IDENTIFICATION OF ISOFORMS OF THE EXOCYTOSIS-SENSITIVE PHOSPHOPROTEINPP63 PARAFUSIN IN PARAMECIUM-TETRAURELIA AND DEMONSTRATION OF PHOSPHOGLUCOMUTASE ACTIVITY/

PP63 (parafusin) is a 63 kDa phosphoprotein which is very rapidly (wit hin 80 ms) dephosphorylated (to P63) during triggered trichocyst exocy tosis; this occurs selectively in exocytosis-competent Paramecium tetr aulelia strains. In the present work, two cDNAs coding for PP63/parafu sin have been isolated, one of which is a new isoform, These isoforms are 99.6% identical and are derived from two different genes. Similari ty searches revealed 43-51% identity of the deduced amino acid sequenc es with known phosphoglucomutases from yeast and mammals. The sequence s of two proteolytic peptides obtained from PP63/parafusin isolated fr om Paramecium are identical to parts of the amino acid sequence deduce d from the major cDNA. The major cDNA was mutated from the macronuclea r ciliate genetic code into the universal genetic code and expressed i n 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 k inase from Paramecium, as is the originally isolated P63/parafusin. Po lyclonal antibodies against recombinant P63-1/parafusin 1 were raised which recognized phosphoglucomutases from different sources. Thus we s how that PP63/parafusin and phosphoglucomutase in Paramecium are ident ical.