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Trafficking of plasmepsin II to the food vacuole of the malaria parasite Plasmodium falciparum.

Klemba M, Beatty W, Gluzman I, Goldberg DE - J. Cell Biol. (2004)

Bottom Line: A family of aspartic proteases, the plasmepsins (PMs), plays a key role in the degradation of hemoglobin in the Plasmodium falciparum food vacuole.To study the trafficking of proPM II, we have modified the chromosomal PM II gene in P. falciparum to encode a proPM II-GFP chimera.Our data support a model whereby proPM II is transported through the secretory system to cytostomal vacuoles and then is carried along with its substrate hemoglobin to the food vacuole where it is proteolytically processed to mature PM II.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Microbiology, Washington University School of Medicine, 660 S. Euclid Ave., Box 8230, St. Louis, MO 63110, USA.

ABSTRACT
A family of aspartic proteases, the plasmepsins (PMs), plays a key role in the degradation of hemoglobin in the Plasmodium falciparum food vacuole. To study the trafficking of proPM II, we have modified the chromosomal PM II gene in P. falciparum to encode a proPM II-GFP chimera. By taking advantage of green fluorescent protein fluorescence in live parasites, the ultrastructural resolution of immunoelectron microscopy, and inhibitors of trafficking and PM maturation, we have investigated the biosynthetic path leading to mature PM II in the food vacuole. Our data support a model whereby proPM II is transported through the secretory system to cytostomal vacuoles and then is carried along with its substrate hemoglobin to the food vacuole where it is proteolytically processed to mature PM II.

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ImmunoEM localization of GFP to the food vacuole and cytostomal vacuoles. (A) A B7 trophozoite displaying prominent food vacuole labeling. Bar, 200 nm. (B and C) Cross sections of cytostomal vacuoles formed during the uptake of erythrocyte cytosol showing labeling of the vacuole membrane. In both panels, the “neck” of the cytostome (arrows) is clearly visible. (D and E) Cross sections of cytostomal vacuoles or hemoglobin transport vesicles displaying membrane labeling. Low magnification images of parasites in B–E are provided in Fig. S2. fv, food vacuole; n, nucleus; pvm, parasitophorous vacuole membrane; cv, cytostomal vacuole; ec, erythrocyte cytoplasm; ct, cytostomal vacuole or hemoglobin-containing transport vesicle. (B–E) Bars, 100 nm.
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fig5: ImmunoEM localization of GFP to the food vacuole and cytostomal vacuoles. (A) A B7 trophozoite displaying prominent food vacuole labeling. Bar, 200 nm. (B and C) Cross sections of cytostomal vacuoles formed during the uptake of erythrocyte cytosol showing labeling of the vacuole membrane. In both panels, the “neck” of the cytostome (arrows) is clearly visible. (D and E) Cross sections of cytostomal vacuoles or hemoglobin transport vesicles displaying membrane labeling. Low magnification images of parasites in B–E are provided in Fig. S2. fv, food vacuole; n, nucleus; pvm, parasitophorous vacuole membrane; cv, cytostomal vacuole; ec, erythrocyte cytoplasm; ct, cytostomal vacuole or hemoglobin-containing transport vesicle. (B–E) Bars, 100 nm.

Mentions: To define the distribution of GFP at the ultrastructural level, B7 parasites were fixed and analyzed by immunoEM with an affinity-purified anti-GFP polyclonal antibody. To first assess the specificity of the antibody, untransfected 3D7 trophozoites were subjected to the labeling protocol. Approximately one colloidal gold particle per parasite was observed, with no clear labeling pattern in evidence. In contrast, sections of B7 trophozoites exhibited heavily labeled food vacuoles (Fig. 5 A). The membranes of cytostomal vacuoles were also frequently labeled (Fig. 5, B and C), an observation that provided the first indication that proPM II–GFP populates the cytostomal vacuolar membrane (presumably the outer membrane; see Discussion) en route to the food vacuole. Although the protein collar of the cytostomal pore itself is poorly visible in these sections, the neck of the cytostomal vacuole, the continuity between the erythrocyte cytosol and the vacuole lumen, and the two membranes making up the vacuole are all evident in Fig. 5 (B and C). Double-membrane structures that may be cross sections of the cytostomal vacuole or hemoglobin transport vesicles are shown in Fig. 5 (D and E). Given the location of cytostomes at the periphery of the parasite and the apparent paucity of immunogold label elsewhere in the vicinity of the parasite plasma membrane, we presume that the peripheral GFP foci seen in live parasites (Fig. 4 B) correspond to cytostomal vacuoles.


