THE ENDOCYTIC PATHWAY

Endocytosis of nutrients in trypanosomatid protozoa is restricted to the flagellar pocket membrane (Webster & Russel 1993, Radek & Hausmann 1994, Overath et al. 1997). However, epimastigotes of T. cruzi present an additional site for the uptake of macromolecules: the cytostome, a deep invagination of the cell plasma membrane close to the flagellar pocket region. It appears that the cytostome is physically linked to the flagellum (Okuda et al. 1977) and represents the main site for both receptor-mediated-endocytosis and fluid-phase-pinocytosis in epimastigotes (Soares & De Souza 1991, Porto-Carreiro et al. 1998). Endocytic vesicles bud off from the cytostome and the flagellar pocket membranes and then deliver their cargo to the reservosomes (Soares & De Souza 1991, Soares et al. 1992). Reservosomes contain tyrosine-phosphorylated proteins, suggesting that protein kinases play a role in the internalization process (Vieira et al. 1996). Incubation of epimastigotes with ATP (50 mM, for 24 hr) prior to the addition of horseradish peroxidase (as a marker of the endocytic pathway) affected the formation of normal reservosomes (Bogitsh et al. 1997), possibly acting on the (still unknown) translocation system governing the traffic of the small endocytic vesicles that cargo proteins from the cell surface to the storage organelles.

Reservosomes are acidic organelles, containing cruzipain (a cysteine proteinase) and ingested proteins. On this basis, it has been proposed that these structures are pre-lysosomal compartments (Soares et al. 1992). Imunocytochemical quantification using DAMP as a probe showed that reservosomes have a luminal pH of about 6.0 (Soares et al. 1992). However, it is still a matter of speculation how these organelles are acidified, as they were not labeled with antibodies against a vacuolar-type H⁺-ATPase, which however recognized other intracellular vacuoles, possibly the acidocalcisomes (Benchimol et al. 1998). A 52-kDa protein sharing sequence homology with glutathione S-transferase (Tc52) has been also localized in reservosomes (Ouassi et al. 1995). It has been postulated that Tc52 is released from the parasite to the external milieu, in order to scavenge glutathione (GSH). The Tc52-GSH complex could be then internalized (by receptor mediated endocytosis?) and accumulated in the reservosomes. As GSH may serve as a storage and transport form of cysteine moieties, it was suggested that the Tc52-GSH complexes might act as a cysteine delivery system. Accordingly, TC52 is developmentally regulated, being fully expressed only by the epimastigotes.

The presence of an early endosomal compartment in T. cruzi epimastigotes is still controversial. It is well known that incubation of the parasites at 28°C with gold labeled proteins results in labeling inside cytoplasmic vesicles and tubules, as well as in the reservosomes (Soares & De Souza 1991, Soares et al. 1992). Figueiredo and Soares (1996) showed that incubation of the parasites at 12°C (a condition that hinders the fusion of endocytic vesicles with early endosomes in mammalian cells) blocked the pinching of endocytic vesicles at the cytostome, inhibiting the uptake of nutrients by the cells. Labeling could be found in the cytostome, but not inside the flagellar pocket or intracytoplasmic vesicles. When the temperature was raised to 28°C, labeling could be then again found in the reservosomes. From these experiments, it was concluded that early endosomes are lacking; cargo vesicles coming from the cell surface (cytostome and flagellar pocket membranes) should shuttle their content directly to the reservosomes. On the other hand, three-dimensional reconstruction of cytoplasmic tubules and vesicles located close to the flagellar pocket showed that they are interconnected, forming a branched network at the anterior end of the cell, morphologically similar to the typical mammalian early endosomes (Porto-Carreiro et al. 1998).

Bogitsh et al. (1996) presented some data demonstrating that, although containing cysteine proteinase, reservosomes are unlikely to be lysosomes (albeit lysosomes have not yet been clearly morphologically and biochemically defined in trypanosomatids). The authors showed that incubation of epimastigotes with ammonium chloride (a weak base that accumulates in acidic compartments) resulted in swelling of reservosomes and electron-lucent vacuoles (considered as lysosomes). However, the exposure period required for swelling of reservosomes was significantly greater than that required for the same effect in lysosomes, probably due to the different pH inside these compartments. Furthermore, methyl esters of aminoacids (which accumulate in eukaryotic lysosomes) had little effect upon reservosomes, precluding their being lysosomes and suggesting that the proteolytic enzymes, such as cysteine proteinases, can be in an inactive state during a life period of the parasites.

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