Cell Biology of Human Thiamine Transporters

说明（由申请人提供）：硫胺素（维生素B1）是人体健康所必需的重要营养素。最近，两个硫胺素转运基因SLC19A2和SLC19A3（分别编码hTHTR1和hTHTR2）已经从许多人体组织中被克隆和鉴定。对这些硫胺素转运体的膜靶向和细胞内运输尚不清楚。蛋白质在质膜上的靶向已被证明涉及特定的靶向信号，如酪氨酸、二亮氨酸、二酸、二酸、组氨酸和多肽编码的PDZ结构域。hTHTRI& 2多肽具有许多这样的潜在分选信号。在初步研究中，我们将hTHTR1与EGFP融合，并在HuTu-80细胞中证实了稳定表达的融合蛋白（hTHTR1-EGFP）的功能和膜定位。为了研究特异性靶向信号在引导hthtr&2到达人肠上皮细胞质膜中的作用，我将比较野生型、截断型和定点突变型hthtr&2与EGFP融合的构建体，并通过共聚焦显微镜成像。我的目的还在于确定hthtri和2在给定细胞膜结构域的表达是否具有细胞类型特异性，使用野生型构建的hthtri和2蛋白与EGFP融合。为了进一步研究hthtri和2蛋白的细胞内运输，我将研究微管网络和基于肌动蛋白的细胞骨架的作用，对新合成的硫胺素转运体向质膜的输送进行动力学分析，并研究蛋白质向质膜的囊泡运输（插入/提取）的作用。

DESCRIPTION (provided by applicant): Thiamine (vitamin B1) is an important nutrient that is essential for human health. Recently two thiamine transporter genes SLC19A2 and SLC19A3 (encoding hTHTR1 and hTHTR2, respectively) have been cloned and characterized from a number of human tissues. Nothing is known about the membrane targeting and intracellular trafficking of these thiamine transporters. Targeting of a protein to the plasma membrane has been shown to involve specific targeting signals such as tyrosine, di-leucine, diacidic, dibasic, histidine, and PDZ domains encoded in the polypeptide. The hTHTRI& 2 polypeptides have a number of such potential sorting signals. In preliminary studies, we fused hTHTR1 to EGFP and demonstrated functionality and membrane localization of the stably expressed fusion protein (hTHTR1-EGFP) in HuTu-80 cells. In order to investigate the role of specific targeting signals in directing hTHTRI& 2 to the plasma membrane of human intestinal epithelial cells, I will compare wild-type, truncated, and site-directed mutant constructs of hTHTRI& 2 fused to EGFP, and image by confocal microscopy. I aim to also determine if the expression of hTHTRI& 2 at a given cell membrane domain is cell-type specific using wild-type constructs of hTHTRI& 2 proteins fused to EGFP. To further examine the intracellular trafficking of hTHTRI& 2 proteins I will examine the role of the microtubule network and the actin-based cytoskeleton, perform kinetic analysis of the delivery of newly synthesized thiamine transporters to the plasma membrane, and investigate the role of vesicular transport of the proteins to the plasma membrane (insertion/retrival).