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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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Related in: MedlinePlus

BFA induces a reversible accumulation of proPM II–GFP in the ER. (A and B) GFP fluorescence in live B7 parasites treated for 2 h with BFA: (A) a trophozoite and (B) a schizont undergoing nuclear division. The arrowhead in B indicates food vacuole fluorescence. (C) Redistribution of GFP 10 min after release of the BFA block. Fluorescent spots reappear at the periphery of the parasite (arrowheads). 100 μg/ml cycloheximide was present to inhibit protein synthesis after BFA washout. Similar results were obtained in the absence of cycloheximide. In A–C, fluorescence from the nuclear stain Hoechst 33342 is pseudocolored red. Bar, 2 μm. (D) Cryosection of a BFA treated B7 trophozoite double-labeled with an antibody against GFP (18-nm colloidal gold) and an antibody recognizing the ER marker BiP (12-nm colloidal gold). Most of the 18-nm gold label is associated with the nuclear envelope (arrowhead), whereas the 12-nm gold label is associated with the peripheral ER (asterisk) extending away from the nucleus. A low magnification image of this parasite is provided in Fig. S2. n, nucleus. Bar, 200 nm. (E) B7 trophozoites were 35S-labeled for 2 h in the presence of 5 μg/ml BFA (“BFA” lane). Both BFA and unincorporated 35S were washed out either in the absence (no inhib) or presence (ALLN) of an inhibitor of PM II maturation. proPM II–GFP and GFP were immunoprecipitated with an anti-GFP antibody. The low intensity of the GFP band in “no inhib” lane relative to proPM II–GFP in the “BFA” lane is likely due to two factors: GFP contains one third of the label present in proPM II–GFP, and may be slowly degraded in the food vacuole. Sizes of molecular mass markers are indicated in kD.
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fig6: BFA induces a reversible accumulation of proPM II–GFP in the ER. (A and B) GFP fluorescence in live B7 parasites treated for 2 h with BFA: (A) a trophozoite and (B) a schizont undergoing nuclear division. The arrowhead in B indicates food vacuole fluorescence. (C) Redistribution of GFP 10 min after release of the BFA block. Fluorescent spots reappear at the periphery of the parasite (arrowheads). 100 μg/ml cycloheximide was present to inhibit protein synthesis after BFA washout. Similar results were obtained in the absence of cycloheximide. In A–C, fluorescence from the nuclear stain Hoechst 33342 is pseudocolored red. Bar, 2 μm. (D) Cryosection of a BFA treated B7 trophozoite double-labeled with an antibody against GFP (18-nm colloidal gold) and an antibody recognizing the ER marker BiP (12-nm colloidal gold). Most of the 18-nm gold label is associated with the nuclear envelope (arrowhead), whereas the 12-nm gold label is associated with the peripheral ER (asterisk) extending away from the nucleus. A low magnification image of this parasite is provided in Fig. S2. n, nucleus. Bar, 200 nm. (E) B7 trophozoites were 35S-labeled for 2 h in the presence of 5 μg/ml BFA (“BFA” lane). Both BFA and unincorporated 35S were washed out either in the absence (no inhib) or presence (ALLN) of an inhibitor of PM II maturation. proPM II–GFP and GFP were immunoprecipitated with an anti-GFP antibody. The low intensity of the GFP band in “no inhib” lane relative to proPM II–GFP in the “BFA” lane is likely due to two factors: GFP contains one third of the label present in proPM II–GFP, and may be slowly degraded in the food vacuole. Sizes of molecular mass markers are indicated in kD.

Mentions: BFA has enjoyed wide application due to its ability to inhibit anterograde protein trafficking from the ER; however, it has been shown to affect protein transport to and from organelles other than the ER, such as the endosomal–lysosomal system (Klausner et al., 1992). BFA inhibits processing of proPM II to mPM II (Francis et al., 1997a), presumably because proPM II cannot reach the food vacuole. To better understand the root cause of inhibition of PM maturation, and to characterize the morphology of the BFA-induced structure(s) in live parasites, we analyzed the distribution of GFP fluorescence in B7 parasites treated with 5 μg/ml BFA for 2 h. In trophozoites, a prominent perinuclear ring of fluorescence was observed (Fig. 6 A). The morphology of this structure closely resembled that observed in untreated trophozoites (Fig. 4 A), but the intensity of fluorescence was greatly increased by BFA treatment. The number of fluorescent foci was greatly diminished (0.14 ± 0.35 per trophozoite, range 0–1, n = 29), which suggests a depletion of cytostomal fluorescence upon BFA treatment. In early schizonts, the fluorescent compartment developed greater complexity and consisted of multiple perinuclear rings (Fig. 6 B). The perinuclear position of this compartment suggested that it was the nuclear envelope, a structure that in many eukaryotic cells is continuous with the ER lumen (Franke et al., 1981). To confirm this, B7 trophozoites were treated with BFA for 2 h and then fixed and sectioned for immunoEM. Both GFP and the resident ER protein BiP were localized in the same sections using secondary antibodies conjugated to 18- and 12-nm colloidal gold, respectively. Much of the GFP label was associated with the nuclear envelope, but some was also observed in elements of the peripheral ER, which consists of tubulovesicular structures extending away from the nucleus toward the parasite plasma membrane (Fig. 6 D). The bulk of the BiP label was associated with the peripheral ER, although it could also be detected in the nuclear envelope (Fig. 6 D and unpublished data). In some sections, continuity between the nuclear envelope and tubulovesicular elements of the peripheral ER could be observed (unpublished data).


