Plant Protein Farnesyltransferase: Molecular and FunctionalAnalyses

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Protein farnesyltransferase (FTase) is a heterodimeric enzyme that post-translationally modifies proteins by attaching an isoprenyl moiety to a C-terminal cysteine. This modification, identified in yeast and animals, is required for membrane association and in vivo activities of many regulatory proteins involved in cell cycle control and differentiation. The major focus of the proposed project is to elucidate the role(s) of FTase and protein prenylation in mediating plant developmental processes. In recent experiments, a cDNA clone encoding a pea FTase beta subunit has been cloned and characterized, protein prenylation has been demonstrated in plant cells, and FTase transcript levels have been analyzed in apical buds of pea seedlings and suspension-cultured tomato cells. In both experimental systems, a rapid, transient increase in FTase mRNA was detected preceding the onset of active cell division. These results are consistent with the hypothesis that FTase mediates the activity of critical components of the signal transduction pathways controlling cell division in plants, analogous to its key role in cell cycle control in fungal and mammalian systems. To test this hypothesis, a cDNA for the FTase alpha subunit will be cloned from pea and biochemical and physiological properties of pea FTase will be delineated using both in vitro assays and in vivo plant cells transiently expressing protein prenylation substrates. Detailed analyses of the temporal and spatial patterns of pea Ftase gene expression will be performed to provide clues as to its functional significance in plant development. Plant cells and stably transformed plants, inhibited in Ftase expression by antisense RNAs or competitive peptide inhibitor, will be monitored for phenotypic, cellular, and molecular changes to directly assess the role of FTase in regulating plant cell division and differentiation. %%% It has recently been discovered that certain proteins are modified, under some physiological conditions, by covalent attachment of isoprenyl groups to a specific C-terminal amino acid. Isoprenyl groups are a type of organic molecule that tends to be more soluble in fats or oils than in water. This modification, then, has the effect of allowing proteins that would otherwise remain dissolved in the watery cytoplasm of a cell to attach to cellular membranes. which are composed of fatty materials. It is now becoming clear that the association of particular proteins with membranes is a critical event in a chain of biochemical events that regulate cell division, membrane fusion, and a variety of other cell biological processes. This important regulatory mechanism has been described and is actively being studied in mammalian cells and in yeast, but until now nothing has been known of its possible existence or role in higher plants. This is the subject of this research. These investigators have identified the gene for one part of the enzyme that carries out addition of isoprenyl groups to proteins in peas and will use techniques of molecular biology to characterize the enzyme further and to study its possible function in plants.