A stomatin dimer modulates the activity of acid-sensing ion channels

Stomatins govern membrane trafficking and ion channel activity. The banana-shaped stomatin-domain dimmers oligomerize into a cylindrical structure. A dynamic hydrophobic pocket at the concave side of the dimer mediates repression of acid-sensing ion channel 3 (ASIC3) activity.


Introduction
The 32-kDa peripheral membrane protein stomatin is the founding member of the stomatin family, which in mammals includes stomatin-like proteins (STOMLs) 1-3 and the kidney-specific podocin (reviewed in Lapatsina et al, 2012a). These proteins are characterized by a conserved domain of B120 residues, the stomatin domain, which is a member of the SPFH (Stomatin, Prohibitin, Flotillin, HflK/C) domain family found in many proteins throughout all kingdoms of life. Membrane targeting of stomatins is mediated by a 20 amino-acid amino (N)-terminal membrane insertion domain featuring a conserved proline (Kadurin et al, 2009). An exception is STOML-2, which lacks this hairpin anchor. The N-and carboxy (C)-terminal regions of stomatins are cytoplasmic and unique for each family member (Salzer qet al, 1993;Seidel and Prohaska, 1998;Boute et al, 2000;Owczarek et al, 2001). Stomatin has been found to be phosphorylated (Salzer et al, 1993), palmitoylated (Snyers et al, 1999b) and to associate with lipid rafts (Snyers et al, 1999a) where it forms higher-order oligomers (Snyers et al, 1998). Furthermore, STOML-1 was shown to form heterooligomers with stomatin at endosomal membranes (Mairhofer et al, 2009). Stomatin can also form heterooligomers with STOML-3 in sensory neurons and this interaction requires the presence of the N-terminal membrane anchoring domain (Lapatsina et al, 2012b). Stomatin is absent in the erythrocyte membrane of patients suffering from a specific form of haemolytic anaemia (Stewart et al, 1992). Since the erythrocyte membrane of affected individuals is leaky to monovalent cations, it was proposed that stomatin regulates the activity of ion channels (Gallagher and Forget, 1995;Unfried et al, 1995). Subsequent studies, however, showed that loss of stomatin is not directly responsible for the observed membrane leakiness (Zhu et al, 1999;Fricke et al, 2003). A central function for the Caenorhabditis elegans stomatin homologue MEC-2 in touch sensation has been convincingly demonstrated (Huang et al, 1995). MEC-2 is present in a complex with the acidsensing ion channel (ASIC)-related, Na þ selective, MEC-4/ MEC-10 ion channel complex (Goodman et al, 2002;Cueva et al, 2007), and all three subunits of this complex are required to transduce mechanical signals in C. elegans (O'Hagan et al, 2005). When co-expressed with MEC-4/ MEC-10, MEC-2 increases the amplitude of amiloridesensitive currents 40-fold, suggesting a positive regulation (Goodman et al, 2002). Strikingly, mouse models lacking STOML-3 also show complete loss of mechanosensitivity in 40% of cutaneous mechanoreceptors , indicating a conserved function of stomatin family members in mammalian mechanosensation. In contrast, overexpression of mouse stomatin inhibits the amplitude of proton-gated currents in cells overexpressing mouse ASIC3 and increases the speed at which ASIC2a channels inactivate (Price et al, 2004).
No high-resolution structural data are available for the mammalian stomatin family, but the crystal structure of a homologue from Pyrococcus horikoshii (ph) has recently been determined (Yokoyama et al, 2008). The analysis of ph stomatin has revealed a crucial role for the stomatin domain in the formation of trimers and possibly higherorder oligomers. Interestingly, ASICs were also shown to have a trimeric channel architecture (Jasti et al, 2007;Gonzales et al, 2009), suggesting a symmetric interaction with a putative stomatin trimer. To understand the role of oligomerization in ion channel regulation, we have used X-ray crystallography to characterize mammalian stomatin and performed a structure-based analysis of its modulation of ASIC family members. Here, we demonstrate that, unlike in ph stomatin, mammalian stomatin forms a banana-shaped dimer and higher-order oligomers that are crucial for its regulation of ion channel function.

Structure of the stomatin dimer
A construct comprising the mouse stomatin domain (amino acids 86-213, stomatin 86-213 ; Figure 1A) was expressed in bacteria and purified to homogeneity. Crystals of this construct diffracted up to 2.4 Å . The phase problem was solved by molecular replacement using the ph stomatin domain as a search model, and the model was refined to an R work /R free of 21.7/26.8% (Table I).
