Role of a novel Golgi export signal in intracellular trafficking of the reovirus p14 protein

细胞包含许多隔间，这些隔间由膜相互隔开，允许不同的隔间执行不同的功能。例如，线粒体为细胞新陈代谢产生能量，细胞核控制基因表达，内质网(ER)合成膜蛋白，溶酶体作为细胞的消化系统发挥作用，内酶体可以吸收细胞外物质并将其引导到溶酶体或将其循环回到细胞周围的质膜。所有这些膜隔间相互通信并协同工作，以维持正常的细胞功能。在任何特定细胞中的大约10,000种不同的蛋白质中，大约有三分之一是膜蛋白。这些膜蛋白中的每一种都必须被分选并运输到它们正确的亚细胞膜隔室。这种膜蛋白运输是所有细胞中必不可少的基本过程，必须受到严格的监管和控制。 大多数蛋白质运输发生在小的膜囊中，它们可以在一个膜室和另一个膜室之间穿梭，在两个膜室之间运送货物。对于要进入质膜的膜蛋白，它们的旅程始于内质网，从那里它们被运输到高尔基复合体，高尔基复合体是将蛋白质定向到溶酶体、内体或质膜的主要分选中心。决定哪种蛋白质去哪里是基于组成运输小泡的不同蛋白质对每种货物蛋白质中存在的特定信号的识别。虽然近年来对调节蛋白质在内质网和高尔基体之间穿梭的信号和载体蛋白进行了很好的描述，但我们对是什么调控了膜蛋白从高尔基体到质膜的运输却知之甚少。我的NSERC资助的研究项目的目标是更清楚地定义细胞如何调控这一重要过程。 以一种简单的膜蛋白(命名为p14)作为模型系统，我们在四年前开始探索这种蛋白是如何运输到质膜的。我们在p14中发现了一个短的氨基酸序列，这是质膜定位所必需的；当这个序列发生突变时，p14无法从高尔基体中退出。我们进一步发现，这个高尔基体输出信号的功能是基于多碱基序(PBM)中包含的三个碱性(即带正电的)氨基酸的存在。这是第一个基于PBM的高尔基输出信号的例子。我们还发现，p14与三种不同的细胞蛋白相互作用，这三种不同的细胞蛋白以前被认为是循环内体途径的组成部分。基于这些结果，我们假设p14PBM通过将高尔基体中的p14分离到循环内体中，然后将p14输送到质膜上，从而发挥高尔基体-质膜转运信号的作用。 NSERC这项提案的目标是通过确定p14 PBM在这一过程中的作用，进一步确定这一被低估但必不可少的贩运途径是如何发挥作用的。为了实现这一目标，我们建议表征p14与这些内吞循环蛋白的相互作用，识别参与这一过程的其他蛋白质，并在活细胞上使用视频显微镜直接研究这一过程是如何发生的。P14蛋白提供了一个简单、易处理的系统来定义高尔基体质膜运输所涉及的途径，高尔基体质膜运输是细胞运输途径中一个鲜为人知的阶段。我们建议的研究有可能更清楚地辨别这些必要的蛋白质运输途径的性质，并提供对这一过程的机械性见解。

Cells contain numerous compartments that are separated from each other by membranes, allowing different compartments to perform distinct functions. For example, mitochondria generate energy for cell metabolism, the nucleus controls gene expression, the endoplasmic reticulum (ER) synthesizes membrane proteins, lysosomes function as the digestive system of the cell, and endosomes can take up extracellular material and direct it to lysosomes or recycle it back to the plasma membrane that surrounds the cell. All of these membrane compartments communicate with each other and work in conjunction to maintain normal cell function. Of the ~10,000 different proteins in any given cell, about one-third of these proteins are membrane proteins. Each of these membrane proteins must be sorted and transported to their correct sub-cellular membrane compartment. Such membrane protein trafficking is an essential, fundamental process in all cells, and must be tightly regulated and controlled. Most protein trafficking occurs in small membrane vesicles that can shuttle between one membrane compartment and another, delivering cargo between compartments. For membrane proteins destined for the plasma membrane, their journey starts in the ER from where they are transported to the Golgi complex, which serves as major sorting hub to direct proteins to lysosomes, endosomes or the plasma membrane. Deciding which protein goes where is based on the recognition of specific signals present in each cargo protein by different proteins that make up the transport vesicle. While sorting signals and carrier proteins that regulate protein shuttling between the ER and Golgi have been well characterized in recent years, we have a very poor understanding of what regulates membrane protein trafficking from the Golgi to the plasma membrane. The goal of my NSERC-funded research program is to more clearly define how cells regulate this essential process. Using a simple membrane protein (named p14) as a model system, we began four years ago to explore how this protein is transported to the plasma membrane. We discovered a short sequence of amino acids in p14 that was required for plasma membrane localization; when this sequence was mutated, p14 failed to exit from the Golgi. We further discovered that the function of this Golgi export signal was based on the presence of three basic (meaning positively charged) amino acids contained within a polybasic motif (PBM). This was the first example of a Golgi export signal based on a PBM. We also discovered that p14 interacted with three different cellular proteins previously identified as components of the recycling endosome pathway. Based on these results, we hypothesize the p14 PBM functions as Golgi-plasma membrane trafficking signal by sorting p14 from the Golgi into recycling endosomes that then deliver p14 to the plasma membrane. The goal of this NSERC proposal is to more determine how this underappreciated, yet essential, trafficking pathway functions by determining the role of the p14 PBM in this process. To achieve this goal, we propose to characterize p14 interactions with these endocytic recycling proteins, to identify additional proteins involved in this process, and to directly examine how this process occurs using video microscopy on live cells. The p14 protein provides a simple, tractable system to define pathways involved in Golgi-plasma membrane trafficking, a poorly understood stage of cellular trafficking pathways. Our proposed studies have the potential to more clearly discern the nature of these essential protein trafficking pathways and to provide mechanistic insights into this process.