Stomatin in Lipid Rafts

Stomatin was originally identified as one of the major proteins of the red cell membrane, however, it is also found in virtually every human tissue. Because it is widely associated with secretory granules, phagosomes, and various endosomes, we think that stomatin is essential for the function of these vesicles. There are patients with a rare hereditary anaemia (Overhydrated Hereditary Stomatocytosis, OHSt), whose red cells lack stomatin and show a high K+/Na+-permeability. For this reason, it was postulated that stomatin may function as an ion channel regulator. Our own results indicate that stomatin plays a role as a membrane-bound, oligomeric, structural protein, which is associated with cholesterol- and sphingolipid-rich membrane domains (lipid rafts). This classification as a lipid raft protein implicates an important role for stomatin as a signal transduction component. In the project "Stomatin in Lipid Rafts" we plan to investigate in detail the role of stomatin in the lipid raft complexes. For this, we shall use biochemical, biophysical, molecular biological and cell biological methods. Our preliminary data indicate that various specific raft populations exist independently. The study of stomatin-specific rafts has only just begun. In this project, the basic characteristics of stomatin-rafts will be determined, that is the biosynthesis, protein and lipid composition, stability, and role in signal transduction. Important questions will be, whether stomatin interacts with other lipid raft proteins and lipids, and with cytoskeletal components, respectively. In addition, the prerequisites to the formation and organization of these stomatin-rafts will be investigated, primarily the regulation of oligomerization, palmitoylation, and phosphorylation of stomatin. As a medically relevant topic, we discuss a probable interaction partner of stomatin as a candidate for Overhydrated Hereditary Stomatocytosis.

In the project P15486 "Stomatin in Lipid Rafts" we have investigated some important aspects of the membrane protein stomatin in the context of cholesterol- and sphingolipid-rich membrane domains (lipid rafts). Stomatin has been described as a major protein of erythrocyte lipid rafts and therefore we analysed these extensively. During this work we found that lipid rafts are associated with the cytoskeleton and that the association depends on the cholesterol level in the cell membrane. Several results that we obtained from different blood samples suggest that the cholesterol dependence of the cytoskeleton-lipid raft association is also valid in living organisms. However, these results are preliminary and have to be validated by a larger study of patient blood samples. In another study, we analysed the enrichment of stomatin in membrane vesicles that are formed and shed from the membrane under different circumstances like elevated calcium levels in the cell, ageing of red cells in the circulation, and storage of blood bags under blood banking conditions. The mechanism of this enrichment is not known but it could be due to the particular structure of stomatin and its capability to form high molecular complexes (oligomers). We investigated stomatin also in other cell types like blood platelets, neutrophils, and epithelial cells. It is interesting that in these cells stomatin is not so much located at the plasma membrane but rather at internal vesicles. These vesicles also contain lipid rafts. During a study of fluorescently labelled stomatin in living epithelial cells, we found an association with lipid droplets/bodies. In a cooperation with the Univ. Linz we used a high sensitivity video microscopy system to study the multiple interactions of stomatin-positive vesicles with positive lipid bodies. These interactions suggest that stomatin may play a role in lipid transport and/or the regulation of lipid metabolism. The proteomic analysis of lipid bodies isolated from stomatin-positive and negative cells did not show a clear difference, however, the result itself was surprising enough and showed that lipid bodies are not merely lipid storage droplets but play an active role in lipid metabolism. In an extensive mutation analysis of stomatin we identified a domain that is essential for the formation of the oligomeric complexes. Within this domain we identified 3 amino acids that are necessary for lipid raft association. Our finding that several mutants were not able to form oligomers but nevertheless associated with lipid rafts led us to the conclusion that oligomerization is not a prerequisite to lipid raft association. One deletion mutant that lacked the ability to oligomerize and associate with lipid rafts was nevertheless expressed at the apical membrane of epithelial cells. It is not clear which signal is necessary for the apical targeting of stomatin, however, it is neither oligomerization nor lipid raft association, two conditions that were previously thought to be important for apical sorting.