Plasma Membrane Trafficking in Polarized Hepatocytes

描述(申请人提供)：我们将研究分子基础 极化的跨高尔基网络(TGN)到质膜(PM)的运输 肝细胞。我们将比较和对比根尖和基底外侧的差异 PM蛋白以及分泌蛋白被包装、靶向和 输送到两个质膜上。大鼠体内肝细胞和大鼠-人肝细胞 体外培养的WiF-B细胞是我们的极化细胞模型。许多精选的质膜蛋白 已通过重组腺病毒在这些细胞中表达。在目标1中，我们 将对选定的TGN后膜载体进行鉴定和表征 顶端和基侧PM蛋白和可溶性蛋白。免疫荧光 检测单一跨膜、糖脂锚定和 多面体PM蛋白从TGN中出现时将识别 是分开携带的而不是一起携带的。我们将免疫分离选定的携带者 然后比较大鼠肝脏中表达这些蛋白质的群体 用抗体探查膜载体的生化性质 贩运分子的存在(例如RABS、T型和V型陷阱、马达)和 质谱分析(MALDI-TOF/电喷雾MS)以确定已知和 新的蛋白质。这种生化比较将扩展到携带囊泡的情况 TGN之间的铜转运P型ATPase(ATP7B) 和肝细胞顶端PM以铜调节的方式，当缺陷时， 导致威尔逊氏病。在目标2中，我们将研究细胞质的作用 多面体顶端蛋白靶向/保留中的蛋白质。几个 ABC转运蛋白C亚家族的顶端蛋白含有C末端 识别的PDZ蛋白识别的三肽，而它们的基侧 同行们缺乏这些主题。我们将使用定点突变，细胞 表达和免疫荧光以确定其必要性和充分性 顶端蛋白定位的基序，然后是异位表达 选定的PDZ域以测试每个PDZ候选者在此 本地化。对于威尔逊蛋白ATPase，我们将同时表达和抑制 不同的域，以确定其铜监管涉及哪些部分 贩卖人口。在目标3中，我们将使用活细胞成像来确定 受已知因素调控的选定载体的TGN-PM后传输 运输蛋白(如RABS 8、11和17以及T形圈套)以及 目标1、B和2中确定的候选人。我们的总体目标是了解 极化肝细胞中PM蛋白运输的分子基础。

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.