Raft and cytoskeleton associations of an ABC transporter: P-glycoprotein.
BACKGROUND
A novel flow cytometric assay has been described in an accompanying report (Gombos et al.,


METHODS
The kinetics of the decrease in immunofluorescence intensity was analyzed after the addition of the raft-preserving Triton X-100 or Nonidet P-40, both of which disrupt the entire membrane. Mild treatments by both detergents leave cells attached to only those proteins that are anchored to the cytoskeleton by rafts or independent of rafts. Agents that affect microfilaments and modulate membrane levels of cholesterol by cyclodextrin were used to distinguish between the raft-mediated and non-raft-related associations of the Pgp. Confocal microscopy and flow cytometric fluorescence energy transfer measurements were used to confirm colocalization of Pgp with raft constituents.


RESULTS
The assay was proved to be sensitive enough to resolve differences between the resistance of UIC2-labeled cell-surface Pgps to Triton X-100 versus Nonidet P-40. Approximately 34% of the UIC2 Fab-labeled Pgp molecules were associated with the cytoskeleton through detergent-resistant, cholesterol-sensitive microdomains or directly, whereas approximately 15% were found to be directly linked to the cytoskeleton. Accordingly, confocal microscopy showed that Pgps colocalize with raft markers, mainly in microvilli. Fluorescence resonance energy transfer efficiency data indicating molecular proximity between Pgp and the raft markers CD44, CD59, and G(M1)-gangliosides also suggested that a significant fraction of Pgps resides in raft microdomains. Raft association of Pgp appears to be of functional significance because its modulation markedly affected drug pumping.


CONCLUSIONS
By using the flow cytometric detergent resistance assay in kinetic mode, we were able to assess the extent of raft association and actin cytoskeleton anchorage of Pgp expressed at physiologically relevant levels. We demonstrated that a significant fraction of Pgp is raft associated on LS-174-T human colon carcinoma cells and that this localization may influence its transporter function. The kinetic flow cytometric detergent resistance assay presented in this report is considered to be generally applicable for the analysis of molecular interactions of membrane proteins expressed at low levels.
Bacso
Zsolt
Z
Department of Biophysics and Cell Biology, University of Debrecen, Medical Faculty, Research Centre for Molecular Medicine, Debrecen, Hungary.
Nagy
Henrietta
H
Goda
Katalin
K
Bene
László
L
Fenyvesi
Ferenc
F
Matkó
János
J
Szabó
Gábor
G
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000502:physiology*; D018528:ATP-Binding Cassette Transporters / Q000502:physiology*; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry; D001692:Biological Transport; D018957:CD59 Antigens / Q000096:biosynthesis; D045744:Cell Line, Tumor; D002784:Cholesterol / Q000378:metabolism; D003505:Cyclodextrins / Q000494:pharmacology; D003599:Cytoskeleton / Q000378:metabolism*; D003902:Detergents / Q000494:pharmacology; D005434:Flow Cytometry; D005452:Fluoresceins / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D005677:G(M1) Ganglioside / Q000378:metabolism; D006801:Humans; D018960:Hyaluronan Receptors / Q000096:biosynthesis; D007700:Kinetics; D021962:Membrane Microdomains / Q000378:metabolism / Q000502:physiology*; D051379:Mice; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D041681:NIH 3T3 Cells; D017830:Octoxynol / Q000494:pharmacology; D021381:Protein Transport; D013997:Time Factors
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D018528:ATP-Binding Cassette Transporters; D000911:Antibodies, Monoclonal; D018957:CD59 Antigens; D003505:Cyclodextrins; D003902:Detergents; D005452:Fluoresceins; D018960:Hyaluronan Receptors; D005677:G(M1) Ganglioside; D017830:Octoxynol; D002784:Cholesterol; C007740:fluorexon