Structural and Functional Characterization of the Endoplasmic Reticulum Lipid Raft-Associated Proteins (Erlins)

Using an antibody screen against isolated lipid raft proteins from human myelomonocytic cells, our laboratory previously discovered two novel, highly related proteins. Based on their enrichment in detergent resistant membrane fractions (DRMs) and their localization to the endoplasmic reticulum (ER), they were named ER lipid raft-associated protein (erlin) -1 and -2. Erlins belong to the group of Stomatin/ Prohibitin/ Flotillin/ HflK/C (SPFH) domain-containing proteins. Members of this protein group differ in respect to subcellular distribution and function; yet all of them are associated with DRMs and form oligomers, suggesting that these properties are important for their function. Erlins are evolutionary conserved with homologues in the nematode Caenorhabditis elegans, plants and vertebrates. The function of the erlin proteins was completely unexplored at the beginning of this study.;While this study was being conducted, a different group reported that erlins function in ER-associated protein degradation (ERAD) of activated inositol-1,4,5-trisphosphate receptors (IP3Rs) and thus might negatively regulate IP3R signalling. We tested this model in C. elegans, which contains one erlin homologue that we found was highly similar to human erlins. However, loss of erlin protein had no effect on IP3Rdependent processes in C. elegans. Using C. elegans as well as mammalian cells, we also tested if erlins play a more general role in ERAD, but our data did not support a major function for erlins in ERAD. Our data indicate that erlins do not play an important role in the regulation of IP3R signalling or in the ERAD pathway.;The first part of this study was aimed at characterizing complex formation of the erlins and identifying domains required for complex formation and DRM association. We determined that erlins form homo- and hetero-oligomers as well as high MW complexes, and each type of complex formation is dependent on distinct regions within the erlin proteins. While oligomerization and DRM association are mediated by a region immediately downstream of the SPFH domain (residues 228-299), formation of high MW complexes is absolutely dependent on a phenylalanine residue C-terminal to this region (F305). Erlin mutants lacking F305 disrupt multimeric erlin complexes and likely have a dominant negative effect on erlin function.