Biochemical studies of the excitable membrane of Paramecium tetraurelia VI. Endogenous protein substrates for in vitro and in vivo phosphorylation in cilia and ciliary membranes.

Abstract

The endogenous protein kinases of isolated P. tetraurelia cilia phosphorylated .apprx. 30 ciliary polypeptides in vitro. Labeling with [.gamma.-32P]ATP was not proportional to the amount of each protein in cilia; some minor polypeptides (e.g., 67,000 and 180,000 MW) were more heavily labeled than some major polypeptides. Certain of the endogenous substrates for protein kinase were localized in the ciliary membrane (130,000, 86,000, 67,000 and 45,000 MW); others were found in axonemes or in both fractions. With cilia from bacterized cultures in the undefined Cerophyl medium, the labeling of specific endogenous phosphate acceptors was altered by pH, cAMP and cGMP, but the labeling pattern was unaffected by the presence of Na+ or K+ (15 mM), Ba++ (5 mM), Ca++ (10-5 or 10-4 M) or EGTA [ethylene glycol bis (.beta.-amino ethyl ether) N,N,N'',N''-tetraacetic acid]. Very similar results were obtained with cilia from cells grown axenically in a semidefined medium; the MW and the extent of phosphorylation of the phosphopolypeptides were comparable to those of cilia from bacterized Cerophyl cultures, although no significant cyclic nucleotide effects were observed in the axenic cilia. Most of the phosphopolypeptides labeled in vitro also turned over rapidly in vitro. The phosphoprotein phosphatase responsible for turnover was partially inhibited by 5 mM NaF. The pattern of ciliary polypeptides labeled in vivo was similar to that observed in the in vitro experiments, although the relative intensities of labeling differed. Behavioral mutants [6] of Paramecium, known to have defects in the excitable membrane that regulates the ciliary beat, showed normal patterns of ciliary protein phosphorylation in vitro, with and without added cyclic nucleotides, at both pH 6.0 and pH 8.0. The mutants also had apparently normal phosphoprotein phosphatase. The Paranoiac A mutant showed a reduction in cGMP-stimulated protein kinase activity.