A biochemical approach to stomatin function

Stomatin is an ancient membrane protein conserved from archaea to humans. This suggests important functions for this protein, however, they are still unclear. Stomatin localizes to the cytoplasmic face of cellular membranes, binds cholesterol, and forms high molecular complexes in cholesterol-rich membrane microdomains, also known as lipid rafts. Moreover, it interacts with various ion channels and the glucose transporter GLUT1 and thus modulates their activities, with cholesterol playing a crucial role. Due to these characteristics and in analogy to the topologically similar protein, caveolin, it is assigned to the integral scaffolding proteins. Because of new results on the structure and function of stomatin, it is now possible to ask detailed questions on the molecular level regarding the relevance of stomatin structure, high molecular complex formation, cholesterol- binding, and lipid raft association, for membrane organization and the interaction with GLUT1. In this project we plan to investigate these aspects primarily with biochemical and proteomic methods. However, the in vitro results will be complemented by in vivo single-molecule microscopic studies in collaboration with the Institute of Biophysics in Linz (Prof. Gerhard Schütz). During this investigation we will study the role of stomatin palmitoylation, cholesterol-binding, association with negatively charged membrane phospholipids, association with cytoskeletal proteins and lipid raft components. Moreover, we want to continue our studies on the stomatin- GLUT1 interaction that have already revealed a role for stomatin as a switch for GLUT1 specificity from glucose to dehydroascorbate (DHA) in human erythrocytes. In these cells, glucose transport is depressed and DHA transport enhanced leading to better utilization of vitamin C thus compensating the lack of vitamin C synthesis in humans. The functional studies complementing the biochemical stomatin-GLUT1 interaction data will be performed within a collaboration with the group of Naomi Taylor in Montpellier (F). In summary, this project will shed light on the function of stomatin in membrane organization and in GLUT1 regulation.

This project on the biochemical investigation of stomatin function used straight-forward methods to exchange putative, crucial positions in this molecule, to express the mutated proteins in an appropriate human cell line, and to analyze these mutants by microscopic and biochemical methods. A major topic of this project was the question, whether stomatin and which of its domains would interact with the glucose transporter GLUT1 and may cause depression of its activity. GLUT1 was reported to be depressed by stomatin-association in a cholesterol-dependent fashion. Technical problems and lack of manpower hampered this subproject and therefore we focused on chemical cross-linking studies to identify the associated protein(s). This approach was successful and we identified GLUT1/SLC2A1 in isolated stomatin-complexes from erythrocyte membranes. Interestingly, several other membrane proteins were also identified, particularly the anion exchanger band 3/SLC4A1 and the water channel aquaporin-1. Moreover, the iron transporter ferroportin-1/SLC40A1, the urea transporter-1/SLC14A1, nucleoside transporter/SLC29A1, the calcium-pump/ Ca-ATPase-4, the integrin-associated protein CD47, and flotillin-1 and -2 were identified as stomatin-interacting proteins. These data suggest that stomatin may play a role as an integral scaffolding protein like the homologous flotillins and topologically similar caveolins. To prove this hypothesis, the identified transporters may now be tested in presence and absence of stomatin and their activity measured. Moreover, the influence of membrane lipid composition, particularly cholesterol and sphingolipid levels, may be studied in future to elucidate the function of stomatin-dependent membrane rafts.Cell biological and biochemical analyses of the selected point and deletion mutants, respectively, largely confirmed our hypotheses regarding stomatin oligomerization and association with membrane rafts. Particularly the C-terminus of stomatin was crucial for both oligomerization and raft-association. Truncation of this domain dramatically increased the mobility of this protein in the membrane, independent of cholesterol-levels. On the other hand, treatment of wild-type stomatin-expressing cells with cytoskeleton-disrupting reagents also led to high mobility. Similarly, a point mutation of a C-terminal proline residue also resulted in a cholesterol-independent, high-mobility mutant. These results suggest that the C-terminus of stomatin is involved in the interaction with cytoskeletal components.