Characterization of Cytoplasmic Factors Which Complement Ca²⁺ Channel Mutations in Paramecium tetraurelia

Abstract

The analysis of Ca^{2 +}-channel function in the single-celled eukaryote Paramecium can be extended to a biochemical level based on recent observations that transfer of cytoplasm from wild-type cells into mutants lacking Ca^{2 +} -channel function (“pawn” mutants) causes the mutant cells to regain Ca^{2 +} -channel activity. Using a convenient behavioral assay for Ca^{2 +} -channel function, we have used microinjection of cytoplasmic fractions into mutant cells to enrich for and characterize those components from wild-type cytoplasm which can “cure” cells carrying mutations in the 3 different pawn genes affecting Ca²⁺ -channel activity (pwA,pwB, and pwC). In each case, the curing factor appears to be a protein component of an intracellular membrane. They are distinguishable on the basis of thermal, pH and divalent ion sensitivities. In addition, the factor curing IhepwC mutational defect has been purified more than 180-fold. Furthermore, thepwB curing activity appears to be amplified during sequential transfer between pwB cells.

Key Words:

• Ca^{2 +} channel
• behavioral mutants
• microinjection
• Paramecium tetraurelia