INTRACELLULAR TRANSGLUTAMINASE--GENE ISOLATION, CHARACTERIZATION, AND FUNCTION

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The long-term objectives of this project are to identify and characterize the in vivo functional role(s) for the intracellular, cytosolic form of the enzyme, transglutaminase (TGase). TGase catalyzes amine-conjugations of polyamines and other primary amines to select endogenous protein substrates through an amine-exchange reaction at gamma-glutaminyl amino acid residues. We have discovered that TGase in plants introduces an epsilon- (gamma-glutaminyl) -lysine isopeptide crosslink between large polypeptide subunits (L) of the enzyme, ribulose 1, 5-bisphosphate carboxylase-oxygenase (rubisco). TGase catalyzes crosslinkage of L monomers to form dimers (L2) and tetrameres (L4), which may be intermediates in a chaperone-mediated pathway leading to the assembly of the only catalytically functional form of rubisco, L8S8. TGase also has a second catalytic activity, that being quanosine 5'-triphosphate (GTP)- binding and GTP hydrolysis. These findings inspire two hypotheses: The first hypothesis is that intracellular TGase, through its capacity to catalyze intermolecular crosslinks between some protein molecules, may function as a catalyst in the assembly of complex quaternary subunit structures of select enzymes. The second hypothesis is that intracellular TGase, through its capacity to hydrolyze GTP, may function as a G-protein in signal transduction processes in plants. These hypotheses will be tested by investigating four specific aims, which are: (1) To characterize recombinant phage clones containing the TGase gene derived from a lambdagt11 cDNA expression library prepared from mRNA isolated from Medicago sativa L (alfalfa) seedlings, isolate the gene for TGase, and determine its nucleotide sequence. (2) To transfer the isolated gene for alfalfa TGase into an Escherichia coli host which simultaneously contains cloned plasmid gene copies of plant large (L) and small (S) polypeptide subunits for rubisco. These recombinants will permit an investigation of the putative chaperone role of plant cellular TGase in mediating the in vivo assembly of L and S into enzymatically functional hexadecameric, L8S8 rubisco. (3) To evaluate putative G-protein properties of TGase by determining the expression of its enzymatic activity and its mRNA synthesis in plant seedlings after administration of agents that perturb signal transduction processes. (4) To utilize knowledge of the nucleotide sequence of the TGase gene in order to exploit anti-sense RNA methodology for suppression of the expression of the TGase enzyme in transformed plants. The results of this project will be relevant to the basic understanding of virtually all biological systems where oligomeric protein subunit assemblies are constructed. It will also advance knowledge of G-protein function(s) in eukaryotic cell systems.