Mechanism of Targeting of a Luminal KDEL-containing Protein to the Golgi Complex

描述（由申请人提供）：本论文项目的目的是定义一种含有KDEL的腔内蛋白ERp44定位于高尔基复合体（GC）的机制、机制和调节模式。了解胃GC定位是如何实现的，将代表一种重要的、新颖的调节运输和分泌蛋白质量控制模式。这项研究的结果将有助于确定内质网质量控制疾病（如2型糖尿病）的新治疗靶点。具体来说，我们提出了三个实验目的。1. 确定ERp44靶向GC所需和足够的序列。我们假设ERp44中存在一个靶向序列，该序列覆盖其KDEL内质网（ER）检索序列。我们建议通过创建截断，内部缺失和点突变，加上嵌合充分性构建和测定它们的亚细胞定位来识别该序列。2. 确定和描述ERp44定位到GC的基本机制。我们将挑战当前的模型，该模型断言ERGIC53将ERp44定位到GC。我们假设GC中存在受体机制，它对ERp44上的靶序列比对KDEL受体的KDEL序列具有更高的亲和力。我们将使用交联和突变结构来定义GC保留蛋白。活细胞荧光显微镜、光漂白和生物化学技术将用于区分GC定位是通过保留还是回收发生的。3. 确定并表征ERp44定位到GC的调控机制。我们假设ERp44以一种受调控的方式定位于内质网或GC。我们将研究a) ERp44的伴侣功能对GC定位是否必要，b)定位如何随内质网应激而变化，c) KDEL-R的调节是否影响ERp44的定位。确定细胞如何调节分泌蛋白的产生对于理解突变或不适当的分泌蛋白如何逃避细胞质量控制并导致糖尿病等重要疾病至关重要。我们的研究将定义在促进或防止蛋白质质量控制中发挥关键作用的新机制。

DESCRIPTION (provided by applicant): The objective of this thesis proiect is to define the machinery, mechanism and mode of regulation by which a luminal KDEL- containing protein, ERp44, localizes to the Golgi complex (GC). Understanding how lumenal GC localization is achieved will represent a significant and novel mode of regulatory trafficking and secretory protein quality control. The outcomes of this study will aid in the identification of new therapeutic targets for ER quality control diseases, such as type 2 diabetes. Specifically, we propose three experimental aims. 1. Identify the necessary and sufficient sequence for targeting of ERp44 to the GC. We hypothesize that a targeting sequence exists within ERp44, which overrides its KDEL Endoplasmic Reticulum (ER) retrieval sequence. We propose to identify this sequence by creating truncation, internal deletion, and point mutations, plus chimeric sufficiency constructs and assay their subcellular localization. 2. Identify and characterize the essential machinery for localization of ERp44 to the GC. We will challenge the current model, which asserts ERGIC53 localizes ERp44 to the GC. We postulate receptor machinery exists in the GC that has a higher affinity for a targeting sequence on ERp44 than the KDEL sequence for the KDEL receptor. We will use cross-linking and mutant constructs to define the GC retention protein(s). Live cell fluorescence microscopy photobleaching and biochemistry techniques will be employed to distinguish whether GC localization occurs via retention or retrieval. 3. Determine and characterize the mechanism of regulation of ERp44 localization to the GC. We hypothesize that ERp44 is localized to the ER or GC in a regulated manner. We will investigate a) whether the chaperone function of ERp44 is necessary for GC localization, b) how localization changes with ER stress and c) whether modulation of KDEL-R affects ERp44 localization. Determining how cells regulate the production of secreted proteins is essential to understanding how mutant or inappropriate secretory proteins escape cellular quality control and cause important diseases such as diabetes. Our research will define new machinery that play a key role in promoting or preventing protein quality control.