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CALCIUM 245
parasites. Organisms such as Entamoeba are
capable of storing intracellular Ca 2 in the
absence of mitochondria.
Endoplasmic reticulum
Functions of stored Ca 2
In mammalian cells, exchangeable Ca 2 in the
endoplasmic reticulum (ER) has been extensively
studied. The ER Ca 2 pool is sufficient to
trigger cell events, while the stored Ca 2 appears
to be essential for translation and proper
folding of proteins. In contrast with the mammalian
ER, much less is known about the corresponding
Ca 2 pool in protozoan parasites.
In mammalian cells, the level of Ca 2 within
the ER has been quantified with mutated
aequorins, aequorin-Sr 2 reconstitution systems,
sequestered fura-2, and chameleons.
The average concentration of Ca 2 in the ER
vastly exceeds the exchangeable pool in mitochondria.
Concentrations in the millimolar
range have been measured. Interestingly, Ca 2
is not uniformly dispersed throughout the ER,
and visualization of the different subcompartments
with sequestered fura-2 reveals that
variable amounts of the stored Ca 2 can be
released in response to inhibitors of the Ca 2 -
ATPase or agonists of ER Ca 2 channels. High
levels of stored Ca 2 appear to be necessary for
protein translation, since depletion of the ER
pool decreases the rate of translation. Much of
the stored Ca 2 in ER is bound to high capacity,
low affinity binding proteins such as calsequestrin,
calreticulin, calnexin and BiP. When
the expression level of BiP is increased by
changes in copy number or upon induction of
the unfolded protein response, the level of
stored ER Ca 2 also increases. BiP, calreticulin
and calnexin serve as Ca 2 -dependent chaperones.
In T. cruzi, the calreticulin gene has been
cloned and its ability to modulate glycoprotein
folding has been demonstrated. The T. cruzi
calreticulin is contained in a microsomal fraction
where it associates with the monoglucosylated
carbohydrate moiety of cruzipain. The
calreticulin from L. donovani has also been
cloned and the recombinant protein binds
Ca 2 . Although the recombinant calreticulin
can be phosphorylated in vitro, it binds to
leishmanial RNAs independent of its phosphorylation
state. Along with the low affinity,
high capacity Ca 2 -binding chaperones, an
unusual protein which appears to be a fusion
of sarcalumenin and an Eps15-like domain
has recently been described in P. falciparum.
In mammalian cells, Eps-15 functions as a
tyrosine kinase substrate. It associates with AP-2
of coated vesicles, and plays a role in vesicle
sorting within the endomembrane system.
Sarcalumenin from mammalian cells resides
within the ER lumen and modulates sensitivity
of the ryanodine-sensitive Ca 2 release channel.
The role of this unusual fusion protein in
P. falciparum is not known. A separate family of
Ca 2 -binding proteins referred to as the CREC
family resides in the secretory pathway. Proteins
in this family contain multiple repeats of the
EF-hand Ca 2 -binding motif (see below) and
may be involved in protein trafficking. Unlike
cytoplasmic EF-hand proteins, members of
the CREC family have a low affinity for Ca 2 .
Reticulocalbin, a member of this family, is
essential, based on homozygous deletion
studies in mice. The reticulocalbin homolog of
P. falciparum has been cloned and is referred to
as Pfs40/PfERC. The protein contains six EFhand
Ca 2 -binding motifs and an ER retrieval
sequence at its carboxy terminus. The protein
localizes to a reticular network within trophozoites,
and its expression level increases during
trophozoite maturation. Overall, these observations
indicate that the ER of protozoan parasites
contains Ca 2 -binding proteins capable
of mediating lumenal activities including translation,
protein folding and protein trafficking.
BIOCHEMISTRY AND CELL BIOLOGY: PROTOZOA