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

细胞包含大量的隔室，这些隔室通过膜相互隔开，从而允许不同的隔室执行不同的功能。例如，线粒体为细胞代谢提供能量，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，内体可以吸收细胞外物质并将其引导到溶酶体或将其再循环回包围细胞的质膜。所有这些膜间室相互沟通，共同维持正常的细胞功能。在任何给定的细胞中大约有10,000种不同的蛋白质，其中大约三分之一是膜蛋白。每一种膜蛋白都必须被分类并运输到它们正确的亚细胞膜区室。这种膜蛋白运输是所有细胞中必不可少的基本过程，必须受到严格的调节和控制。大多数蛋白质运输发生在小的膜泡中，它们可以在一个膜室和另一个膜室之间穿梭，在不同的膜室之间运送货物。对于到达质膜的膜蛋白，它们的旅程从内质网开始，在那里它们被运送到高尔基复合体，高尔基复合体作为主要的分选中心，将蛋白质引导到溶酶体、核内体或质膜。决定哪一种蛋白质去哪是基于构成运输囊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。虽然近年来已经很好地描述了调节内质网和高尔基体之间蛋白质穿梭的分类信号和载体蛋白，但我们对调节膜蛋白从高尔基体到质膜的运输的机制知之甚少。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节这一基本过程。使用一种简单的膜蛋白（命名为p14）作为模型系统，我们从四年前开始探索这种蛋白是如何运输到质膜的。我们在p14中发现了质膜定位所需的短序列氨基酸；当该序列发生突变时，p14不能从高尔基体中退出。我们进一步发现，这个高尔基输出信号的功能是基于一个多基基基序（PBM）中包含的三个碱性（即带正电荷）氨基酸的存在。这是基于PBM的高尔基输出信号的第一个例子。我们还发现p14与三种不同的细胞蛋白相互作用，这些蛋白先前被确定为循环内体途径的组成部分。基于这些结果，我们假设p14 PBM作为高尔基-质膜运输信号，通过将p14从高尔基体中分选到再循环的内体中，然后将p14传递到质膜。NSERC提案的目标是通过确定p14 PBM在这一过程中的作用，进一步确定这一未被充分认识但至关重要的贩运途径的功能。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，鉴定参与这一过程的其他蛋白质，并使用活细胞视频显微镜直接检查这一过程是如何发生的。p14蛋白提供了一个简单、易于处理的系统来定义高尔基-质膜运输途径，这是细胞运输途径的一个鲜为人知的阶段。我们提出的研究有可能更清楚地辨别这些基本蛋白质运输途径的性质，并为这一过程提供机制见解。

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.