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

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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.