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J Biol Chem, Vol. 275, Issue 14, 9978-9985, April 7, 2000
From the Department of Experimental, Environmental Medicine and
Biotechnologies, Medical School, University of Milano-Bicocca,
Hospital S. Gerardo, 20052 Monza, Italy, the ¶ Department of
Medical Chemistry and Biochemistry and the § Institute
of Veterinary Physiology and CH-8092 Biochemistry, University of
Milano, 20133 Milano, Italy, and the After incubation of intact living cultured rat
cerebellar granule cells at 37 °C with a new GM1 ganglioside analog,
carrying a diazirine group and labeled with 125I 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 °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 125I 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.
Tubulin Anchoring to Glycolipid-enriched, Detergent-resistant
Domains of the Neuronal Plasma Membrane*
,
, and Eidgenössische
Technische Hochschule, Zurich, Switzerland
*
This work was supported by Grant Cofin 1998 from Ministero
dell'Università e della Ricerca Scientifica e Tecnologica (Rome, Italy) (to M. M.) and Grant CT98.00488.CT04.115.33097 from the CNR
(Rome, Italy) (to P. P.).The costs of publication of this article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: Medical School,
University of Milano-Bicocca, via Saldini 50, 20133 Milano, Italy. Tel.: 39-02-70645238; Fax: 39-02-70645254; E-mail: paola.palestini@ unimib.it.
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