Plasma Membrane Trafficking in Polarized Hepatocytes

Johns Hopkins University, Baltimore MD

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Abstract

DESCRIPTION (provided by applicant): We will study the molecular basis of trans-Golgi-network (TGN) to plasma membrane (PM) trafficking in polarized hepatic cells. We will compare and contrast how diverse apical and basolateral PM proteins, as well as secretory proteins, are packaged, targeted and delivered to the two plasma membranes. Rat hepatocytes in vivo and rat-human WIF-B cells in vitro are our polarized cell models. Many selected PM proteins have been expressed in these cells using recombinant adenovirus. In Aim #1, we will identify and characterize the post-TGN membrane carriers of selected apical and basolateral PM proteins and soluble proteins. Immunofluorescence detection of combinations of single transmembrane, glycolipid-anchored and polytopic PM proteins as they emerge from the TGN will identify cargoes that are carried separately versus together. We will immuno-isolate selected carrier populations from livers of rats expressing these proteins then compare biochemical properties of the membrane carriers using antibodies to probe for the presence of trafficking molecules (e.g., rabs, t- and v-SNAREs, motors) and mass spectrometric analysis (MALDI-TOF/ electrospray MS) to identify known and novel proteins. This biochemical comparison will extend to vesicles carrying the copper-transporting P-type ATPase (ATP7B), which traffics between the TGN and apical PM of hepatocytes in a copper-regulated manner and, when defective, causes Wilson's disease. In Aim #2, we will study the roles of cytoplasmic proteins in the targeting/retention of polytopic apical proteins. Several apical proteins of the ABC transporter subfamily C contain C-terminal tripeptides recognized by identified PDZ proteins, whereas their basolateral counterparts lack these motifs. We will use site-directed mutagenesis, cell expression and immunofluorescence to determine the necessity and sufficiency of the motifs for apical protein localization, followed by ectopic expression of selected PDZ domains to test the role of each PDZ candidate in this localization. For the Wilson protein ATPase, we will both express and inhibit different domains to identify which parts are involved in its copper-regulated trafficking. In Aim #3, we will use live-cell imaging to determine the steps in the post-TGN-to-PM transport of selected carriers that are regulated by known trafficking proteins (eg. rabs 8, 11 and 17 and the t-SNAREs) as well as candidates identified in Aims #1 B and #2. Our overall goal is to understand the molecular basis of PM protein traffic in polarized hepatocytes.