Functional characterization and localization of protein phosphatase type 2C from Paramecium.
We cloned a protein phosphatase 2C gene from Paramecium (PtPP2C), which 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 ciliate Q codons (TAA) to CAA PtPP2C was expressed as an active protein in 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 demonstrate that the carboxy end is in fact needed for activity, we generated an enzymatically active PtPP2C containing a C-terminally located tobacco etch virus-protease site. Upon proteolytic truncation enzyme activity was lost, i.e. the C terminus of PP2C is indispensable for enzyme activity. During these experiments isoleucine 214 was fortuitously identified to be essential for PP2C catalysis. Mutation of the hydrophobic amino acid to glycine in the ciliate or bovine isoforms resulted in inactive protein. Because Ile214 is in a loop region without defined 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 PtPP2C was demonstrated by producing an active chimera, i.e. the PP2C from Paramecium has no obvious regions which may be specifically involved in subcellular localization or substrate recognition. Using antibodies against recombinant PtPP2C we localized the enzyme by immunogold labeling in the cytosol and nucleus and very distinctly on the ciliary microtubule/dynein complex. The data suggest a role for PtPP2C in the regulation of dyneins, i.e. in cellular cargo transport and ciliary motility.
Grothe
K
K
Pharmazeutisches Institut der Universität Tübingen, D-72076 Tübingen, Germany.
Hanke
C
C
Momayezi
M
M
Kissmehl
R
R
Plattner
H
H
Schultz
J E
JE
D000595:Amino Acid Sequence; D000818:Animals; D002384:Catalysis; D002417:Cattle; D002923:Cilia / Q000201:enzymology; D003062:Codon; D004398:Dyneins / Q000502:physiology; D015723:Gene Library; D006801:Humans; D008870:Microtubules / Q000201:enzymology; D008969:Molecular Sequence Data; D010247:Paramecium / Q000201:enzymology*; D010749:Phosphoprotein Phosphatases / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D016415:Sequence Alignment
D003062:Codon; D011993:Recombinant Fusion Proteins; D010749:Phosphoprotein Phosphatases; D004398:Dyneins