The integral membrane proteins of the DP1 (deleted in polyposis) and reticulon families are responsible for maintaining the high membrane curvature required for both smooth endoplasmic reticulum (ER) tubules and the edges of ER sheets, and mutations in these proteins lead to motor neuron diseases, such as hereditary spastic paraplegia. Reticulon/DP1 proteins contain reticulon homology domains (RHDs) that have unusually long hydrophobic segments and are proposed to adopt intramembrane helical hairpins that stabilize membrane curvature. We have characterized the secondary structure and dynamics of the DP1 family protein produced from the YOP1 gene (Yop1p) and identified a C-terminal conserved amphipathic helix (APH) that, on its own, interacts strongly with negatively charged membranes and is necessary for membrane tubule formation. Analyses of DP1 and reticulon family members indicate that most, if not all, contain C-terminal sequences capable of forming APHs. Together, these results indicate that APHs play a previously unrecognized role in RHD membrane curvature stabilization.T he endoplasmic reticulum (ER) is the largest membranebound organelle in the eukaryotic cell and adopts diverse morphologies, including tubules that extend outward to the cell periphery and sheet-like structures closer to the nucleus (1, 2). The regions of high membrane curvature that are found in the ER tubules and sheets are stabilized by the DP1 (deleted in polyposis) and reticulon classes of integral membrane proteins (3-5). Consistent with their role in ER morphology, similar proteins have not been found in prokaryotes (6).There are six human DP1 proteins originally identified as accessory proteins facilitating the expression of odorant receptors, and thus termed receptor expression-enhancing proteins (REEPs) (7). The ability of REEPs to facilitate receptor trafficking has recently been related back to their ER shaping activities (8). The importance of REEPs in ER morphology is also highlighted by their implication in human diseases that are associated with neurons having long axons and requiring an extended tubular ER (9, 10). For example, mutations in the transmembrane domain of REEP1, which is primarily expressed in neurons (11), lead to pure forms of hereditary spastic paraplegia (HSP) (12, 13).The reticulon family in humans comprises four members, Rtn1-Rtn4 (6). Rtn4 is of particular interest because it is primarily responsible for generating or stabilizing the tubular ER in mammalian cells. Rtn4 is critical in neuronal cell processes because it inhibits spontaneous neurite outgrowth (14) and restricts neuronal plasticity (15), and it has been implicated in several neural diseases, including schizophrenia and motor neuron disease (16-18).The DP1 and reticulon proteins have in common a region containing two, unusually long hydrophobic segments (â35 aa in length each) known as a reticulon homology domain (RHD) (6). In humans, REEPs 1-6 are similar overall; however, the RHD of REEPs 1-4 is truncated (Fig. S1). REEPs 1-4 also possess...