However, it remains unclear whether this is unique to RTNLB13 or is conserved across all reticulons-to date, experimental evidence has only been reported for two reticulons. Here we used biophysical methods to characterize a minimal library of putative APH peptides from across the 21 A. We found that reticulons with the closest evolutionary relationship to RTNLB13 contain curvature-sensing APHs in the same location with sequence conservation. Our data reveal that a more distantly-related branch of reticulons developed a ~ 20-residue linker between the transmembrane domain and APH. This may facilitate functional flexibility as previous studies have linked these isoforms not only to ER remodeling but other cellular activities. The reticulons (RTNs) are a large family of integral membrane proteins present in all eukaryotes and first discovered in mammalian neurons 1, 2, where they are involved in endocytosis and intracellular transport 3, 4. In addition, they have been linked to apoptosis, axonal growth and regeneration 5, 6. RTNs are also localized to the endoplasmic reticulum (ER), where they are involved in ER shaping from sheets to tubules 7, 8, 9. In many species, deletion of RTNs leads to defects in ER tubule generation, whereas their overexpression increases both ER tubulation and fragmentation 8, 10, 11, 12. Abnormal ER morphology caused by mutations in ER-shaping proteins, such as the RTNs, has been implicated in neurological diseases and viral infections 5, 13. ER shaping is therefore not only necessary for maintaining correct ER function, but for the overall health of the cell itself 14, 15. The function of RTNs is uniquely coupled with generation and recognition of membrane curvature, and the structure of these proteins is exquisitely adapted to this role via a membrane-embedded domain and membrane-peripheral domain acting in concert.

The membrane-embedded domain is a highly conserved ~ 200-amino-acid region termed the reticulon homology domain (RHD). This domain is present in all RTNs and is composed of two hydrophobic regions separated by a soluble loop of approximately 60 amino acids 16, 17, 18. These hydrophobic regions are believed to form wedges in the membrane that promote curvature, and this curvature is further stabilized through RTN oligomerization (or scaffolding) 10, 19, 20, 21.

The membrane-peripheral domain is an amphipathic α-helix (APH) immediately following the RHD which sits on the surface of the membrane at the interface between the polar head-group regions and the hydrophobic core. Interestingly, despite widely-accepted speculation that the C-terminal APH is conserved across all reticulons, experimental evidence for the presence of this APH has been provided for only two RHD-containing proteins, RTNLB13 (Reticulon-Like Protein subfamily B 13) from Arabidopsis thaliana and Yop1p from yeast 22, 23, 24.

Dmpc Pro