Abstract
The endoplasmic reticulum (ER) network consists of tubules with high membrane curvature in cross-section, generated by the reticulons and REEPs. These proteins have two pairs of trans-membrane (TM) segments, followed by an amphipathic helix (APH), but how they induce curvature is poorly understood. Here, we show that REEPs form homodimers by interaction within the membrane. When overexpressed or reconstituted at high concentrations with phospholipids, REEPs cause extreme curvature through their TMs, generating lipoprotein particles instead of vesicles. The APH facilitates curvature generation, as its mutation prevents ER network formation of reconstituted proteoliposomes, and synthetic L- or D-amino acid peptides abolish ER network formation in Xenopus egg extracts. In Schizosaccharomyces japonicus, the APH is required for reticulon’s exclusive ER-tubule localization and restricted mobility. Thus, the TMs and APH cooperate to generate high membrane curvature. We propose that the formation of splayed REEP/reticulon dimers is responsible for ER tubule formation.
Highlights
The endoplasmic reticulum (ER) network consists of tubules with high membrane curvature in cross-section, generated by the reticulons and REEPs
The nonsedimentable population reproducibly gave higher cross-linking yields, suggesting that most molecules are more tightly associated in lipoprotein particles (LPPs) than in low-curvature membranes. These results indicate that REEPs generate LPPs when present at high concentrations, likely because REEP dimers generate so much curvature that they break up phospholipid bilayers into monolayers
We have analyzed the molecular mechanism by which the REEPs generate the high membrane curvature that is characteristic of ER tubules in cross-section
Summary
The endoplasmic reticulum (ER) network consists of tubules with high membrane curvature in cross-section, generated by the reticulons and REEPs. When purified Rtn or REEP proteins are reconstituted into liposomes, they form short, narrow tubules[7] These proteins localize exclusively to tubules and sheet edges, the high-curvature regions of the ER3,5,6. The high membrane curvature of tubules and sheet edges is stabilized by the Rtns and REEPs by an unknown mechanism All of these proteins contain pairs of closely spaced transmembrane (TM) segments followed by an amphipathic helix[6,13,14] (APH; Fig. 1a and Supplementary Fig. 1a). Scaffolding would involve the formation of intrinsically curved arc-shaped protein oligomers on the membrane surface, which would mold the lipid bilayer into tubules Whether these mechanisms are responsible for the membrane-shaping activity of the Rtns and REEPs remains unclear. We use in vitro and in vivo experiments to show that the TMs and APH cooperate to generate high membrane curvature and to derive a model for ERtubule formation
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