# ER Morphology in the Pathogenesis of Hereditary
## 3 ER Shaping Proteins
### REEP1 (SPG31) and REEP2 (SPG72)  
Mutations in _REEP1_ give rise to the third most common dominant form of HSP, accounting for 6.5% of cases [84], while _REEP2_ mutations are rare. The most common mutations described for REEP1 are missense mutations, insertion, or deletions that cause premature stop codons [85]. The REEP family of proteins was first identified for their role in trafficking odorant receptors [86] and G-protein coupled receptors to the plasma membrane [87]. They are part of REEPs/DP1/Yop1p family of proteins, which shares a RHD (see above) believed to be responsible for their association with ER membranes. The yeast homolog Yop1p is one of the best characterized members of the superfamily. Yop1p reconstituted into liposomes has been shown to generate tubules [70; 88], leading to the hypothesis that this family of proteins is responsible for ER membrane tubulation. Six mammalian REEPs (REEP1-6) have been identified. However, while REEP5-6 displays the highest homology with Yop1p, REEP1-4 is characterized by the presence of a shorter first hydrophobic segment and the lack of a \(N\)-terminal cytoplasmic domain [89], likely resulting in the absence of the first transmembrane domain [88]. REEP1 and REEP2 also possess a \(C\)-terminal tubulin binding domain, which has not been identified in REEP5-6 [89]. These structural divergences are found also in lower-complexity organisms. _Caenorhabditis elegans_, _Strongylocentrotus purpuratus_ (sea urchin) and _Drosophila melanogaster_ have two REEP orthologs, one with high similarity to REEP1-4 and one with greater similarity to REEP5-6 [89], supporting the notion that REEP1-4 proteins may have functions partially distinct from those performed by REEP5-6/Yop1p. Structural studies on Yop1p indicated that the transmembrane domains in the RHD contain enough helical residues to fully traverse the hydrophobic bilayer of the ER. In addition, a \(C\)-terminal amphiphelix (APH) was identified, whose deletion abolishes membrane tubule formation in vitro [83; 88]. The APH region is widely conserved among REEP1-6 and is likely shared also by RTNs [83; 88], suggesting a conserved role in membrane shaping. Nevertheless, tubule formation has been reported in vitro upon reconstitution into liposomes only for Yop1p [70; 88]. Curiously, _Xenopus laevis_ REEP4 and REEP5 homologs or _Drosophila melanogaster_ REEP5-6 homolog ReepB, have been reported to form tubules in vitro only in the presence of the atlastin yeast homolog Sey1p and GTP [83; 90]. Moreover, Yop1p has been shown to generate extreme curvature at high concentrations by converting phospholipid bilayers into micelle-like lipoprotein particles (LPVs) [83], while _Xenopus_ REEP5 generates LLPs (but never tubules) even at very low concentrations. Unfortunately, in vivo evidence corroborating these in vitro observations is sorely missing. Overall, this suggests that although the transmembrane and APH domains of REEP proteins have been broadly associated with ER membrane-shaping properties, the precise role of individual REEP family members in the formation and maintenance of the ER is still poorly understood. Similarly, the functional difference between REEP1-4 and REEP5-6 is still obscure. The expression levels of REEP1-2 are low, compared to REEP5-6, although they are enriched in mammalian neuronal tissues [91; 92]. Indeed, EGFP fusions at the C-termini of all possible isoforms of the _Drosophila_ homolog of REEP1-4 (ReepA) or REEP5-6 (ReepB) revealed a weak expression of ReepA, which was barely detectable in the fly central nervous system and undetectable in the axonal or presynaptic ER. In agreement with this, the loss of ReepB caused ER sheet expansion and partial loss of ER in _Drosophila_ distal motor axons, while these effects are essentially undetectable after ReepA knock-out [93].