Identification of isoforms of the exocytosis-sensitive phosphoprotein PP63/parafusin in Paramecium tetraurelia and demonstration of phosphoglucomutase activity.
PP63 (parafusin) is a 63 kDa phosphoprotein which is very rapidly (within 80 ms) dephosphorylated (to P63) during triggered trichocyst exocytosis; this occurs selectively in exocytosis-competent Paramecium tetraurelia strains. In the present work, two cDNAs coding for PP63/parafusin have been isolated, one of which is a new isoform. These isoforms are 99.6% identical and are derived from two different genes. Similarity searches revealed 43-51% identity of the deduced amino acid sequences with known phosphoglucomutases from yeast and mammals. The sequences of two proteolytic peptides obtained from PP63/parafusin isolated from Paramecium are identical to parts of the amino acid sequence deduced from the major cDNA. The major cDNA was mutated from the macronuclear ciliate genetic code into the universal genetic code and expressed in Escherichia coli. The recombinant protein shows the same biochemical and immunological characteristics as the (P)P63/parafusin originally isolated from Paramecium. It has the same specific phosphoglucomutase activity as phosphoglucomutase from chicken muscle. We also show that recombinant P63-1 parafusin 1 is a substrate of an endogenous casein kinase from Paramecium, as is the originally isolated P63/parafusin. Polyclonal antibodies against recombinant P63-1/parafusin 1 were raised which recognized phosphoglucomutases from different sources. Thus we show that PP63/parafusin and phosphoglucomutase in Paramecium are identical.
Hauser
K
K
Fakultät für Biologie, Lehstuhl für Zellbiologie und Ultrastrukturforschung, Universität Konstanz, P.O. Box 5560, D-78434, Kontstanz, Federal Republic of Germany.
Kissmehl
R
R
Linder
J
J
Schultz
J E
JE
Lottspeich
F
F
Plattner
H
H
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000276:immunology; D001483:Base Sequence; D002645:Chickens; D003001:Cloning, Molecular; D003429:Cross Reactions; D018076:DNA, Complementary / Q000737:chemistry; D008969:Molecular Sequence Data; D016805:Paramecium tetraurelia; D010733:Phosphoglucomutase / Q000276:immunology / Q000378:metabolism*; D010750:Phosphoproteins / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism; D015800:Protozoan Proteins / Q000737:chemistry* / Q000378:metabolism; D011817:Rabbits; D016415:Sequence Alignment
D000906:Antibodies; D018076:DNA, Complementary; D010750:Phosphoproteins; D015800:Protozoan Proteins; C105860:parafusin-2; C087273:parafusin protein, Paramecium; D010733:Phosphoglucomutase