Mouse stomatin 86-213 has a mixed a/b-fold ( Figure 1B; Supplementary Figure 1A). The N-terminal b-strand 1 is subdivided into b1a and b1b by a short loop and together with b2 and b3 forms an anti-parallel, curved b-sheet. Helices a2 and a4 extend in parallel and occupy the groove of the sheet, whereas the short a1 and a3 helices are oriented perpendicularly at both ends of the sheet. N-and C-termini are located at opposing sides of the molecule. The core of the mouse stomatin domain is very similar to that of ph stomatin (root mean square deviation (r.m.s.d.) of 1.5 Å along 107 aligned residues), with only minor deviations in a1, a2 and b1a ( Figure 1B). Ph stomatin harbours an additional unique b-strand (b0) at the N-terminus ( Figure 1B). The corresponding region in stomatin 86-213 forms a loop. b0 mediates trimerization of ph stomatin by forming an interstrand contact to the neighbouring molecule (Supplementary Figure 1B) (Yokoyama et al, 2008). Also, flotillin2 (pdb 1WIN) and the SPFH domain of the major vault protein (Tanaka et al, 2009) share a similar fold to the mouse stomatin domain (r.m.s.d. of 2 Å along 106 amino acids and of 2.6 Å along 109 aligned residues, respectively, Figure 1B, Supplementary Figure 1C).
The mouse stomatin domain dimerized in the crystal via the C-terminal four residues of b3 (amino acids 196-199), which form a symmetric intermolecular b-sheet with the opposing molecule burying 600 Å 2 surface area per molecule (Figure 2A and B). The interaction between the monomers features only main chain, no side chain interactions. The resulting stomatin dimer has a banana-shape, where a2-a4 form the outer and the b-sheet the inner surface of the banana. The N-termini are located at opposite ends of the dimerization interface.
To analyse the relevance of the stomatin dimer for assembly in solution, analytical ultracentrifugation (AUC) experiments were carried out ( Figure 2C). In these experiments, stomatin 86-213 was observed in a monomer-dimer equilibrium with a dissociation constant (K D ) of 37 mM. Also in analytical gel filtration experiments, coupled to a right-angle light scattering device, stomatin 86-213 showed a monomerdimer equilibrium ( Figure 2D). To exclude the possibility that the C-terminal truncation of the crystallized construct caused artificial dimer formation, we analysed a C-terminal extended construct, stomatin  , which corresponds in length to the crystallized trimeric ph stomatin construct. Stomatin 86-255 also eluted in a monomer-dimer equilibrium in analytical gel filtration ( Figure 2D), indicating that residues 214-255 of mouse stomatin do not influence oligomerization.
To test whether the dimer interface in the crystal corresponds to the observed dimer in solution, a mutagenesis approach was applied. Since the intermolecular b-sheet interface employed only main chain contacts, we initially sought   to disrupt the interface by electrostatic repulsion, using the V197D mutation, where the two negatively charged aspartate side chains from different monomers would directly oppose each other. However, this variant was insoluble. To disrupt b3 locally, we then introduced the V197P mutation into the stomatin 86-213 construct. In circular dichroism (CD) measurements, this mutant showed a very similar spectrum as stomatin  , indicating that the mutation did not grossly disturb the fold of the stomatin domain ( Figure 2E). In analytical ultracentrifugation, however, the V197P mutation completely prevented dimerization ( Figure 2C). Also in analytical gel filtration experiments, stomatin 86-213 V197P was mostly monomeric ( Figure 2D).
To analyse whether the V197P mutation can disrupt selfassociation of full-length stomatin (from here on described as stomatin) in living cells, we used Bimolecular Fluorescence Complementation (BiFC) (Hu et al, 2002). In this assay, protein-protein interactions are detected by monitoring the development of a fluorescent complex comprising the N-and C-terminal fragments of YFP (each fused to the C-terminal end of stomatin or a stomatin variant; Supplementary Figure 2). For stomatin, we previously showed a robust fluorescence signal development at the plasma membrane and in intracellular structures (Lapatsina et al, 2012b). BiFC stomatin constructs carrying the V197P mutation showed a substantially reduced fluorescence signal consistent with the idea that the protein does not efficiently oligomerize in cells (Supplementary Figure 2).
