Interaction in situ of the cytoskeletal protein vinculin with bilayers studied by introducing a photoactivatable fatty acid into living chicken embryo fibroblasts.
The cytoskeletal protein vinculin, a putative actin--plasma-membrane linker, has been shown by hydrophobic photo-labeling to interact in vitro directly with bilayers of acidic phospholipids [Niggli et al. (1986) J. Biol. Chem. 261, 6912-6918]. In order to demonstrate that such an interaction occurs also in intact cells, chicken embryo fibroblasts were incubated for 2 h with a 3H-labeled photoactivatable fatty acid, 11-(4-[3-(trifluoromethyl)-diazirinyl]phenyl)-[2-3H]undecanoic acid. This resulted in biosynthetic incorporation into cellular lipids of a fraction of the fatty acid added. Following photolysis, vinculin was immunoprecipitated from different subcellular fractions using a specific polyclonal anti-vinculin antibody. The protein was recovered from both the cytosolic and the crude membrane fraction. Vinculin from both fractions incorporated label, but the membrane-associated population was at least eight times more strongly photolabeled than the cytosolic protein. Moreover, photolysis increased only labeling of the membrane-bound but not of the cytosolic protein. These results suggest that the direct interaction of vinculin with the hydrophobic core of the phospholipid layer observed in vitro may also be relevant in intact cells, and may be involved in its function as a linker protein.
Niggli
V
V
Department of Biochemistry, University of Basel, Switzerland.
Sommer
L
L
Brunner
J
J
Burger
M M
MM
D000818:Animals; D002458:Cell Fractionation; D002478:Cells, Cultured; D002642:Chick Embryo; D003598:Cytoskeletal Proteins / Q000378:metabolism*; D005227:Fatty Acids / Q000378:metabolism*; D005347:Fibroblasts / Q000378:metabolism; D008051:Lipid Bilayers; D008055:Lipids / Q000096:biosynthesis / Q000302:isolation & purification; D008715:Methionine / Q000378:metabolism; D010782:Photolysis; D013347:Subcellular Fractions / Q000378:metabolism; D013462:Sulfur Radioisotopes; D014316:Tritium; D016596:Vinculin
D003598:Cytoskeletal Proteins; D005227:Fatty Acids; D008051:Lipid Bilayers; D008055:Lipids; D013462:Sulfur Radioisotopes; D014316:Tritium; C062882:11-(4-(3-(trifluoromethyl)diazirinyl)phenyl)undecanoic acid; D016596:Vinculin; D008715:Methionine