Conditional expression of glycosylphosphatidylinositol phospholipase C in Trypanosoma brucei.
Trypanosoma brucei glycosylphosphatidylinositol phospholipase C (GPIPLC) is expressed in the bloodstream stage of the life cycle, but not in the procyclic form. It is capable of hydrolyzing GPI-anchored proteins and phosphatidylinositol (PI) in vitro. Several roles have been proposed for GPIPLC in vivo, in the release of variant surface glycoprotein during differentiation or in the regulation of GPI and PI levels, but none has been substantiated. To explore GPIPLC function in vivo, tetracycline-inducible GPIPLC gene (GPIPLC) conditional knock-out bloodstream form and tetracycline-inducible GPIPLC-expressing procyclic cell lines were constructed. We were unable to generate GPIPLC null mutants. Cleavage of GPI-anchored proteins was abolished in extracts from uninduced conditional knock-outs and was restored upon induction. Despite the barely detectable level of GPIPLC activity in uninduced conditional knock-out bloodstream forms, their growth was not affected. GPI-protein cleavage activity could be induced in procyclic cell extracts, up to wild-type bloodstream levels. Myo-[3H]inositol incorporation into [3H]inositol monophosphate was about 14-fold lower in GPIPLC conditional knock-out bloodstream forms than in the wild type. Procyclic cells expressing GPIPLC showed a 28-fold increase in myo-[3H]inositol incorporation into [3H]inositol monophosphate and a 1.5-fold increase in [3H]inositol trisphosphate levels, suggesting that GPIPLC may regulate levels of inositol phosphates, by cleavage of PI and phosphatidylinositol 4,5-bisphosphate.
Ochatt
C M
CM
Laboratory of Molecular Parasitology, The Rockefeller University, New York, NY 10021, USA.
Bütikofer
P
P
Navarro
M
M
Wirtz
E
E
Boschung
M
M
Armah
D
D
Cross
G A
GA
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D017931:DNA Primers / Q000235:genetics; D017353:Gene Deletion; D015971:Gene Expression Regulation, Enzymologic; D018390:Gene Targeting; D017125:Genes, Protozoan; D007294:Inositol / Q000378:metabolism; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D013752:Tetracycline / Q000494:pharmacology; D014346:Trypanosoma brucei brucei / Q000201:enzymology* / Q000235:genetics* / Q000254:growth & development; D010738:Type C Phospholipases / Q000235:genetics*
D017931:DNA Primers; D007294:Inositol; D010738:Type C Phospholipases; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D013752:Tetracycline