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J. Biol. Chem., Vol. 277, Issue 47, 44731-44739, November 22, 2002
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From the Centro de Investigaciones en Química
Biológica de Córdoba, Departamento de Química
Biológica, Facultad de Ciencias Químicas, Universidad
Nacional de Córdoba, Ciudad Universitaria,
Córdoba 5000, Argentina
Glycosylphosphatidylinositol
(GPI)-anchored proteins are clustered mainly in
sphingolipid-cholesterol microdomains of the plasma membrane. The
distribution of GPI-anchored fusion yellow fluorescent protein
(GPI-YFP) in the plasma membrane of Chinese hamster ovary (CHO)-K1
cells with different glycolipid compositions was investigated. Cells
depleted of glycosphingolipids by inhibiting glucosylceramide synthase
activity or cell lines expressing different gangliosides caused by
stable transfection of appropriate ganglioside glycosyltransferases or
exposed to exogenous GM1 were transfected with GPI-YFP cDNA. The
distribution of GPI-YFP fusion protein expressed at the plasma membrane
was studied using the membrane-impermeable cross-linking agent
bis(sulfosuccinimidyl)suberate. Results indicate that GPI-YFP forms
clusters at the surface of cells expressing GM3, or cells depleted of
glycolipids, or transfected cells expressing mainly GD3 and GT3, or GM1
and GD1a, or mostly GM2, or highly expressing GM1. However, no
significant changes in membrane microdomains of GPI-YFP were detected
in the different glycolipid environments provided by the membranes of
the cell lines under study. On the other hand, wild type CHO-K1 cells
exposed to 100 µM GM1 before cross-linking with
bis(sulfosuccinimidyl)suberate showed a dramatic reduction in the
amount of GPI-YFP clusters. These findings clearly indicate that
manipulating the glycolipid content of the cellular membrane, just by
changing the ganglioside biosynthetic activity of the cell, did not
significantly affect the association of GPI-YFP on the cell surface of
CHO-K1 cells. The effect of exogenous GM1 gangliosides on GPI-YFP
plasma membrane distribution might be a consequence of the ganglioside
level reached in plasma membrane and/or the effect of particular
ganglioside species (micelles) that lead to membrane architecture
and/or dynamic modifications.
Effect of Gangliosides on the Distribution of a
Glycosylphosphatidylinositol-anchored Protein in Plasma Membrane from
Chinese Hamster Ovary-K1 Cells*
,
*
This work was supported in part by Grants 194/00 and 82/01
from Secretaría de Ciencia y Técnología
(SECyT)-Universidad Nacional de Córdoba and Ramon
Carrillo-Arturo Oñativia from Ministerio de Salud de la
Nación Argentina.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.
Fellow of Ramon Carrillo-Arturo Oñativia from Ministerio de
Salud de la Nación Argentina.
§
Fellow of Consejo Nacional de Investigaciones
Científicas y Técnicas (CONICET).
¶
Career Investigator of Consejo Nacional de Investigaciones
Científicas y Técnicas (CONICET). To whom correspondence
should be addressed. E-mail:
daniotti@dqb.fcq.unc.edu.ar.
Copyright © 2002 by The American Society for Biochemistry and Molecular Biology, Inc.
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