Isolation of an immunoreactive analogue of brain fodrin that is associated with the cell cortex of Dictyostelium amoebae

Abstract

We have used a polyclonal affinity-purified antibody made against chicken brain fodrin (both 240 and 235 Kd subunits) as a probe to determine if a fodrinlike protein exists in amoebae of Dictyostelium discoideum. In Western blots of whole cells and the isolated cell cortex, polypeptides measuring 220 and 70 Kd are recognized by the fodrin antibodies. In situ localization by indirect immunofluorescence with antifodrin indicates that the immunoreactive polypeptides are cortical. The immunoreactive analogues copatch and cocap with concanavalin A. At the level of resolution of the electron microscope, immunocytochemistry with antifodrin and colloidal gold confirms that the immunoreactive analogues are cortical proteins associated with microfilaments on the cytoplasmic side of the plasma membrane. We have isolated and characterized the 220 Kd protein to determine if it is similar to fodrin and to investigate its relationship to the 70 Kd polypeptide. The 220 Kd protein can be extracted from the cortex in the absence of detergent and isolated by gel filtration and sucrose density gradient sedimentation. The 220 Kd is a rod-shaped protein 118 +/- 17.8 nm (N = 37) in length. It has a sedimentation coefficient of 9.3 S and Stokes' radius of 13 nm and exists as a dimer of approximately 500,000 daltons (Mr). Isolated 220 Kd binds to actin filaments in vitro when assayed by rotary shadowing. Morphological criteria distinguish 220 Kd from Dictyostelium myosin II heavy chain (215 Kd) and the filaminlike protein at 240 Kd. The 70 Kd polypeptide appears to be a cleavage fragment of the 220 Kd, since it is found after prolonged storage when formerly only the 220 Kd was present. Furthermore, the 220 and 70 Kd polypeptides exhibit similar one-dimensional peptide maps when treated with TPCK trypsin. On the basis of its physical and immunoreactive characteristics, and location in the cell, the 220 Kd may be a fodrinlike protein.