucleic Acids Res. 26:4413-4421. 2. Fogl C, Puckey L, Hinssen U, Zaleska M, El-Mezgueldi M, Croasdale R, Bowman A, Matsukawa A, Samana NJ, Savva R, Pfuhl M. 2011. A structural and functional dissection of the cardiac stress response factor MS1. Proteins. 3. Mahadeva H, Brooks G, Lodwick D, Chong NW, Samani NJ. 2002. ms1, a novel stress-responsive, muscle specific gene that is up-regulated in the early stage of pressure overload-induced left ventricular hypertrophy. FEBS Lett. 521:100-104 4. Wallace MA, Lamon S, Russell AP. 2012. The regulation and function of the striated muscle activator of rho signaling (STARS) protein. Frontiers in Physiology. Vol. 3 Art. 469:1-5 5. Zaleska, M., et al. 2013. Structural and Functional Characterization of MS1. EBSA Poster Presentation.
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