title: Ca2+ Regulation of Trypanosoma brucei Phosphoinositide Phospholipase C.
abstract: We characterized a phosphoinositide phospholipase C (PI-PLC) from the procyclic form (PCF) of Trypanosoma brucei. The protein contains a domain organization characteristic of typical PI-PLCs, such as X and Y catalytic domains, an EF-hand calcium-binding motif, and a C2 domain, but it lacks a pleckstrin homology (PH) domain. In addition, the T. brucei PI-PLC (TbPI-PLC) contains an N-terminal myristoylation consensus sequence found only in trypanosomatid PI-PLCs. A peptide containing this N-terminal domain fused to green fluorescent protein (GFP) was targeted to the plasma membrane. TbPI-PLC enzymatic activity was stimulated by Ca(2+) concentrations below the cytosolic levels in the parasite, suggesting that the enzyme is constitutively active. TbPI-PLC hydrolyzes both phosphatidylinositol (PI) and phosphatidylinositol 4,5-bisphosphate (PIP2), with a higher affinity for PIP2. We found that modification of a single amino acid in the EF-hand motif greatly affected the protein's Ca(2+) sensitivity and substrate preference, demonstrating the role of this motif in Ca(2+) regulation of TbPI-PLC. Endogenous TbPI-PLC localizes to intracellular vesicles and might be using an intracellular source of PIP2. Knockdown of TbPI-PLC expression by RNA interference (RNAi) did not result in growth inhibition, although enzymatic activity was still present in parasites, resulting in hydrolysis of PIP2 and a contribution to the inositol 1,4,5-trisphosphate (IP3)/diacylglycerol (DAG) pathway.
authors.affiliation: Center for Tropical and Emerging Global Disease, University of Georgia, Athens, Georgia, USA Department of Cellular Biology, University of Georgia, Athens, Georgia, USA.
authors.forename: Sharon
authors.initials: S
authors.lastname: King-Keller
authors.affiliation: Center for Tropical and Emerging Global Disease, University of Georgia, Athens, Georgia, USA Department of Cellular Biology, University of Georgia, Athens, Georgia, USA.
authors.forename: Christina A
authors.initials: CA
authors.lastname: Moore
authors.affiliation: Center for Tropical and Emerging Global Disease, University of Georgia, Athens, Georgia, USA Department of Cellular Biology, University of Georgia, Athens, Georgia, USA.
authors.forename: Roberto
authors.initials: R
authors.lastname: Docampo
authors.affiliation: Center for Tropical and Emerging Global Disease, University of Georgia, Athens, Georgia, USA Department of Cellular Biology, University of Georgia, Athens, Georgia, USA smoreno@uga.edu.
authors.forename: Silvia N J
authors.initials: SN
authors.lastname: Moreno
mesh_terms: D002118:Calcium / Q000378:metabolism*; D002462:Cell Membrane / Q000201:enzymology; D055785:Gene Knockdown Techniques; D010716:Phosphatidylinositols / Q000378:metabolism; D054801:Phosphoinositide Phospholipase C / Q000235:genetics / Q000378:metabolism*; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism; D014346:Trypanosoma brucei brucei / Q000201:enzymology*
chemical_list: D010716:Phosphatidylinositols; D010727:Phosphoric Diester Hydrolases; D054801:Phosphoinositide Phospholipase C; C021491:glycerophosphoinositol glycerophosphodiesterase; D002118:Calcium