To determine the function of the stomatin dimer in the modulation of ASICs, electrophysiological recordings were performed. For this purpose, Chinese hamster ovary (CHO) cells were used because they are devoid of endogenous proton-gated ASIC-like currents (Garcia-Anoveros et al, 2001;Cadiou et al, 2007;Smith et al, 2007) and are easier to handle than other cells such as primary mouse fibroblasts (Supplementary Figure 3). In CHO cells overexpressing ASIC3, application of a low pH solution immediately evokes an inward current, which rapidly inactivates and is followed by a smaller, sustained current throughout the period of low pH stimulation ( Figure 3A). In agreement with previous data (Price et al, 2004), co-expression of stomatin with ASIC3 was associated with a large inhibition of ASIC3 peak current amplitude. Accordingly, we observed that at both pH 6 and pH 4, CHO cells co-expressing ASIC3 and stomatin had significantly smaller currents compared with ASIC3 alone ( Figure 3A). In contrast, stomatin V197P showed no inhibitory action on ASIC3 proton-gated currents. This effect was not caused by different expression levels, since stomatin and stomatin V197P were expressed at similar levels (Supplementary Figure 4A).
Using mCherry-tagged stomatin and eGFP-tagged ASIC3 constructs, stomatin appeared to co-localize with ASIC3 in intracellular structures and, to a lesser extent, at the plasma membrane (Supplementary Figure 4B Figure 4B). Also in BiFC assays, stomatin and the V197P mutant co-localized with ASIC3 at intracellular structures and at the plasma membrane (Supplementary Figure 5).
To unequivocally show a physical interaction between ASIC3 and stomatin, pull-down studies were performed ( Figure 3B). In agreement with previous data (Price et al, 2004; Lapatsina et al, 2012b), ASIC3 was co-immuno-precipitated with Myc-tagged stomatin and, to a similar extent, with the V197P mutant. These results indicate that dimerization of stomatin is not a prerequisite for a physical interaction with ASICs.
Stomatin is also known to accelerate the inactivation of ASIC2a proton-gated currents (Price et al, 2004), a phenomenon that we also observed using a shift to pH 5 ( Figure 3C), but not to pH 4 (Supplementary Figure 6). Unlike current amplitude, which is dependent on the amount of functional ion channels present in the plasma membrane, the inactivation time of ASIC2a-mediated currents is a mechanistic feature of ion channel gating. Therefore, faster ASIC2a inactivation times in the presence of stomatin are likely representative of a direct interaction between ASIC2a and stomatin or a modulation of the local membrane environment.
Similarly to the loss of inhibitory action upon ASIC3, stomatin V197P had no significant effect on the inactivation time of ASIC2a proton-gated currents ( Figure 3C; Supplementary Figure 6). Taken together, these experiments indicate that the functional minimal building block of eukaryotic stomatin involved in the modulation of ASIC2a and ASIC3 currents is a banana-shaped dimer.

A hydrophobic pocket is important for stomatin function
Analysis of the crystal packing revealed that Leu91 and Ile92 in the N-terminal loop preceding the stomatin domain protrude into a hydrophobic pocket at the concave face of an opposing stomatin 86-213 dimer ( Figure 4A). The hydrophobic pocket had a volume of 350 Å 3 and its inner surface was formed by the partially conserved residues Phe101, Tyr124, Val126, Ile136, Leu178 and Thr182 ( Figure 4B). This assembly resulted in a linear oligomeric structure, which was further intertwined with an opposing oligomer resulting in a double helical architecture (Supplementary Figure 7A). In a second crystal form, a similar double helical structure containing a stomatin 86-213 dimer was observed (Table I; Supplementary Figure 7B), and the hydrophobic pocket was also occupied by Leu91 and Ile92 of a neighbouring molecule.
To analyse the architecture of the pocket in the absence of Leu91 and Ile92, we mutated the two residues to alanine. According to the structure, this mutation completely removes the interaction site between the two dimers. Stomatin 86-213 LI91,92AA was crystallized and the structure solved by molecular replacement (Table I). Also in this crystal form 3, a banana-shaped dimer built via b3 was found, but the hydrophobic pocket was empty and greatly reduced in size (volume of 150 Å 3 ; Figure 4C). In particular, the binding site for Leu91 was completely occupied by Tyr124 and Asp140, whereas the cleft occupied by Ile92 in crystal form 1 and 2 was still present and even widened by a movement of Ile136 ( Figure 4B and C). Pocket closure of the Leu91-binding site was caused by rearrangements in a1, a2 and b2, which moved towards the pocket ( Figure 4B).
