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 VSG 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 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 PIP2. (C) 1999 Elsevier Science B.V. All rights