Structural and functional analysis of stomatin and stomatin-like proteins

In this project, the structure and function of the integral membrane protein stomatin was investigated by biochemical, molecular and cell biological methods. Our results show that stomatin has structural determinants which allow its association with membrane microdomains, the socalled "lipid rafts". Lipid rafts are characterized by a specific, cholesterol- and sphingolipid-rich composition, and by their association with acylated proteins, like glycosyl-phosphatidylinositol (GPI)-linked proteins and Src-family kinases. A special type of lipid rafts are caveolae, invaginations of the plasma membrane, which contain caveolin-1 as a major coat protein. Stomatin has many features in common with caveolin-1, like a long hydrophobic domain with cytoplasmic orientation ("hairpin loop"), oligomerization, palmitoylation, detergent insolubility, and low, buoyant density, however, stomatin is not associated with caveolae but with another type of lipid rafts. We studied the subcellular distribution of stomatin in epithelial, endothelial, and haematopoietic cells, using immunofluorescence and immunoelectron microscopy. In epithelial cells, we identified two, roughly equal pools of stomatin expression, the plasma membrane, particularly membrane protrusions, and juxtanuclear vesicles, which co-localized with a marker for late endosomes. When we aggregated GPI-linked proteins at the plasma membrane, they were endocytosed and co-localized with stomatin, implicating that stomatin is a coat protein of a specific subset of endosomes. In haematopoietic cells, we found stomatin mainly on cytoplasmic granules and little in the plasma membrane. Whereas stomatin was identified on all types of neutrophil granules, it was specifically associated with the alpha-granules of resting platelets. It is known that, upon activation, these granules fuse with the plasma membrane and expel their contents. We found that activated platelets contain a higher amount of stomatin in the plasma membrane, suggesting a role for stomatin in granule targeting and/or fusion. These granules contain lipid rafts, with stomatin as a major raft component. Interestingly, we found a striking difference in stomatin subcellular localization in polarized and non-polarized epithelial cells. In non-polarized cells, stomatin was mainly expressed in juxtanuclear vesicles, whereas in polarized cells, it was predominantly expressed at the apical surface. This finding suggests that these stomatin- coated vesicles may be targeted to the apical membrane upon polarization and that stomatin may play a role in the targeting and/or fusion process, as discussed for the granules of haematopoietic cells. Another interesting aspect of stomatin function was revealed upon Ca2+-induced vesiculation of red cells. After induction, we found that the shed microvesicles contained lipid rafts, with highly enriched stomatin and the GPI- linked protein acetylcholine esterase. This result implicates a role for stomatin in membrane blebbing and, again, in membrane fusion. The enrichment of stomatin is specific, because similar raft proteins, like flotillin-1 and -2 are excluded. In summary, these results clearly show that stomatin is a specific lipid raft-associated protein residing in a subset of rafts, different from those containing caveolins and/or flotillins. Starting from this basis, we will be able to proceed and study the specific microdomains in detail, thus contributing to the general discussion about lipid raft assembly and organization.