To test the importance of this pocket for the inhibition of ASICs, we introduced a mutation expected to close the pocket (T182W). In density gradient centrifugation experiments, a similar mutation, T182A, was shown not to alter oligomerization of human stomatin (Umlauf et al, 2006). Stomatin 86-213 T182W showed no apparent folding defect in CD measurements ( Figure 4D) and eluted as monomer/dimer in analytical gel filtration experiments ( Figure 4E), indicating that dimer formation is not affected by this mutation. When overexpressed in CHO cells, stomatin T182W showed similar expression levels as stomatin and displayed a similar cellular distribution when it was fluorescently tagged ( Supplementary  Figures 4 and 5). Furthermore, stomatin T182W still interacted with ASIC3 in pull-down experiments ( Figure 3B). However, in contrast to stomatin, stomatin T182W lost the ability to modulate proton-gated ASIC3 as shown in wholecell recordings ( Figure 4F). Furthermore, this T182W mutant no longer caused a significant decrease in the inactivation time of ASIC2a proton-gated currents at pH 5.0 ( Figure 4G). At pH 4.0, it even prolonged inactivation times (Supplementary Figure 6).
As expected from the crystal structure, the fold and dimer formation of stomatin 86-213 LI91,92AA was not affected ( Figure 4D and E). Also the cellular localization of fluorescently tagged stomatin LI91,92AA was comparable to stomatin ( Supplementary Figures 4 and 5). Furthermore, we still observed the typical modulation of ASIC3 and ASIC2a currents for stomatin LI91,92AA ( Figure 4F and G; Supplementary Figure 6). These results indicate that the hydrophobic pocket is crucial for the function of stomatin in inhibiting ASIC2 and ASIC3. However, Leu91 and Ile92 in the loop preceding the stomatin domain do not appear to be the physiological targets of this pocket for inhibition of ASIC function. Accordingly, the helical filaments observed in crystal form 1 and 2 have no apparent functional relevance.
The C-terminus of ASIC3 contains a Leu488, Leu489 motif resembling the N-terminal Leu91, Ile92 in stomatin ( Figure 5A). Assuming that these residues might interact with the stomatin hydrophobic pocket, we mutated both to aspartic acid (LL488,489DD) to disrupt such a putative interaction. The ASIC3 Leu488, Leu489 mutant still co-localized with stomatin at internal membranes and the plasma membrane ( Supplementary Figures 4 and 5). It also interacted with stomatin in pull-down assays (Supplementary Figure 8A), indicating that these two amino acids are not crucial for the interaction with stomatin. Interestingly, the LL488,489DD mutant showed a substantial increase in proton-gated transient and sustained current amplitudes ( Figure 5B). The regulation of this mutant by stomatin was partially lost: Transient amplitudes of this mutant at pH 6.0 were still affected by the presence of stomatin ( Figure 5C), but stomatin did not cause an effect on sustained current amplitudes at pH 6.0 or transient and sustained current amplitudes at pH 4.0 ( Figure 5C; Supplementary Figure 8). These data suggest that the C-terminus of ASIC3 is important for modulation by stomatin. Mutations in a second hydrophobic motif in the C-terminus of ASIC3 (VL470,471DD) resulted in a non-functional ASIC3 channel ( Figure 5B).

Higher-order oligomerization of stomatin
When analysing the crystal packing of crystal form 3 (Table I), we noticed that the stomatin 86-213 LI91,92AA dimers assembled in tubular structures via two conserved symmetric interfaces ( Figure 6A and B; Supplementary Figure 7C). Interface-1 has a buried surface area per molecule of 700 Å 2 ( Figure 6C). In particular, Trp185 and Leu145 form a hydrophobic surface at the periphery of interface-1, to which Leu109 of the opposing molecule binds. In the centre of interface-1, Arg152 forms a hydrogen bond with Thr149 of the opposing molecule. Interestingly, Trp184 in human stomatin corresponding to Trp185 in mouse stomatin has previously been shown to be important for oligomerization of stomatin (Umlauf et al, 2006). Interface-2 has a buried surface area of 300 Å 2 and features an interaction of the invariant Arg97 with a conserved surface-exposed amino acid triplet formed by Tyr123, Glu193 and Arg191 ( Figure 6C). (F) Electrophysiological recordings of CHO cells co-transfected with ASIC3 and the indicated stomatin constructs at pH 4.0 and pH 6.0, as described in Figure 3A. (G) Inactivation times for CHO cells transfected with ASIC2a and the indicated stomatin constructs as described in Figure 3C.