Trafficking of plasmepsin II to the food vacuole of the malaria parasite Plasmodium falciparum.

Klemba M, Beatty W, Gluzman I, Goldberg DE - J. Cell Biol. (2004)

ImmunoEM localization of GFP to the food vacuole and cytostomal vacuoles. (A) A B7 trophozoite displaying prominent food vacuole labeling. Bar, 200 nm. (B and C) Cross sections of cytostomal vacuoles formed during the uptake of erythrocyte cytosol showing labeling of the vacuole membrane. In both panels, the “neck” of the cytostome (arrows) is clearly visible. (D and E) Cross sections of cytostomal vacuoles or hemoglobin transport vesicles displaying membrane labeling. Low magnification images of parasites in B–E are provided in Fig. S2. fv, food vacuole; n, nucleus; pvm, parasitophorous vacuole membrane; cv, cytostomal vacuole; ec, erythrocyte cytoplasm; ct, cytostomal vacuole or hemoglobin-containing transport vesicle. (B–E) Bars, 100 nm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2171955&req=5

fig5: ImmunoEM localization of GFP to the food vacuole and cytostomal vacuoles. (A) A B7 trophozoite displaying prominent food vacuole labeling. Bar, 200 nm. (B and C) Cross sections of cytostomal vacuoles formed during the uptake of erythrocyte cytosol showing labeling of the vacuole membrane. In both panels, the “neck” of the cytostome (arrows) is clearly visible. (D and E) Cross sections of cytostomal vacuoles or hemoglobin transport vesicles displaying membrane labeling. Low magnification images of parasites in B–E are provided in Fig. S2. fv, food vacuole; n, nucleus; pvm, parasitophorous vacuole membrane; cv, cytostomal vacuole; ec, erythrocyte cytoplasm; ct, cytostomal vacuole or hemoglobin-containing transport vesicle. (B–E) Bars, 100 nm.
Mentions: To define the distribution of GFP at the ultrastructural level, B7 parasites were fixed and analyzed by immunoEM with an affinity-purified anti-GFP polyclonal antibody. To first assess the specificity of the antibody, untransfected 3D7 trophozoites were subjected to the labeling protocol. Approximately one colloidal gold particle per parasite was observed, with no clear labeling pattern in evidence. In contrast, sections of B7 trophozoites exhibited heavily labeled food vacuoles (Fig. 5 A). The membranes of cytostomal vacuoles were also frequently labeled (Fig. 5, B and C), an observation that provided the first indication that proPM II–GFP populates the cytostomal vacuolar membrane (presumably the outer membrane; see Discussion) en route to the food vacuole. Although the protein collar of the cytostomal pore itself is poorly visible in these sections, the neck of the cytostomal vacuole, the continuity between the erythrocyte cytosol and the vacuole lumen, and the two membranes making up the vacuole are all evident in Fig. 5 (B and C). Double-membrane structures that may be cross sections of the cytostomal vacuole or hemoglobin transport vesicles are shown in Fig. 5 (D and E). Given the location of cytostomes at the periphery of the parasite and the apparent paucity of immunogold label elsewhere in the vicinity of the parasite plasma membrane, we presume that the peripheral GFP foci seen in live parasites (Fig. 4 B) correspond to cytostomal vacuoles.

Bottom Line: A family of aspartic proteases, the plasmepsins (PMs), plays a key role in the degradation of hemoglobin in the Plasmodium falciparum food vacuole.To study the trafficking of proPM II, we have modified the chromosomal PM II gene in P. falciparum to encode a proPM II-GFP chimera.Our data support a model whereby proPM II is transported through the secretory system to cytostomal vacuoles and then is carried along with its substrate hemoglobin to the food vacuole where it is proteolytically processed to mature PM II.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Microbiology, Washington University School of Medicine, 660 S. Euclid Ave., Box 8230, St. Louis, MO 63110, USA.

ABSTRACT
A family of aspartic proteases, the plasmepsins (PMs), plays a key role in the degradation of hemoglobin in the Plasmodium falciparum food vacuole. To study the trafficking of proPM II, we have modified the chromosomal PM II gene in P. falciparum to encode a proPM II-GFP chimera. By taking advantage of green fluorescent protein fluorescence in live parasites, the ultrastructural resolution of immunoelectron microscopy, and inhibitors of trafficking and PM maturation, we have investigated the biosynthetic path leading to mature PM II in the food vacuole. Our data support a model whereby proPM II is transported through the secretory system to cytostomal vacuoles and then is carried along with its substrate hemoglobin to the food vacuole where it is proteolytically processed to mature PM II.

Show MeSH
Related in: MedlinePlus