FUNCTIONAL-CHARACTERIZATION AND LOCALIZATION OF PROTEIN PHOSPHATASE TYPE 2C FROM PARAMECIUM

me cloned a protein phosphatase 2C gene from Paramecium (PtPP2C), whic h codes for one of the smallest PP2C isoforms (Klumpp, S,, Hanke, C,, Donella-Deana, A., Beyer, A., Kellner, R., Pinna, L. A., and Schultz, J. E. (1994) J. Biol. Chem. 269, 32774-32780). After mutation of 9 cil iate Q codons (TAA) to CAA PtPP2C was expressed as an active protein i n Escherichia coli, The catalytic core region contains 284 amino acids as defined by C- and N-terminal deletions, The C terminus from amino acid 200-300 of PP2C isoforms has only about 20% similarity, To demons trate that the carboxy end is in fact needed for activity, we generate d an enzymatically active PtPP2C containing a C-terminally located tob acco etch virus-protease site. Upon proteolytic truncation enzyme acti vity was lost, i.e. the C terminus of PP2C is indispensable for enzyme activity. During these experiments isoleucine 214 was fortuitously id entified to be essential for PP2C catalysis, Mutation of the hydrophob ic amino acid to glycine in the ciliate or bovine isoforms resulted in inactive protein. Because Ile(214) is in a loop region without define d secondary structure, our data clearly go beyond the x-ray structure, The functional equivalence of the 180 amino acid long C terminus from the bovine PP2C with the 100 amino acid long carboxy end of the PtPP2 C was demonstrated by producing an active chimera, i,e, the PP2C from Paramecium has no obvious regions which may be specifically involved i n subcellular localization or substrate recognition. Using antibodies against recombinant PtPP2C we localized the enzyme by immunogold label ing in the cytosol and nucleus and very distinctly on the ciliary micr otubule/dynein complex, The data suggest a role for PtPP2C in the regu lation of dyneins, i,e, in cellular cargo transport and ciliary motili ty.