Biochemical and Genetic Evidence for the Presence of Multiple Phosphatidylinositol- and Phosphatidylinositol 4,5-Bisphosphate-Specific Phospholipases C in Tetrahymena

Abstract

, hydrolyze PtdIns and glycosyl-PtdIns, and they are considered important virulence factors. All unicellular eukaryotes studied so far contain a single PI-PLC-like gene. In this report, we show that ciliates are an exception, since we provide evidence that Tetrahymena species contain two sets of functional genes coding for both bacterial and eukaryotic PLCs. Biochemical characterization revealed two PLC activities that differ in their phosphoinositide substrate utilization, subcellular localization, secretion to extracellular space, and sensitivity to Ca 2؉ . One of these activities was identified as a typical membrane-associated PI-PLC activated by low-micromolar Ca 2؉ , modestly activated by GTP␥S in vitro, and inhibited by the compound U73122 [1-(6-{[17␤-3-methoxyestra-1,3,5(10)-trien-17-yl]amino}hexyl)-1H-pyrrole-2,5-dione]. Importantly, inhibition of PI-PLC in vivo resulted in rapid upregulation of PtdIns(4,5)P 2 levels, suggesting its functional importance in regulating phosphoinositide turnover in Tetrahymena. By in silico and molecular analysis, we identified two PLC genes that exhibit significant similarity to bacterial but not trypanosomal PLC genes and three eukaryotic PI-PLC genes, one of which is a novel inactive PLC similar to proteins identified only in metazoa. Comparative studies of expression patterns and PI-PLC activities in three T. thermophila strains showed a correlation between expression levels and activity, suggesting that the three eukaryotic PI-PLC genes are functionally nonredundant. Our findings imply the presence of a conserved and elaborate PI-PLC-Ins(1,4,5)P 3 -Ca 2؉ regulatory axis in ciliates.Phosphoinositides (PIs), i.e., phosphatidylinositol (PtdIns) and its phosphorylated derivatives, are ubiquitous constituents of eukaryotic membranes, with many and diverse functions in receptor signaling, membrane trafficking, and cytoskeleton organization. Among PI-metabolizing enzymes, phospholipases C (PLCs) that hydrolyze PtdIns and phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P 2 ] are of particular importance because they generate second messengers such as inositol 1, 4,5-trisphosphate [Ins(1,4,5)P 3 ] and participate in the replenishment of the inositol pool available for phosphoinositide synthesis. These phospholipases fall into distinct families: (i) the well-established family of eukaryotic phosphoinositide-specific PLCs (PI-PLCs) (EC 3

Signalling in ciliates: long- and short-range signals and molecular determinants for cellular dynamics

“…Constitutive activity explains why other efforts to monitor InsP 3 increase during stimulation have failed. Formation of InsP 3 generally requires a phospholipase C (PLC) which, in ciliates, occurs in different isoforms (Klöppel et al, ; Leondaritis et al, ) some of which may fulfill this task. For comparison, phospholipase C subfamilies β, γ, δ, ϵ, η and ζ occur in animals and only type ζ in plants (Munnik & Testerink, ), whereas orthologs in ciliates are addressed in Section IX.1.…”

Evolutionary Cell Biology of Proteins from Protists to Humans and Plants

“…From bacterial precursors and from a small number of PL‐C paralogs (Leondaritis et al. ), only two appear GPI‐specific—a situation which is even less clear in mammals (Müller et al. ; Staudt et al.…”

Intracellular calcium channels in protozoa

“…This is not surprising considering the largely aberrant pharmacology of ciliates (Plattner et al , 2009) and the absence of any evolutionary pressure for this plant toxin. Also the inhibitory effect of Li + on phenomena related to IP 3 in Paramecium (Ladenburger et al , 2006) are no stringent argument considering its pleiotropic effects, whereas the usefulness of compound U73122 as an inhibitor of phosphoinositide-specific phospholipase C in ciliates (Leondaritis et al , 2011) has not yet been known at the time of those analyses.…”