Abstract
The group of stomatin/prohibitin/flotillin/HflK/C (SPFH) domain-containing proteins comprise members of diverse subcellular localization and function. Association with detergent-resistant membranes (DRMs) and the propensity to form oligomers are two common properties of SPFH domain proteins and likely important for the function of these proteins. Our laboratory recently discovered two novel members of this protein group, which, based on their endoplasmic reticulum (ER) localization and association with DRMs, were named ER lipid raft-associated protein (erlin)-1 and -2. Here we characterized erlin oligomerization and identified domains within the erlins responsible for oligomerization and DRM association. Using co-immunoprecipitation and sucrose density gradient centrifugation approaches on endogenous and ectopically expressed erlin proteins, we found that they formed homo- and hetero-oligomers and were part of large multimeric complexes. These properties were independent of their DRM association. By analyzing truncation and point mutants of erlin-2 we discovered that interaction between erlin monomers (oligomerization) and association with high molecular weight complexes require distinct regions within the protein. Although oligomerization and DRM association were mediated by a region immediately downstream of the SPFH domain (residues 228-300), integration into high molecular weight complexes was absolutely dependent on a phenylalanine residue C-terminal of this region (Phe-305), which lies within a short stretch of hydrophobic residues. Our data demonstrate that lower order oligomerization and incorporation into multimeric complexes are two separate biochemical properties of the erlins, because they are mediated by distinct regions.
Highlights
Proteins containing the stomatin/prohibitin/flotillin/HflK/C (SPFH)4 domain comprise a large protein group with members in many different species from prokaryotes to eukaryotes (1)
Erlin-1 and -2 Form Homo- and Hetero-oligomers—We previously reported that, in addition to the 43-kDa band corresponding to the monomeric form of ectopically expressed HAtagged erlin-2, a ϳ80-kDa band, which might represent a dimeric version of the protein, can be detected in detergent-resistant membranes (DRMs) fractions isolated from NIH-3T3 murine fibroblasts (6)
We demonstrated that the erlins are part of high molecular weight complexes
Summary
Proteins containing the stomatin/prohibitin/flotillin/HflK/C (SPFH) domain comprise a large protein group with members in many different species from prokaryotes to eukaryotes (1). The function of the erlins is still relatively unexplored, but the latter study suggests that erlin-2 acts as a substrate recognition factor during ER-associated protein degradation of activated inositol 1,4,5-trisphosphate receptors and other substrates (8) Both erlins are closely related as they share ϳ80% identity at the amino acid level, whereas the extreme N and C termini show the lowest conservation (2, 9, 10). In addition to DRM association, another common characteristic of SPFH domain-containing proteins is the propensity to form oligomers, the size of these complexes varies somewhat between members Stomatin and podocin both form large homo-oligomers of ϳ9 –12 and ϳ20 –50 monomers, respectively (5, 11), whereas prohibitin-1 and -2 form multimeric complexes of ϳ1.2 MDa, which were shown to have a ringlike structure (12). The oligomerization domains of most SPFH domaincontaining proteins have only been defined in relatively broad terms, the domain necessary for oligomerization and DRM association of stomatin was mapped to a 9-amino acid stretch downstream of the SPFH domain (16)
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