PURIFICATION AND CHARACTERIZATION OF AN ALPHA(1)BETA(2) ISOFORM OF CAPZ FROM HUMAN ERYTHROCYTES - CYTOSOLIC LOCATION AND INABILITY TO BIND TO MG2-FILAMENTS ARE CAPPED BY ADDUCIN( GHOSTS SUGGEST THAT ERYTHROCYTE ACTIN)

CapZ (''capping protein'') is a heterodimeric actin capping protein th at blocks actin filament assembly and disassembly at the fast growing (barbed) filament ends and is proposed to function in regulating actin filament dynamics as well as in stabilizing actin filament lengths in muscle and nonmuscle cells. We show here that erythrocytes contain a nonmuscle isoform of capZ (EcapZ) that is present exclusively in the c ytosol and is not associated with the short actin filaments in the ery throcyte membrane skeleton. This is unlike other cell types where capZ is associated with cytoskeletal actin filaments and suggests that cyt osolic EcapZ may be inactive, or alternatively, that the barbed ends a re capped by adducin, a membrane skeleton protein that was shown recen tly to cap actin filament barbed ends in vitro [Kuhlman, P. A., Hughes , C. A., Bennett, V., & Fowler, V. M. (1996) J. Biol. Chem. 271, 7986] . To distinguish between these possibilities, we purified EcapZ from e rythrocyte cytosol and characterized its biochemical and functional pr operties. Two-dimensional gel electrophoresis and western blotting rev eals the EcapZ subunit composition to be alpha(1) beta(2), as describe d for capZ from many other nonmuscle cells, with no evidence for postt ranslational modifications. Purified EcapZ is fully functional in bloc king actin elongation from barbed filament ends (K-cap similar to 1-5 nM) as well as in nucleating actin polymerization. Furthermore, cytoso lic EcapZ binds to actin filament barbed ends, indicating that sequest ering of EcapZ by a cytosolic inhibitory factor or insufficient amount s of EcapZ in cytosol also cannot account for its absence from the mem brane skeleton. To test directly whether the barbed ends of the erythr ocyte actin filaments were already capped, we measured binding of puri fied EcapZ to isolated membranes. Purified EcapZ does not cosediment w ith membranes prepared by hypotonic lysis in the presence of magnesium , suggesting that the barbed ends of the erythrocyte actin filaments a re capped under these conditions but not by EcapZ. In contrast, purifi ed EcapZ stoichiometrically reassociates with all the actin filament b arbed ends in membranes prepared by hypotonic lysis in 5 mM sodium pho sphate, pH 8.0 (5P8), conditions in which the barbed filament ends wer e previously reported to be uncapped. Comparison of the amounts of add ucin associated with membranes prepared in the presence and absence of magnesium reveals that 60-80% of the adducin dissociates from the mem brane during hemolysis and washing in 5P8 buffer, suggesting that the barbed ends become artifactually uncapped due to loss of adducin. The erythrocyte actin filaments may thus represent a specialized class of membrane-associated actin filaments that are capped by adducin instead of capZ.