Plasma Membrane Trafficking in Polarized Hepatocytes

描述（由申请人提供）：我们将研究 跨高尔基体网络（TGN）到质膜（PM）的运输 肝细胞我们将比较和对比不同的顶端和基底外侧 PM蛋白以及分泌蛋白被包装、靶向和 传递到两个质膜。大鼠体内肝细胞和大鼠-人肝细胞 体外WIF-B细胞是我们的极化细胞模型。许多选定的PM蛋白 已经使用重组腺病毒在这些细胞中表达。在目标#1中，我们 将识别和表征选定的TGN后膜载体 顶侧和基底侧PM蛋白和可溶性蛋白。免疫荧光 检测单跨膜、糖脂锚定和 当它们从TGN中出现时，多位PM蛋白将识别出 分别携带而不是一起携带。我们将对选定的携带者进行免疫隔离 表达这些蛋白质的大鼠肝脏中的细胞群， 使用抗体探测膜载体的生物化学性质， 运输分子的存在（例如，rabs、t型和v型SNARE、电机）和 质谱分析（MALDI-TOF/电喷雾MS），以鉴定已知和 新型蛋白质这种生化比较将扩展到携带 铜转运P型ATP酶（ATP 7 B），它在TGN之间运输 和肝细胞的顶端PM以铜调节的方式，当有缺陷时， 导致威尔逊氏病在目标#2中，我们将研究细胞质的作用， 蛋白质在多位顶端蛋白的靶向/保留中的作用。几 ABC转运蛋白亚家族C的顶端蛋白含有C-末端 三肽识别的PDZ蛋白，而他们的基底外侧 而对手则缺乏这些主题。我们将使用定点突变，细胞 表达和免疫荧光，以确定的必要性和充分性， 顶端蛋白定位的基序，随后异位表达 选择PDZ域来测试每个PDZ候选者在此 本地化对于Wilson蛋白ATP酶，我们将同时表达和抑制 不同的领域，以确定哪些部分涉及其铜管制 贩卖人口在目标3中，我们将使用活细胞成像来确定 所选载体的后TGN至PM运输由已知的 运输蛋白（例如，rab 8、11和17以及t-SNARE）以及 目标1 B和目标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.