Structure and membrane remodelling mechanism of the DP1/Reticulon family of ER proteins.

内质网（ER）是真核细胞内最大的细胞器，也是形态学上最引人注目的细胞器。内质网是由一个高度动态的脂质膜网络与核膜邻接，并向外延伸到细胞周边。ER是关键细胞功能的枢纽，是膜蛋白、分泌蛋白和脂质的合成位点;它也是细胞内钙的储存库，并参与毒素灭活。靠近细胞核的ER在形态上主要由提供大表面积的片层组成，所述片层被细胞的蛋白质产生组分（核糖体）包覆。在离细胞核更远的地方，ER包含大量快速且连续重塑的小管，即它们在秒的时间尺度上形成和消失。这些不同的ER形态在真核生物中是保守的，它们如何形成和稳定是细胞生物学中的一个基本问题，对人类健康和疾病有影响，特别是神经元，它比大多数其他类型的细胞更依赖于高度弯曲的ER。DP 1和reticulon蛋白质家族负责产生和稳定在管状ER和ER片边缘中发现的高度弯曲的膜，并且称为受体表达增强蛋白（REEP）的人类DP 1蛋白质中的突变导致运动神经元疾病遗传性痉挛性截瘫（HSP）。DP 1和reticulon蛋白共享一个嵌入膜中的同源区域，称为reticulon同源结构域（RHD），然而，对RHD的结构没有分子见解，并且通过扩展，不了解RHD中的突变如何导致HSP等疾病。RHD功能的当前范例是蛋白质的该区域含有螺旋发夹，其不完全穿过膜脂质双层从胞质溶胶到ER，并且以这种方式挤压膜的胞质溶胶一半以诱导曲率。然而，我们已经建立了一个实验系统，研究一个代表性的RHD从酵母，并发现它包含四个跨膜螺旋足够长，以充分穿越膜。我们还发现了一种与膜强烈相互作用的两亲性螺旋（APH），去除酵母RHD的这一区域会消除其弯曲膜的能力。APH通常存在于参与膜重塑的蛋白质中，这支持了APH插入膜双层的胞质小叶是RHD弯曲膜的中心机制的可能性。我们将通过确定RHD的完整三维结构并评估其与膜的相互作用来验证这一假设。然后，我们将介绍HSP引起的突变，并确定它们如何破坏RHD的结构和功能，并有助于HSP的发展。利用我们对DP 1 RHD和APH的结构研究结果，我们将通过对参与神经元生长和修复的人类reticulon家族的靶向研究来测试它们的普遍性。

The endoplasmic reticulum (ER) is the largest and morphologically most striking of the organelles found inside eukaryotic cells. The ER is composed of a highly dynamic network of lipid membranes contiguous with the nuclear membrane and extending outward to the cell periphery. The ER is a hub for critical cellular functions, being the site of synthesis for membrane proteins, secreted proteins, and lipids; it is also a store for intracellular calcium, and involved in toxin inactivation. The ER close to the cell nucleus is morphologically composed mostly of sheets that provide a large surface area that is coated with protein-producing components of the cell (ribosomes). Further from the nucleus, the ER contains a large number of tubules that are rapidly and continuously remodelled, i.e. they form and disappear on the timescale of seconds. These distinct ER morphologies are conserved across eukaryotes and how these are formed and stabilised is a fundamental question in cell biology with implications for human health and disease, especially that of neurons, which have a greater reliance on highly curved ER than most other types of cells. The DP1 and reticulon families of proteins are responsible for generating and stabilising the highly curved membranes that are found in the tubular ER and the edges of ER sheets, and mutations in the human DP1 proteins called Receptor Expression Enhancing Proteins (REEPs) cause the motor neuron disease Hereditary Spastic Paraplegia (HSP). DP1 and reticulon proteins share a homologous region that is embedded in the membrane called a reticulon homology domain (RHD), however, no molecular insight into the architecture of the RHD exists, and by extension there is no understanding of how mutations in the RHD can lead to diseases like HSP. The current paradigm for RHD function is that this region of the protein contains helical hairpins that do not fully cross the membrane lipid bilayer from the cytosol to the ER, and in this way crowd the cytosolic half of the membrane to induce curvature. However, we have established an experimental system for studying a representative RHD from yeast and have found that it contains four transmembrane helices long enough to fully traversing the membrane. We have also discovered an amphipathic helix (APH) that interacts strongly with membranes, and removal of this region of the yeast RHD abolishes its ability to curve membranes. APHs are commonly found in proteins involved in membrane remodelling, lending support to the possibility that insertion of this APH into the cytosolic leaflet of the membrane bilayer is the central mechanism by which RHDs curve membranes. We will test this hypothesis by determining the complete three-dimensional structure of the RHD and evaluating its interactions with membranes. We will then introduce HSP-causing mutations and determine how they disrupt the RHD structure and function and contribute to HSP development. Using our structural findings for the DP1 RHD and APH, we will test their generality through targeted studies of the human reticulon family, which are involved in neuronal growth and repair.