Huang Liping, Kirschke Catherine P
United States Department of Agriculture, Agriculture Research Service, Western Human Nutrition Research Center, Davis, CA, USA.
FEBS J. 2007 Aug;274(15):3986-97. doi: 10.1111/j.1742-4658.2007.05933.x. Epub 2007 Jul 16.
It has been demonstrated that the plasma membrane expression of ZIP1 is regulated by endocytic mechanisms. In the zinc-replete condition, the level of surface expressed ZIP1 is low due to the rapid internalization of ZIP1. The present study aimed to identify a sorting signal(s) in ZIP1 that mediated endocytosis of ZIP1. Four potential sorting signals (three di-leucine-and one tyrosine-based) were found by searching the eukaryotic linear motif resource for functional sites in proteins (http://elm.eu.org). Site-directed mutagenesis and immunofluorescence microscopic analyses demonstrated that the di-leucine sorting signal, ETRALL144-149, located in the variable loop region of ZIP1, was required for the ZIP1 internalization and lysosomal degradation. Substitutions of alanines for the di-leucine residues (LL148,149/AA) severely impaired the internalization of ZIP1 and subsequent protein degradation, leading to an accumulation of the mutant ZIP1 on the cell surface, as well as inside the cell. Using chimeric proteins composed of an alpha-chain of interleukin-2 receptor fused to the peptides derived from the variable loop region of ZIP1, we found that the di-leucine sorting signal of ZIP1 was required and sufficient for endocytosis of the chimeric proteins.
已证明ZIP1的质膜表达受内吞机制调控。在锌充足的条件下，由于ZIP1的快速内化，表面表达的ZIP1水平较低。本研究旨在确定ZIP1中介导ZIP1内吞作用的分选信号。通过在真核线性基序资源中搜索蛋白质的功能位点（http://elm.eu.org），发现了四个潜在的分选信号（三个基于双亮氨酸和一个基于酪氨酸的信号）。定点诱变和免疫荧光显微镜分析表明，位于ZIP1可变环区域的双亮氨酸分选信号ETRALL144 - 149是ZIP1内化和溶酶体降解所必需的。将双亮氨酸残基替换为丙氨酸（LL148,149/AA）严重损害了ZIP1的内化及随后的蛋白质降解，导致突变型ZIP1在细胞表面以及细胞内积累。使用由白细胞介素 - 2受体的α链与源自ZIP1可变环区域的肽融合而成的嵌合蛋白，我们发现ZIP1的双亮氨酸分选信号对于嵌合蛋白的内吞作用是必需且足够的。
