The intracellular localization of recombinant GFP-histatin 3 variants in saccharomyces cerevisiae
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Salivary histatins exhibit potent killing activities through inhibition of mitochondrial function in Candida ablicans. However, the detailed molecular mechanism still remains unclear. In this study, we employed Saccharomyces cerevisiae (a pathogenic fungus which shares a similar genetic background with C. albicans) as a model system to study the intracellular localization of histatins. Histatin 3, a 32-amino acid antifungal protein, and numerous length variants of this protein were studied. A MET3 promotor was adapted to control the expression level of green fluorescent protein (GFP) and histatin fusion proteins through different concentrations of methionine. Fluorescence generated by GFP allowed real-time monitoring of histatin 3 expression through confocal microscopy. Our kinetic study showed that intracellularly expressed histatin 3 exclusively exists in the cytoplasm with no preference to any organelle including the mitochondria. The larger histatin variants appeared to aggregate in the cytoplasm. Our results demonstrate that histatin 3 does not directly target the mitochondria after expression, suggesting that the killing mechanism via the mitochondrial pathway may not occur in S. cerevisiae. Recent developments of a C. albicans specific vector for the expression of GFP fusion proteins will further advance this line of research involving histatin localization in C. albicans.
PLEASE NOTE: This work is protected by copyright. Downloading is restricted to the BU community: please click Download and log in with a valid BU account to access. If you are the author of this work and would like to make it publicly available, please contact firstname.lastname@example.org.Thesis (MSD)--Boston University, Goldman School of Dental Medicine, 2005.Includes bibliographical references: leaves 57-61.
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