Our hypothesis is that there are unique sets of molecules and structures involved in moving proteins and lipids out of the hepatocyte TGN. We have identified six molecules functioning in the constitutive pathway. These are essential for the formation of a distinct vesicle population containing the pIgA-receptor (pIgA-R): (1) TGN38, the transmembrane receptor for the p62cplx; (2) p62; (3) the 25 kD GTPase associated with p62 (together called p62cplx); (4) a approximately 100 kD catalytic PI 3-kinase subunit; (5) phosphatidylinositol transfer protein (PITP) and (6) dynamin. In this proposal we extend our studies of these molecules to examine their roles in exit from the Golgi in the clathrin-mediated pathway (S.A.1). To date, the molecules shown to be essential for pIgA- R budding appear to be signaling molecules. A goal of S.A. 2 will be to search for traditional coat-proteins associated with pIgA-R vesicles using 2D gel analysis. Potential coat-proteins will be sequenced and functionally characterized. The 2D gel analysis extends the power of our cell-free assay, providing the capacity to follow sorting of many proteins. Two sorting assays, pre-sorting of exiting cargo between distinct trans-cisternae and sorting into two vesicle/tubule populations upon exit from the TGN will be developed. A major signaling molecule is the p62cplx associated PI 3-kinase which acts synergistically with PITP in the formation of PI(3)P. PITP is predicted to be involved in the regulation of diacylglycerol (DAG) balance in Golgi membranes. S.A. 3 focuses on understanding the mechanism of how these lipids, PI(3)P and DAG, function in exit from the Golgi. This includes testing the hypothesis that the function of the modified ER on the trans-face of the Golgi (formerly known as the GERL) is to accept DAG produced in the Golgi. In S.A. 4 we will test whether tubules, rather than vesicles, leave the trans-Golgi carrying constitutive cargo. The specialized equipment and expertise for high resolution, optimal preservation EM morphological studies of labile structures (i.e. tubules) are available to us through our collaboration with the Boulder Laboratory for 3D fine structure. In addition, the interplay between signaling lipids and dynamin at the TGN will be examined. Finally, the possibility that dynamin is the molecule controlling tubule versus vesicle production at the TGN will be evaluated. Together, the data obtained using diverse approaches will help us reach our ultimate goal of understanding the structure function of the trans-Golgi and the molecular details of the process of secretion.