To test the relevance of these interfaces for the function of stomatin, mutagenesis studies were performed. To disrupt interface-1, we generated a stomatin 86-213 L109D,L145D mutant, which behaved like stomatin 86-213 in CD spectroscopy ( Figure 6D) and eluted as stomatin 86-213 in analytical gel filtration experiments ( Figure 6E). In CHO cells, stomatin L109D,L145D also showed similar expression levels compared with stomatin and localized similarly ( Supplementary  Figures 4 and 5). However, in whole-cell recordings, stomatin L109D,L145D failed to inhibit ASIC3 at pH 4.0 and peak current density was significantly greater than in the presence of stomatin ( Figure 6F). At pH 6.0, stomatin L109D,L145D had an intermediate inhibitory action; although peak current density was not significantly lower than the peak current density of ASIC3 expressed alone, it was also not significantly greater than the peak current density of ASIC3 co-expressed with stomatin ( Figure 6F). Disruption of interface-2 with stomatin R97D led to an intermediate phenotype so that ASIC3 proton-gated current amplitudes at both pH 4.0 and pH 6.0 were not significantly different from wild-type ASIC3 alone or ASIC3 co-expressed with stomatin ( Figure 6F). Interestingly, the R184C mutation in MEC-2 corresponding to Arg97 in mouse stomatin leads to reduced touch sensitivity (Chalfie and Sulston, 1981).
A combined triple mutant in both interfaces, stomatin 86-213 R97D, L109D, L145D, showed a similar CD spectrum compared with stomatin 86-213 ( Figure 6D) and a similar gel filtration profile ( Figure 6E). Stomatin R97D, L109D, L145D also co-localized with ASIC3 in CHO cells ( Supplementary  Figures 4 and 5) and could be co-immunoprecipitated with ASIC3 ( Figure 3B). However, this triple mutant failed to inhibit ASIC3 currents at both pH 4.0 and pH 6.0, suggesting a synergistic action of these two interfaces in the inhibition of ASIC currents. These data are consistent with a model whereby both interface-1 and interface-2 participate in higher-order stomatin oligomer formation in cells that can regulate ion channel activity.

Discussion
Stomatin-domain proteins exert powerful physiological effects on ion channels (Goodman et al, 2002;Price et al, 2004;Martinez-Salgado et al, 2007;Wetzel et al, 2007;Lapatsina et al, 2012a, b). Proton-gated ASIC3-mediated current amplitude is suppressed and ASIC2a current inactivation time is accelerated in the presence of stomatin (Price et al, 2004), and we have used this as an assay of stomatin function. By solving the core structure of the eukaryotic stomatin domain and performing a structurefunction analysis, we were able to define three key regions of the stomatin molecule necessary for its ability to modulate ASIC2a and 3. First, we show that the basic building block of stomatin is a banana-shaped dimer, which is formed by an intermolecular b-sheet at the C-terminus of the stomatin domain and is necessary for the modulation of ASICs. Second, we have identified a dynamic hydrophobic pocket in the stomatin domain, the closure of which prevents modulation of ASICs without affecting dimerization. This pocket can accommodate a dipeptide of two amino acids with branched aliphatic side chains (Leu, Ile). We have also (F) Electrophysiological recordings of CHO cells transfected with ASIC3 and the indicated stomatin constructs at pH 4.0 and pH 6.0, as described in Figure 3A.
Structure of the stomatin dimer J Brand et al provided evidence that a leucine-leucine dipeptide in the C-terminus of ASIC3 could contribute to channel modulation by stomatin. Finally, we found two conserved surfaces in the stomatin domain, which could be involved in the formation of ring-like structures. Disruption of the potential interaction sites in this oligomer rendered the stomatin molecule ineffective in modulating ASICs. This finding raises the interesting possibility that these two interfaces are used in plasma membrane-associated stomatin oligomers.
