PILTO STUDY--PARAFUSIN IN LIVER AND HEPATOCYTES

Exocytosis is a common problem invented by eukaryotic cells for release of most protein-rich secretory products. In regulative exocytosis, these secretory products are in vesicles docked on sites below the plasma membrane until reception of an appropriate stimulus. In the search for specific proteins involved in regulation of exocytosis, we found a 63 kDa phosphoprotein in the protozoan, Paramecium tetraurelia. This phosphoprotein, named parafusin, undergoes dephosphorylation upon stimulation of exocytosis. We have raised antibodies to parafusin and have found that it is present in normal rat hepatocytes. The aims of our proposed studies are to (1) immunolocalize parafusin in liver and cultured hepatocytes via light and electron microscopy; (2) perform biochemical localization of the phosphorylated and dephosphorylated forms of parafusin in subcellular fractions; and (3) clone cDNA of the parafusin molecule using a liver expression library. This will allow investigation of the regulation of this protein in states in which its function may be perturbed (e.g., development, regeneration).