Proteomic analysis of the membrane skeleton of Tetrahymena thermophila

Abstract

The membrane skeleton of the Tetrahymena is likely to determine many aspects of the cell surface structure of this ciliated protozoan. The availability of the genomic sequence of Tetrahymena thermophila has facilitated a proteomic analysis of the non-microtubular portion of the cortical cytoskeleton of Tetrahymena thermophila. Potassium iodide-Triton X-100 insoluble residues of these cells retain the cell surface organization of this ciliate, while being depleted in microtubular proteins. Proteins in these membrane skeletal residues have been subsequently fractionated into distinct sets by means of selective extraction and precipitation steps. We have initiated a proteomic analysis of one group of membrane skeletal proteins: the set containing epiplasmin C and associated proteins. Bands containing each of the specific proteins were excised from preparative SDS-polyacrylamide gels. These proteins were reduced, alkylated and digested in-gel with trypsin with the resulting enzymatic peptide fragments analyzed by LC-MS using electrospray ionization. The observed peptide molecular weights were used to search a database of predicted Tetrahymena proteins in order to identify the protein in the original gel band. Further confirmation was obtained by sequencing several enzymatically-derived peptides in the mass spectrometer. Two major components of these structures were identified – epiplasmin C and epiplasmin A – both of which are predicted to be coiled coil proteins. The other major protein to be identified was TCBP-25, an EF-hand containing calcium-binding protein. Several other minor proteins were identified, including epiplasmin B protein, another coiled coil protein. Intriguingly, among the minor proteins present in these preparations were proteins predicted to be related to lipid or protein kinases. Another minor protein was found to be related to a centromeric calcium-binding protein. These minor proteins suggest the possibility of the linkage of these cytoskeletal proteins to cellular signaling processes. We are currently working to analyze protein-protein interactions within the membrane skeleton by means of non-denaturing gel electrophoresis of protein complexes, as well as far-Western blotting experiments.