The candidate will study the non-classical secretion of plasma trans-glutaminase (TG) in transfected cell lines. Transglutaminase cDNA in an expression vector driven by the cytomegalovirus (CMV) promoter will be transiently transfected into several cell lines. To distinguish the transfected transglutaminase from the endogenous protein, the transfected form will be made with the addition of an histidine tag or a c-myc epitope at the C-terminus, recognizable by the commercially available reagents. The secretion of this protein without the signal peptide will be characterized kinetically by pulse-chase labeling, immunoprecipitation, and autoradiographic analysis of SDS-PAGE. Stable transfectants of the selected cell lines will be examined for possible association between the newly synthesized protein and the cell surface using an impermeant reagent for biotinylation, immunoprecipitation, immunoblotting, and staining with streptavidin conjugate. In order to identify transglutaminase protein complexes in the cytosol, expressing cells that secrete TG will be labeled with 35S-amino acids and fractionated on percoll gradients. Fractions will be analyzed by immunoprecipitation to test whether nascent TG occurs in a high-molecular-weight complex in the cytosol. The transglutaminase secretion will next be analyzed with the addition of selected inhibitors for protein secretion or with the heat shock treatment, which increases the fraction of partially folded protein intermediates in the cytosol. In addition, the candidate will test whether plasma transglutaminase crosses the membrane as a folded protein or as a linear polypeptide. For this purpose, plasma transglutaminase will be expressed with a fusion protein with the transmembrane anchor of VSV G protein. Alternatively, plasma transglutaminase will be fused to dihydrofolate reductase; such a fusion protein can cross membrane transit sites only in the absence of methotrexate.