There is plentiful evidence that stomatin and other stomatin domain containing proteins can form higher-order oligomers (Snyers et al, 1998;Umlauf et al, 2006). These oligomers are thought to create microenvironments at membranes required for specific cellular functions, for example, the control of ion channels (Huber et al, 2006;Lapatsina et al, 2012b). In our crystal form 3, we observed ring-like stomatin oligomers with an outer diameter of 8 nm. Slight alterations in the assembly interfaces might lead to oligomers of different diameter in the cell. Furthermore, the C-terminal region of stomatin was shown to contribute to oligomerization (Umlauf et al, 2006). For cyanobacterial stomatin (Boehm et al, 2009) and yeast prohibitin (Tatsuta et al, 2005), ring-like oligomers were identified by singleparticle electron microscopy analysis, supporting the idea that stomatin-domain proteins may assemble at the membrane in ring-like structures with a banana-shaped dimer as the building block. Also most of the other SPFHdomain proteins, such as prohibitin, erlins and podocin localize to distinct membrane microdomains in the cell (Browman et al, 2007). Thus, our model of stomatin oligomerization might be relevant for many other SPFH domain containing proteins, although the SPFH domain can also be engaged in different oligomerization modes (Supplementary Figure 1B and C).
Striking parallels to the well-known membrane scaffolds of the BAR (Bin/Amphiphysin/Rvs) family are apparent. BARdomain also assemble into banana-shaped dimers with similar mM affinities to that shown for the stomatin dimer here (Peter et al, 2004;Frost et al, 2009). Furthermore, BARdomain dimers assemble via low affinity interactions into helical structures involving interactions at the tip and the lateral side of the dimer and may thus exert changes in membrane curvature, which often is supported by the insertion of N-terminal amphipathic helices (Shimada et al, 2007;Frost et al, 2008). The membrane insertion domain of stomatins might exert a similar function in targeting and concentrating stomatin at cellular membranes and creating membrane curvature. In the absence of this hydrophobic hairpin, the affinity for membranes and lipid droplets appears to be low and stomatin is cytoplasmic (Umlauf et al, 2004;Lapatsina et al, 2012b). Accordingly, the stomatin construct used in this study did not contain the membrane anchor and also did not bind to liposomes in vitro.
The activity of ASICs can be modulated by phosphorylation, and an interaction of their C-termini with the PDZ (PSD95, Dlg1 and zo-1)-and BAR-domain containing protein PICK1 (protein interacting with C-kinase1) is required for this modulation (Baron et al, 2002;Duggan et al, 2002;Leonard et al, 2003;Deval et al, 2004). Other PDZ-domain containing proteins, like PSD95 and Lin-7b, have also been shown to interact with the C-terminus of ASICs and can exert either positive or negative modulation of pH-gated currents (Hruska-Hageman et al, 2004). In these cases, it is thought that channel activity is primarily modulated by regulating the number of channels on the plasma membrane. In the present study, we have identified a C-terminal di-leucine motif in ASIC3, which appears to be involved in stomatin modulation without affecting ASIC3 membrane targeting. Mutation of the di-leucine motif to aspartic acid greatly increased current amplitudes without affecting the physical interaction with stomatin ( Figure 5). This suggests that stomatin primarily regulates functional properties of the ASIC3 channel via this di-leucine motif. Conversely, closure of the hydrophobic pocket in stomatin with the T182W mutation abolished stomatin's ability to negatively regulate ASIC3 (Figure 4).
MEC-2 is a positive regulator of mechanosensitive ion channels such as MEC-4 and MEC-10 (O'Hagan et al, 2005;Lapatsina et al, 2012a). The mechanosensitive ion channel targets of mammalian stomatin-domain containing proteins are, however, still unknown (Smith and Lewin, 2009;Lumpkin et al, 2010;Poole et al, 2011;Lapatsina et al, 2012a). The functional domains of the stomatin molecule identified here as essential for the negative regulation of ASICs may be equally important for the regulation of mechanotransduction. Interestingly, the conserved surface-exposed Arg97 at interface-2, is at an identical position to a mutant allele u64 of mec-2 that produces some touch insensitivity in worms (Chalfie and Sulston, 1981). Finally, hetero-dimerization of flotillins, and, possibly, higher-order oligomerization, induces membrane curvature and vesicle budding at the plasma membrane during clathrin-independent endocytosis (Frick et al, 2007). Thus, it is conceivable that the architecture of the ring-like stomatin oligomers is fundamental for an understanding of a wide range of different cellular functions involving SPFH-domain proteins.

