------------------------------------------------------------------------ Item No. 1 of 1 ACCESSION NO: 0184592 SUBFILE: CRIS PROJ NO: TEXR-1999-01796 AGENCY: CSREES TEXR PROJ TYPE: NRI COMPETITIVE GRANT PROJ STATUS: TERMINATED CONTRACT/GRANT/AGREEMENT NO: 99-35304-8003 PROPOSAL NO: 1999-01796 START: 01 NOV 1999 TERM: 31 OCT 2001 FY: 2000 GRANT YR: 1999 INVESTIGATOR: Thompson, G. A. PERFORMING INSTITUTION: BOTANY UNIV OF TEXAS AUSTIN, TEXAS 78712 THE BIOCHEMISTRY OF GLYCOSYLPHOSPHATIDYLINOSITOL-ANCHORED PROTEINS IN PLANTS OBJECTIVES: Characterize glycosylphosphatidylinositol (GPI)-anchored proteins that we and others have found to occur in plants. Determine the ways by which GPI anchorage of purple acid phosphatase and other proteins serves a useful function for plants. Study the metabolism of the GPI anchor under different growth conditions. APPROACH: Use proteolytic enzymes, high performance liquid chromatography, and mass spectrometry to identify the components of the partial GPI anchor on certain proteins we have isolated from Spirodela and Arabidopsis. Scale up isolation techniques to obtain complete GPI anchor before its partial degradation by endogenous enzymes. Utilize radioactive precursors to estimate the production and turnover of the GPI-anchored purple acid phosphatase (PAP) of Spirodela and Arabidopsis under differing conditions of phosphate deficiency. Overexpress PAP in Arabidopsis arabinogalactan proteins. NON-TECHNICAL SUMMARY: (see attached page) PROGRESS: 1999/11 TO 2001/10 During the past decade glycosylphosphatidylinositol(GPI)-anchored proteins have been implicated in many important metabolic processes in animals. However, until a very few years ago the presence of GPI-anchored proteins in plants was merely a subject of speculation. This research project originally had as its principal goal the determination of whether GPI-anchored proteins exist and function in plants. Our initial findings in 1996 and our follow-up studies identified for the first time a GPI-anchored phosphatase in the duckweed Spirodela oligorrhiza. During the present renewal our primary aim was to gather more information about the nature and metabolism of GPI-anchored proteins in plants. Because of technical difficulties stemming from the low abundance of the Spirodela phosphatases we have continued our studies of the metabolism and properties of plant GPI-anchored proteins with cultured cells of Arabidopsis thaliana. GPI-anchored arabinogalactan proteins are secreted in abundance by that widely used organism. Using HPLC we were able to resolve the Arabidopsis AGPs into 15 or more individual protein species of increasing molecular weight. Pulse labeling the AGPs with radioactive galactose or acetate led to incorporation of the tracer predominantly into the higher molecular mass AGPs over a 1-8 h period, with the smaller AGPs more gradually becoming radioactive. We conclude that AGPs are made as very large macromolecules and are subsequently processed into smaller homologs. Physiological stress triggered a massive AGP release having a different distributional pattern, but with higher molecular mass AGPs still being most highly radiolabeled. Arabidopsis cells appear capable of releasing higher mass AGP species apparently stored in cell wall sites along with a unique mixture of freshly synthesized AGPs in combinations potentially active in signaling. While the rates of AGP release by cultured cells may be much higher than those found in intact plant tissues, the types of AGP seem to be the same. It is clear that secretion of the AGPs in both cultured plant cells and intact plants involves cleavage of the lipid moiety of the GPI anchor. It is likely that the specialized secretory pathway involved in transporting GPI-anchored proteins to the cell surface is under regulatory control distinct from that exerted on other secretory pathways. Studying rates and patterns of AGP secretion may well shed light on plant cell-cell communication and defense responses. IMPACT: 1999/11 TO 2001/10 At present the functions of AGPs are very poorly understood, although much indirect evidence indicates a role in cell-cell communication. Finding a system in which the quantitative release of AGPs can be measured means that the regulation of the process can be studied. The regulation may well involve some element of the GPI-anchored protein secretion pathway. Thus a greater understanding of plant developmental biology can be attained. PUBLICATIONS: 1999/11 TO 2001/10 1. Thompson, G. A., Jr., and Okuyama, H. 2000. Lipid-linked Proteins of Plants. Progress in Lipid Research, 39:19-39. 2. Darjania,, L., Ichise, N., Ichikawa, S., Okamoto, T., Okuyama, H., and Thompson, G. A., Jr. 2000. Metabolism of Glycosylphosphatidylinositol-anchored Proteins in Arabidopsis. Biochemical Society Transactions, 28: 725-727. 3. Darjania, L., Ichise, N., Ichikawa, S., Okamoto, T., Okuyama, H., and Thompson, G. A., Jr. 2001. Dynamic Turnover of Arabinogalactan Proteins in Cultured Arabidopsis Cells. Plant Physiology and Biochemistry. in press. PROJ CONTACT: Name: THOMPSON, G. A. Phone: 512-471-5036 Fax: 512-471-3878 Email: guythom@utxvms.cc.utexas.edu ------------------------------------------------------------------------