Reticulon Short Hairpin Transmembrane Domains Are Used to Shape ER Tubules

Reticulons are integral membrane proteins that partition into and shape the tubular endoplasmic reticulum (ER). We propose that reticulons use a membrane insertion mechanism to generate regions of high membrane curvature in the ER. A reticulon contains two short hairpin transmembrane domains (TMDs), which could generate membrane curvature by increasing the area of the cytoplasmic leaflet. Here, we test whether the short length of these hairpin TMDs is required for reticulon membrane-shaping functions in mammalian cells. We lengthened the TMDs of reticulon 4 to resemble a typical bi-pass TMD that spans both leaflets. We find that TMD mutants oligomerize like wild type (wt), however, they are not immobilized, do not partition into tubules, do not constrict tubules and no longer suppress peripheral ER cisternae. Therefore, short hairpin TMD length is required for reticulon protein partitioning and membrane-shaping functions. Another membrane protein with a short hairpin TMD is caveolin. We show that an ER-retained caveolin construct also partitions within the ER in a manner that is dependent on it containing a short hairpin TMD. These data suggest that a short hairpin TMD may be a general feature used by membrane-shaping proteins to partition into and shape regions of high membrane curvature.