Protein expression and purification
Constructs of mouse stomatin (amino acids 86-213, stomatin 86-213 , and amino acids 86-255, stomatin 86-255 ) and the indicated point mutants were expressed as N-terminal GST-fusion proteins in Escherichia coli BL21 DE3 phage resistant Rosetta (Novagen), including a PreScission protease cleavage site between the GST and stomatin constructs. In all constructs, Cys87 was mutated to serine to prevent oxidative cross-linking. Bacteria were grown to an OD 600 of 0.4 in TB medium when protein expression was induced with 80 mM IPTG, followed by overnight expression at 181C. Cells were resuspended in ice-cold lysis buffer (50 mM HEPES/NaOH, pH 7.5, 500 mM NaCl, 0.1 mM Pefabloc SC (Roth), 1 mM DNAse (Roth)) and lyzed using a microfluidizer (Microfluidics, Newton, USA). After centrifugation at 100 000 g for 45 min at 41C, the supernatant was applied on a GSH-column pre-equilibrated with lysis buffer and extensively washed with 50 mM HEPES/NaOH, pH 7.5, 500 mM NaCl. In the same buffer, overnight cleavage of the GST tag was performed at 41C in the presence of 250 mg PreScission protease which was directly added to the GSH beads. The protein was eluted, concentrated and further purified by size exclusion chromatography on a Superdex75 26/60 column pre-equilibrated with 10 mM HEPES/NaOH, pH 7.5, 150 mM NaCl. Peak fractions of the protein were pooled and concentrated to B30 mg/ml. The protein was flash-frozen in liquid nitrogen and stored at À 801C. For all constructs, the yield of purified protein was B1.5 mg/l bacteria culture.

Crystallization and structure determination
Before crystallization, proteins were diluted with 10 mM HEPES/ NaOH, pH 7.5, 150 mM NaCl. All crystallization trials were performed at 201C.
The sitting-drop vapour diffusion method in 96-well plate format was used to crystallize stomatin  . In all, 300 nl protein at 6 mg/ml was mixed with an equal volume of reservoir solution containing 50 mM cadmium sulphate, 1 M sodium acetate and 100 mM HEPES/NaOH, pH 7.5. After 6 days, hexagonal crystals appeared. Crystals were transferred in a cryo-solution containing 100 mM HEPES/NaOH pH 7.5, 150 mM NaCl, 50 mM cadmium sulphate, 1 M sodium acetate and 25% glycerol. All crystals were cryo-cooled by plunging them in liquid nitrogen.
Data sets of single crystals were recorded at beamline 14.1, BESSY II, Berlin, Germany with a Rayonics MX-225 CCD detector or at the microfocus setup at Swiss Light Source PX06SA beamline, equipped with a PILATUS M6 detector. Initial indexing and determination of an optimal data collection strategy was done using Mosflm (Leslie, 2006). Recorded intensities were integrated with the programme XDS (Kabsch, 2010) or HKL-2000 (Otwinowski and Minor, 1997). Molecular replacement was carried out using the programs MOLREP (Vagin and Teplyakov, 1997) via the CCP4 graphical interface version 6.0.2 (COLLABORATIVE COMPUTATIONAL PROJECT, 1994), using the ph stomatin domain as a search model. Figures were prepared using PyMOL (Schrödinger LLC, 2003). The plugin VASCo (Steinkellner et al, 2009) was used to illustrate the hydrophobic surfaces. The cavity was analysed using the CASTp server (Dundas et al, 2006). The size of the interfaces was calculated with the help of the PISA server (Krissinel and Henrick, 2007). The 3D figure was prepared with pymol and Adobe Acrobat 9 Pro Extended (Kumar et al, 2010). Use of Adobe Acrobat Reader 9.0 (or a higher version) is required to activate the 3D figure.

Circular dichroism
Protein samples were diluted to 0.2 g/l in a buffer containing 150 mM NaF and 10 mM Na 2 HPO 4 , pH7.4. CD measurements with three replicates were performed using a Chirascan spectrometer (Applied Photophysics) in the wavelength spectrum of 190240 nm. For data analysis, the web DICHROWEB server (Whitmore and Wallace, 2004) with the CDSSTR algorithm was used.

Right-angle light scattering
A coupled RALS-refractive index detector (Malvern) was connected in line to an analytical gel filtration column Superdex75 10/300 to determine absolute molecular masses of the applied proteins. Data were analysed with the provided OmniSec software. The running buffer contained 100 mM HEPES/NaOH, pH 7.5, 150 mM NaCl. For each protein sample, 100 ml of a 1.5 mg/ml protein solution was applied.

