Our long-term goal is a complete molecular definition of Golgi apparatus biogenesis. The Golgi apparatus is the central organelle within the secretory pathway of human cells. How the cell generates apparently stable downstream organelles within the secretory pathway, specifically, the Golgi apparatus, while at the same time ensuring vectorial transport of newly synthesized protein to the plasma membrane, is a major challenge in cell biology. We find so-called resident Golgi apparatus membrane proteins recycle continuously in a coat protein independent manner from the Golgi apparatus to the ER. This recycling occurs in wild type cells in the absence of any genetic manipulation and is of immediate medical significance as it is a key step in the delivery of numerous bacterial and plant protein toxins to their ultimate site of cellular intoxication. The immediate aims of the proposed research are to characterize the molecular mechanisms that produce this novel coat-protein-independent Golgi-to-ER protein recycling pathway. We know that rab6 and rab33b are regulators of the pathway, and assume that rab effectors are important. The proposed Specific Aims are: 1. Characterization of intermediates. Preliminary experiments suggest that expression of late acting, membrane trafficking machinery proteins block Golgi glycosyltransferase recycling to the ER. This and other approaches will be used to characterize intermediates. 2. Determination if rab33b and rab6 act in parallel or in series within a pathway. Rab6 is more trans in localization than rab33b. Do the two rabs act in parallel or in series? 3. Characterization of the functional importance of rab effectors and in particular rab33b effectors in Golgi to ER recycling. Rab33b and rab6 in vitro display a different pattern of effectors yet each seems to regulate Golgi glycosyltransferase cycling to the ER. How does this work in a molecular sense? We propose to use rab33b and rab6 and their effectors as the prime molecular handles to dissect mechanism and pathway of resident Golgi protein recycling to the ER. In conclusion, we propose a mechanistic step forward in our understanding of Golgi apparatus biogenesis.
