Abstract
After incubation of intact living cultured rat cerebellar granule cells at 37 degrees C with a new GM1 ganglioside analog, carrying a diazirine group and labeled with (125)I in the ceramide moiety, followed by photoactivation, a relatively small number of radiolabeled proteins were detected in a membrane-enriched fraction. A protein of about 55 kDa with a pI of about 5 carried a large portion of the radioactivity even if incubation and cross-linking were performed at 4 degrees C and in the presence of inhibitors of endocytosis, suggesting that it is cross-linked at the plasma membrane. Immunoprecipitation and Western blotting experiments showed the positivity of this protein for tubulin. Trypsin treatment of intact cells ruled out the involvement of a plasma membrane surface tubulin. Release of radioactivity from cross-linked tubulin after KOH treatment (but not hydroxylamine treatment) suggested that the photoactivated ganglioside reacts with an ester-linked fatty acid anchor of tubulin. Low buoyancy, detergent-resistant membrane fractions, isolated from cells after incubation with the GM1 analogue and photoactivation, proved their enrichment in endogenous and radioactive GM1 ganglioside, sphingomyelin, cholesterol, signal transduction proteins, and tubulin. It is noteworthy that radioactive tubulin was also detected in this fraction, indicating the presence of tubulin molecules carrying a fatty acid anchor in detergent-resistant, ganglioside-enriched domains of the plasma membrane. Parallel experiments carried out with a phosphatidylcholine analogue, also carrying a diazirine group and labeled with (125)I in the fatty acid moiety, showed the specificity of tubulin interaction with GM1. Taken together, these results indicate that some tubulin molecules are associated with a lipid anchor to detergent-resistant glycolipid-enriched domains of the plasma membrane. This novel feature of membrane domains can provide a key for a better understanding of their biological role.
Highlights
Gangliosides are amphiphilic components of the plasma membrane of vertebrate cells and are known to participate in a number of cell surface-mediated events, such as cell-cell recognition processes, modulation of cell growth and differentiation, receptor function, and membrane-mediated transfer of information [1,2,3,4,5]
Parallel experiments carried out with a phosphatidylcholine analogue, carrying a diazirine group and labeled with 125I in the fatty acid moiety, showed the specificity of tubulin interaction with GM1. These results indicate that some tubulin molecules are associated with a lipid anchor to detergent-resistant glycolipid-enriched domains of the plasma membrane
This wide range of functions is likely attributable to their membrane topology. They are asymmetrically located in the outer leaflet of the plasma membrane bilayer, with the oligosaccharide moiety exposed toward the external medium and the ceramide portion embedded in the hydrophobic core of the bilayer
Summary
Gangliosides are amphiphilic components of the plasma membrane of vertebrate cells and are known to participate in a number of cell surface-mediated events, such as cell-cell recognition processes, modulation of cell growth and differentiation, receptor function, and membrane-mediated transfer of information [1,2,3,4,5]. This wide range of functions is likely attributable to their membrane topology They are asymmetrically located in the outer leaflet of the plasma membrane bilayer, with the oligosaccharide moiety exposed toward the external medium and the ceramide portion embedded in the hydrophobic core of the bilayer. We used this approach in order to identify proteins interacting with gangliosides in neurons, in which these glycosphingolipids are abundant [24], but in which the role of domains has been only partially investigated [13] For this purpose, cultured rat cerebellar granule cells were utilized, coupled with the use of a new GM1 ganglioside analog, TID-GM1,1 carrying a photoactivable diazirine group and labeled with 125I in the ceramide moiety
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