Immunological Characterization of Membrane Skeletal Proteins in Tetrahymena pyriformis

The cell cortex (or pellicle) of Tetrahymena is composed of three closely associated layers: the cell membrane, the alveolar sacs, and the epiplasm. The epiplasm is the innermost layer of the cortex and is thought to be analogous to the membrane skeleton found in eukaryotic cells, since it consists largely of filamentous proteins responsible for maintaining cell stability and shape. Furthermore, the epiplasrnic layer provides a means by which the cell can regulate the location and distribution of structures along its surface. Low salt extraction of Triton-potassium iodide (TKI) cortical residues is an efficient means by which three membrane skeletal proteins (band A (235 kDa), band C (125 kDa), and a 23 kDa protein) can be isolated. Previous immunofluorescence studies have localized bands A and C in the cell cortex and irnrnunoblots have demonstrated that these proteins are major components of the membrane skeleton in Tetrahymena. In this study, polyclonal antibodies were raised in rabbits against three membrane skeletal proteins isolated by low salt extraction of TKI cortical residues: band A and C, and a 23 kDa protein. Immunoblots were performed to characterize the number and size of immunoreactive proteins in total cellular protein as opposed to previous studies which only probed cortical proteins. Furthermore, immunoblots were performed to test the specificity of each antisera prepared against specific proteins in an attempt to clarify apparently contradictory data obtained by previous studies using both polyclonal and monoclonal antibodies. Polyclonal antibodies against band A did not detect the presence of band A at 235 kDa, but did detect three bands between 100 and 140 kDa. Affinity-purified band C antibodies were demonstrated to identify a 125 kDa protein from whole-cell lysates, and no bands above 125 kDa, as previous studies have recognized. Interestingly, the 23 kDa polyclonal antibody recognized not only a 23 kDa protein from the total cellular protein, but a high molecular weight protein (above 125 kDa) as well. munological Characterization of Membrane Skeletal Proteins in Tetrahymena pyrvormis GL