Biochemical characterization of ezrin-actin interaction.
The highly related actin isoforms are thought to have different functions. We recently demonstrated a polarized distribution of actin isoforms in gastric parietal cells and association of gastric ezrin with the cytoplasmic beta-actin isoform (Yao, X., Chaponnier, C., Gabbiani, G., and Forte, J. G. (1995) Mol. Biol. Cell. 6, 541-557). Here we used ultrastructural immunocytochemistry to verify that beta-actin is located within canalicular microvilli and the apical cortex of parietal cells, similar to the localization reported for ezrin. Furthermore, we tested whether ezrin binds preferentially to cytoplasmic beta-actin compared with the skeletal muscle alpha-actin isoform. Purified cytoplasmic beta-actin (from erythrocytes) and skeletal alpha-actin were assembled with gastric ezrin. Co-sedimentation experiments showed that gastric ezrin selectively co-pelleted with the beta-actin isoform and only very poorly with alpha-actin. Binding of erythrocytic beta-actin to ezrin is saturable with a molar ratio of approximately 1:10 (ezrin:actin) and a dissociation constant approximately 4.6 x 10(-8) M. In addition, ezrin promoted pyrene-labeled actin assembly, with predominant effects on filament elongation and a distinct preference for beta-actin compared with alpha-actin. Given these isoform-selective associations, we speculate that actin isoforms might segregate into different functional domains and exert specificity by interacting with isoform-orientated binding proteins.
Yao
X
X
Department of Molecular and Cell Biology, University of California, Berkeley, 94720, USA.
Cheng
L
L
Forte
J G
JG
D000199:Actins / Q000378:metabolism*; D000818:Animals; D003598:Cytoskeletal Proteins; D007150:Immunohistochemistry; D016253:Microscopy, Immunoelectron; D018482:Muscle, Skeletal / Q000378:metabolism / Q000648:ultrastructure; D010295:Parietal Cells, Gastric / Q000378:metabolism / Q000648:ultrastructure; D010750:Phosphoproteins / Q000378:metabolism*; D011485:Protein Binding; D011817:Rabbits; D000099149:Ezrin
D000199:Actins; D003598:Cytoskeletal Proteins; D010750:Phosphoproteins; D000099149:Ezrin