Analytical ultracentrifugation
Molecular mass studies of stomatin variants in 10 mM HEPES/ NaOH, pH 7.5, 150 mM NaCl were performed in an XL-A type analytical ultracentrifuge (Beckman) equipped with UV absorbance optics. Sedimentation equilibrium experiments were carried out using six-channel cells with 12 mm optical path length and the capacity to handle three solvent-solution pairs of about 70 ml liquid. Sedimentation equilibrium was reached after an equilibrium speed of 24 000 r.p.m. for about 30 h at 101C. The radial absorbance in each compartment was recorded at three different wavelengths between 270 and 290 nm, depending on the concentration used in the experiments. Molecular mass determinations employed the global fit of the three radial distributions using the programs POLYMOLE or POLYMOLA (Behlke et al, 1997). When proteins adopt a monomer-dimer equilibrium, the molecular mass, M, can be treated approximately as a weight average parameter (M w ). This value is a composite of the monomer molecular mass (M m ) and that of the dimer (M d ) and the partial concentrations of monomers, c m , and dimers, c d .
Therefore, the equilibrium constant, K d , can be derived with

Cellular localization
CHO cells were grown to a density of 60% in Dulbecco's Modified Eagle's Medium supplemented with 10% bovine serum albumin, 100 units/ml penicillin, 100 mg/ml streptomycin (Roth) and 4.5 mM glucose as supplement. Transfections with plasmids encoding mouse stomatin-mCherry and rat eGFP-ASIC3 were performed using Fugene (Roche). At 24 h post-transfection, cells were fixed with 5% paraformaldehyde at room temperature, washed with PBS, water and mounted on cover slides using Mowiol (Roth). Images were acquired using Leica SP5 confocal microscope and analysed using the ImageJ software.

Immunoprecipitation and immunoblotting
CHO cells were co-transfected with plasmids encoding Myc-Histagged stomatin or of the indicated stomatin variants and plasmids encoding FLAG-tagged ASIC3. After 24 h, cells were solubilized with lysis buffer containing 1% TritonX-100, 0.1% SDS, 10 mM Tris-HCl, pH 7.6, 150 mM NaCl, 100 mM 0.1 mM Pefabloc SC (Roth). Following ultracentrifugation at 100 000 g for 20 min at 41C, 500 mg of detergent-soluble protein was subjected to immunoprecipitation using anti-c-Myc (Millipore catalogue # 06-340) and protein A-Sepharose and then washed three times in lysis buffer. Proteins were eluted from the beads with SDS sample buffer (4% SDS, 0.4% Bromphenol blue, 40% glycerol, 200 mM Tris-HCl, pH 6.8) by incubation for 5 min at 601C and separated on SDS-PAGE. The proteins were transferred to nitrocellulose membrane and blocked by incubation in 5% milk powder in TBST (50 mM TRIS, pH 7.4, 150 mM NaCl, 0.1% Tween 20) for 1 h. Immunoblots were incubated with anti-polyhistidin-peroxidase conjugate (Sigma Aldrich Catalogue # A7058) or anti-FLAG (Sigma Aldrich Catalogue # F3165) antibodies o/n at 41C. The blots were washed three times with TBST in 5% milk powder in TBST and incubated with horseradish-peroxidase conjugate anti-mouse antibody and washed three times with TBST. Bound antibodies were detected using the enhanced chemiluminescence kit (Amersham). For expression tests, a monoclonal mouse antibody directed against the c-Myc tag (sc-40 9E10 Santa Cruz, California), or a-Tubulin (T 6793, Sigma-Aldrich, Munich) was used.

Bimolecular fluorescence complementation assays
BiFC was used to determine the localization of interaction between ASIC3 and stomatin variants in CHO cells. A construct of ASIC3 fused at its C-terminus to a C-terminal fragment of YFP was coexpressed with a construct of stomatin fused at its C-terminus with an N-terminal fragment of YFP according to Hu et al (2002). A fluorescent signal develops if the N-and C-terminal fragments of YFP come within 5 nm of each other. The BiFC signal was visualized 24 h after transfection in living cells using epifluorescent microscopy (Olympus IX71, Â 100 oil-immersion objective). Self-association of stomatin was detected by monitoring BiFC signal development over time. Human embryonic kidney (HEK) cells were transfected with BiFC constructs of stomatin or stomatin V197P fused at their C-termini with N-and C-terminal fragments of YFP. Eight hours after transfection, cells were transferred into a 384well plate and fluorescence development was monitored every 30 min for 16 h at 371C using a TECAN M1000 plate reader (imaging conditions: excitation: 515 nm, bandwidth 8 nm; emission: 535 nm, bandwidth 8 nm). The slope was determined by a linear fit to the increase in relative fluorescence intensity over time (excitation: 515 nm, bandwidth 8 nm; emission: 535 nm, bandwidth 8 nm).