Two studies on post-Golgi trafficking in polarized cells.

摘要

In this work I examine two systems of Golgi-associated trafficking of membrane vesicles. First, I show that synapsin I, a neuronal phosphoprotein, is present in epithelial cells in what seems to be a novel Golgi compartment. In the nervous system, synapsin I associates in a phosphorylation-dependent manner with the cytoplasmic surfaces of synaptic vesicles at nerve terminals. I describe the detection of this protein in epithelial cells and its localization to what appears to be a novel compartment of the Golgi complex. Myosin II also associates with this unique compartment. The putative function of synapsin I as a linker between cytoskeletal elements and synaptic vesicles in brain suggests a role in membrane-cytoskeletal interactions in an organelle like the Golgi. In addition, the distribution of synapsin I in synaptic vesicles and insulin secretory granules of the pancreas suggests a role in secretion in different cell types. In the next study, I analyze the localization of two P-type ATPases in epithelial tissues, the Wilson and Menkes proteins. These copper-transporters control copper metabolism and are implicated in two diseases in which copper metabolism is affected. In addition, they constitute an interesting model for TGN to plasma membrane trafficking and its regulation by a ligand. As deduced from subcellular fractionation studies, I describe the distribution of Wilson Protein in the liver in Golgi and apical plasma membrane. Copper treatments decreased the amount of Wilson Protein found at the Golgi, but did not consistently change the amount at the plasma membrane. Menkes Protein was present in mucosal epithelial cells, a distribution that agrees with its presumed role in copper absorption in the intestine. As seen by immunofluorescence microscopy, copper load or depletion of animals seemed to perturb greatly the localization of the protein, with a tight localization in the Golgi region in the presence of a copper chelator, while present in the basolateral plasma membrane region after treatment with copper. These in vivo studies on the localization of Wilson and Menkes proteins help elucidate the pathways for post-Golgi trafficking and their regulation.