SGER: Use of the Tetracysteine Tag to Study Localization Dynamics of Hydroxyproline-Rich Glycoproteins (HRGP)

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A grant has been awarded to Dr. Maura Cannon of the University of Massachusetts to test the application of the recently developed 'tet-tag' technology for use in plant cells. This technology will be tested with a HRGP (hydroxyproline-rich glycoprotein) in the model plant Arabidopsis. HRGPs are a large family of cell wall proteins that occur in all known plants, and are involved in cell wall structure, cell shape, cell division, and plant growth and development. Although vitally important to plants, very little is known about HRGPs. To identify their localization dynamics at the cellular level, would be a valuable contribution to understanding their functions. In animal cells it has been shown that the biarsenical fluorophores, FLAsH or ReAsH, bind to recombinant proteins containing a tetracysteine sequence, CCXXCC, where X is a non-cysteine amino acid. When bound to the tet-tag, FLAsh and ReAsH fluoresce at different wavelengths to each other, therefore, a tet-tagged protein can be visualized in space and time, and older proteins can be distinguished from younger proteins. Antibodies are not required with these probes, thus making them very useful for localizing products of large gene families, where a specific antibody to an individual gene family member, can be impossible to obtain. Also, due to its small size, the tet-tag is less likely to hinder protein function, which is important in the case of structural proteins such as HRGPs. Results from this project will not only provide data concerning the HRGP in question, but it will enable other cell wall proteins to be probed by the methods developed. This is important because plant cell walls are a major component of food and animal feed, and are the starting material for most fuels. Data on the dynamics of essential components of cell walls will facilitate the improved production of plant biomass. Furthermore, the research in this proposal will provide training for students at the interface of molecular, cellular and developmental biology.