Membrane-microfilament interactions in ascites tumor cell microvilli. Identification and isolation of a large microfilament-associated membrane glycoprotein complex.
[14C]Glucosamine metabolic labeling and concanavalin A blots were used to identify four major glycoprotein species associated with ascites tumor cell microvillar microfilament cores and with a transmembrane complex containing actin. Phalloidin shift analysis of glucosamine-labeled microvilli showed that glycoproteins of 110-120, 80, 65, and 55 kDa are stably associated with the microfilament cores. Analysis of large (greater than 10(6) kDa) transmembrane complexes from microvillar membranes made under microfilament-depolymerizing conditions (Carraway, C. A. C., Jung, G., and Carraway, K. L. (1983) Proc. Natl. Acad. Sci. U. S. A. 80, 430-434) revealed glycoproteins of the same Mr values, showing the same relative staining or labeling patterns as those observed with the microfilament cores. Gel filtration of high salt, high pH extracts of intact microvilli, microfilament cores, or transmembrane complexes showed that in all of these fractions the glycoproteins are associated in a very large, stable complex. The glycoprotein multimer was isolated essentially free of actin and other components by Sephacryl S-1000 chromatography of microvilli, microvillar membranes prepared at pH 11, microfilament cores, or transmembrane complex fractions in Triton X-100, 1 M KCl, glycine, pH 9.5. Purified glycoprotein complex bound actin when incubated under polymerizing conditions. The presence of the glycoprotein heteromultimer in both microfilament cores and transmembrane complex from isolated membranes and the association of the purified glycoprotein complex with actin are consistent with our hypothesis that the glycoprotein-containing transmembrane complex is an association site for microfilaments at the plasma membrane.
Carothers Carraway
C A
CA
Department of Biochemistry and Molecular Biology, University of Miami School of Medicine, Florida 33101.
Fang
H
H
Ye
X H
XH
Juang
S H
SH
Liu
Y C
YC
Carvajal
M E
ME
Carraway
K L
KL
D000199:Actins / Q000378:metabolism; D000230:Adenocarcinoma / Q000378:metabolism; D000818:Animals; D001201:Ascites / Q000378:metabolism; D015153:Blotting, Western; D002850:Chromatography, Gel; D003208:Concanavalin A; D004591:Electrophoresis, Polyacrylamide Gel; D005944:Glucosamine / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000302:isolation & purification / Q000378:metabolism*; D008840:Microfilament Proteins / Q000737:chemistry / Q000378:metabolism*; D008871:Microvilli / Q000737:chemistry / Q000378:metabolism*; D010590:Phalloidine; D051381:Rats; D014407:Tumor Cells, Cultured
D000199:Actins; D008562:Membrane Glycoproteins; D008840:Microfilament Proteins; D003208:Concanavalin A; D010590:Phalloidine; D005944:Glucosamine