Tubulin anchoring to glycolipid-enriched, detergent-resistant domains of the neuronal plasma membrane

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 I-125 in the ceramide moiety, followed by photoactivation, a relatively small number of radiolabeled proteins mere detected in a memb rane-enriched fraction. A protein of about 55 kDa with a pI of about 5 carr ied a large portion of the radioactivity even if incubation and cross-linki ng were performed at 4 degrees C and in the presence of inhibitors of endoc ytosis, suggesting that it is cross-linked at the plasma membrane. Immunopr ecipitation and Western blotting experiments showed the positivity of this protein for tubulin, Trypsin treatment of intact cells ruled out the involv ement of a plasma membrane surface tubulin. Release of radioactivity from c ross-linked tubulin after I(OH treatment (but not hydroxylamine treatment) suggested that the photoactivated ganglioside reacts with an ester-linked f atty acid anchor of tubulin, Low buoyancy, detergent-resistant membrane fra ctions, isolated from cells after incubation with the GM1 analogue and phot oactivation, proved their enrichment in endogenous and radioactive GM1 gang lioside, sphingomyelin, cholesterol, signal transduction proteins, and tubu lin. It is noteworthy that radioactive tubulin was also detected in this fr action, indicating the presence of tubulin molecules carrying a fatty acid anchor in detergent-resistant, ganglioside-enriched domains of the plasma m embrane, Parallel experiments carried out with a phosphatidylcholine analog ue, also carrying a diazirine group and labeled with I-125 in the fatty aci d moiety, showed the specificity of tubulin interaction with GM1. Taken tog ether, these results indicate that some tubulin molecules are associated wi th a lipid anchor to detergent-resistant glycolipid-enriched domains of the plasma membrane. This novel feature of membrane do mains can provide a key for a better understanding of their biological role.