trans

248 INTRACELLULAR SIGNALING
cycle. Exogenous Ca 2 potentiates the release
of InsP 3 from membranes of Plasmodium.
The level of InsP 3 becomes elevated early during
gametogenesis, at the onset of flagellum
development.
InsP 3 release from the membrane is mediated
by members of the phosphatidylinositolspecific
phospholipase C family of proteins
(PLC). Three broad groups of PLC have been
described. PLC- is activated by the alpha
subunit of trimeric G proteins (G q ). PLC- has
an SH2 domain and is recruited to phosphorylated
tyrosine residues on catalytic receptors.
Finally, PLC- lacks binding sites for G q or SH2
domains and may be regulated by association
with small G proteins. To date, T. cruzi is the
only protozoan parasite from which the PLC
has been cloned. Similar to other single-celled
organisms, the T. cruzi enzyme is of the -type.
However, it lacks the PH domain usually found
at the amino terminus of other -type
enzymes. Instead, it is acylated by myristate
and palmitate, and this modification may help
target the enzyme to appropriate membranes.
The enzyme also has an unusual bridge of
charged amino acids (mostly acidic) between
its two catalytic X and Y domains. Enzyme
activity is stimulated with Ca 2 , consistent
with the Ca 2 -dependent rise in InsP 3 observed
in permeabilized cells. Conditions that stimulate
the in vitro transition from trypomastigotes
to amastigote forms simultaneously
elevate the expression level of PLC- and
increase the cellular pool of InsP 3 . Despite the
ability of T. cruzi to regulate InsP 3 levels in the
cell, to date it has not been possible to show
InsP 3 -dependent Ca 2 release from the ER of
this organism. Unlike the situation in T. cruzi,
the addition of InsP 3 to isolated permeabilized
Plasmodium trophozoites causes Ca 2
release from what appear to be two distinct
compartments. One compartment is depleted
by inhibitors of the SERCA pumps, including
thapsigargin and 2,5-di(t-butyl)-1,4 benzohydroquinone,
and thus is likely to derive from
the ER. The other compartment is discharged
with the ion exchanger nigericin or neutralizing
agent chloroquine. These reagents disrupt
pH gradients within acidic organelles,
suggesting that InsP 3 might also release
Ca 2 from acidocalcisomes. Interestingly, in
E. histolytica, where a well defined ER is not
present, permeabilized cells still release Ca 2
from an intracellular compartment in response
to InsP 3 as well as Ins(2,4,5)P 3 . Depletion of the
Ca 2 pool with InsP 3 does not prevent further
Ca 2 release with inositol (1,3,4,5) tetrakisphosphate
(InsP 4 ), and the association of InsP 3 to
membranes could not be removed with excess
unlabeled InsP 4 or vice versa. Taken together,
these data suggest that two Ca 2 pools exist in
these cells, with differing sensitivities to inositol
phosphates. Within Entamoeba, the stored
Ca 2 is likely contained in vacuoles. A plasma
membrane-type PMCA Ca 2 -ATPase has been
localized to vacuoles in this organism. Whether
these vacuoles share other properties with acidocalcisomes
has yet to be determined.
In summary, much of the nuance involved
in ER Ca 2 storage and release has not been
determined for protozoan parasites. Unlike the
mammalian system where the ER Ca 2 pool is
of central importance to the regulation of cell
response, the parasite ER pool is of comparatively
unknown significance. Nonetheless, it is
likely that parasites will use the stored Ca 2 to
mediate ER activities associated with translation
and to modulate [Ca 2 ] i concentrations
during signal production.
Acidocalcisomes
Perphaps the most remarkable aspect of
Ca 2 homeostasis in pathogenic protozoa has
been the discovery of exchangeable Ca 2 in
an acidic compartment, referred to as the
BIOCHEMISTRY AND CELL BIOLOGY: PROTOZOA