Table 7. Comparison of stomatin structural changes and functional consequences referring to wildtype.
| Domain / Motif | Mutant | Structural change | Change of functional characteristics | Conclusion |
|---|---|---|---|---|
| N-terminus | ΔN | Loss of N-terminal domain | Highly similar to WT in all characteristics; slow diffusion, strongly enhanced by cytoD. | Oligomerization and DRM-association as described [51], see structural model (S4 Fig). Interaction with actin cytoskeleton, as WT. |
| IM | Cys30Ser | Loss of palmitoylation | Affected oligomerization; Reduced lateral speed after cholesterol-depletion. | Suggests a structural change impairing oligomerization. Cholesterol-depletion creates a barrier to Cys30Ala diffusion. |
| Pro47Ser | Transmembrane glycoprotein | No PM-staining; no oligomer-formation; no DRM-association. | As to be expected from a normal transmembrane protein [40] (S4 Fig). | |
| CRAC/ CARC | Ile57Ala | Loss of CRAC consensus residue | Weak PM-staining, enhanced oligomerization, enhanced DRM-association. | Suggests that Ile-56 rather than Ile-57 is the real CRAC consensus residue. |
| Tyr60Ala | Loss of CRAC consensus residue | Weak PM-staining; affected oligomerization; aggregation/precipitation; no DRM-association. | Suggests a severe structural change. Loss of DRM-association is in line with a role of Tyr-60 in the CRAC consensus. | |
| Arg62Ala | Loss of CRAC consensus residue | No oligomerization; no DRM-association; large MF, strongly reduced by cholesterol-loading. | Loss of DRM-association is in line with a role of Arg-62 in the CRAC consensus. Large MF suggests low binding to cholesterol-rich membranes or cytoskeleton. | |
| PHB/ SPFH | Cys87Ser | Loss of palmitoylation | Unstable, largely degraded; no PM-staining; affected oligomerization; affected DRM-association. | In line with the role of Cys-87-palmitate being the major anchor for the PHB/SPFH domain to the PM [16] (S4 Fig). |
| Phe91Ala | Loss of putative head-to-head interaction site | Affected oligomerization; aggregation/precipitation; no DRM-association. | Suggests a role of Phe-91 in DRM-association. Tendency for aggregation suggests an unstable structure. | |
| Arg97Ala | Loss of prominent positive charge | No PM-staining; affected oligomerization. | In line with a role of Arg-97 as an interaction partner of (a) negatively charged PM component(s). | |
| Coiled-coil | ΔCC | Loss of coiled-coil domain | No oligomer-formation; strongly enhanced MF after cytoD-treatment. | Lack of oligomerization verifies the proposed function in oligomerization via coiled-coil interaction (S4 Fig). Enhanced MF after cytoA suggests binding to cytoskeleton. |
| Pro200Ala | Loss of structural fixpoint | Weak PM-staining; affected oligomerization; enhanced MF after cytoD-treatment. | Suggests a disability of the mutant to form oligomers via coiled-coil interaction due to higher flexibility (S4 Fig). Enhanced MF after cytoD suggests interaction with cytoskeleton. | |
| Pro245Ala | Loss of structural fixpoint | Weak PM-staining; affected oligomerization; reduced DRM-association; large MF, strongly reduced by cholesterol-loading; MF unchanged by cytoD. | Mutation effect suggests interference with ORA/CARC-binding to DRMs (S4 Fig). Pro-245 is an essential residue for downstream interactions with cortical actin cytoskeleton. | |
| ORA/ CARC | ΔC | Loss of C-terminal domain | No oligomers; no DRM-association; largest MF, unchanged by cytoD; fastest diffusion; low cholesterol-binding (Table 1). | ORA characteristics as described [51] (S4 Fig). FRAP data indicate that the C-terminus is essential for actin-cytoskeleton-binding. Lack of DRM-association is in line with low cholesterol-binding. |
| Phe269Ala | Loss of crucial aromatic residue | No oligomers; reduced DRM-association; reduced cholesterol-binding (Table 1). | ORA characteristics as described [51], except for partial DRM-association that suggests a tag-effect. Reduced DRM-association is in line with reduced cholesterol-binding. | |
| Pro270Ala | Loss of structural fixpoint | No PM-staining; affected oligomerization; enhanced DRM-association; enhanced cholesterol-binding (Table 1). | ORA characteristics as described [51]. Enhanced DRM-association is in line with stronger cholesterol-binding. |
PM, plasma membrane; DRM, detergent-resistant membrane; ΔN, N-terminal deletion; ΔC, C-terminal deletion; ΔCC, coiled-coil deletion; IM, intramembrane domain; CRAC, cholesterol recognition/interaction amino acid consensus sequence; CARC, reverse CRAC motif; PHB, prohibitin homology domain; SPFH, stomatin-prohibitin-flotillin-HflK/C domain; ORA, oligomerization and lipid raft association domain; MF, mobile fraction.