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)

BFA induces a reversible accumulation of proPM II–GFP in the ER. (A and B) GFP fluorescence in live B7 parasites treated for 2 h with BFA: (A) a trophozoite and (B) a schizont undergoing nuclear division. The arrowhead in B indicates food vacuole fluorescence. (C) Redistribution of GFP 10 min after release of the BFA block. Fluorescent spots reappear at the periphery of the parasite (arrowheads). 100 μg/ml cycloheximide was present to inhibit protein synthesis after BFA washout. Similar results were obtained in the absence of cycloheximide. In A–C, fluorescence from the nuclear stain Hoechst 33342 is pseudocolored red. Bar, 2 μm. (D) Cryosection of a BFA treated B7 trophozoite double-labeled with an antibody against GFP (18-nm colloidal gold) and an antibody recognizing the ER marker BiP (12-nm colloidal gold). Most of the 18-nm gold label is associated with the nuclear envelope (arrowhead), whereas the 12-nm gold label is associated with the peripheral ER (asterisk) extending away from the nucleus. A low magnification image of this parasite is provided in Fig. S2. n, nucleus. Bar, 200 nm. (E) B7 trophozoites were 35S-labeled for 2 h in the presence of 5 μg/ml BFA (“BFA” lane). Both BFA and unincorporated 35S were washed out either in the absence (no inhib) or presence (ALLN) of an inhibitor of PM II maturation. proPM II–GFP and GFP were immunoprecipitated with an anti-GFP antibody. The low intensity of the GFP band in “no inhib” lane relative to proPM II–GFP in the “BFA” lane is likely due to two factors: GFP contains one third of the label present in proPM II–GFP, and may be slowly degraded in the food vacuole. Sizes of molecular mass markers are indicated in kD.
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Related In: Results  -  Collection

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fig6: BFA induces a reversible accumulation of proPM II–GFP in the ER. (A and B) GFP fluorescence in live B7 parasites treated for 2 h with BFA: (A) a trophozoite and (B) a schizont undergoing nuclear division. The arrowhead in B indicates food vacuole fluorescence. (C) Redistribution of GFP 10 min after release of the BFA block. Fluorescent spots reappear at the periphery of the parasite (arrowheads). 100 μg/ml cycloheximide was present to inhibit protein synthesis after BFA washout. Similar results were obtained in the absence of cycloheximide. In A–C, fluorescence from the nuclear stain Hoechst 33342 is pseudocolored red. Bar, 2 μm. (D) Cryosection of a BFA treated B7 trophozoite double-labeled with an antibody against GFP (18-nm colloidal gold) and an antibody recognizing the ER marker BiP (12-nm colloidal gold). Most of the 18-nm gold label is associated with the nuclear envelope (arrowhead), whereas the 12-nm gold label is associated with the peripheral ER (asterisk) extending away from the nucleus. A low magnification image of this parasite is provided in Fig. S2. n, nucleus. Bar, 200 nm. (E) B7 trophozoites were 35S-labeled for 2 h in the presence of 5 μg/ml BFA (“BFA” lane). Both BFA and unincorporated 35S were washed out either in the absence (no inhib) or presence (ALLN) of an inhibitor of PM II maturation. proPM II–GFP and GFP were immunoprecipitated with an anti-GFP antibody. The low intensity of the GFP band in “no inhib” lane relative to proPM II–GFP in the “BFA” lane is likely due to two factors: GFP contains one third of the label present in proPM II–GFP, and may be slowly degraded in the food vacuole. Sizes of molecular mass markers are indicated in kD.
Mentions: BFA has enjoyed wide application due to its ability to inhibit anterograde protein trafficking from the ER; however, it has been shown to affect protein transport to and from organelles other than the ER, such as the endosomal–lysosomal system (Klausner et al., 1992). BFA inhibits processing of proPM II to mPM II (Francis et al., 1997a), presumably because proPM II cannot reach the food vacuole. To better understand the root cause of inhibition of PM maturation, and to characterize the morphology of the BFA-induced structure(s) in live parasites, we analyzed the distribution of GFP fluorescence in B7 parasites treated with 5 μg/ml BFA for 2 h. In trophozoites, a prominent perinuclear ring of fluorescence was observed (Fig. 6 A). The morphology of this structure closely resembled that observed in untreated trophozoites (Fig. 4 A), but the intensity of fluorescence was greatly increased by BFA treatment. The number of fluorescent foci was greatly diminished (0.14 ± 0.35 per trophozoite, range 0–1, n = 29), which suggests a depletion of cytostomal fluorescence upon BFA treatment. In early schizonts, the fluorescent compartment developed greater complexity and consisted of multiple perinuclear rings (Fig. 6 B). The perinuclear position of this compartment suggested that it was the nuclear envelope, a structure that in many eukaryotic cells is continuous with the ER lumen (Franke et al., 1981). To confirm this, B7 trophozoites were treated with BFA for 2 h and then fixed and sectioned for immunoEM. Both GFP and the resident ER protein BiP were localized in the same sections using secondary antibodies conjugated to 18- and 12-nm colloidal gold, respectively. Much of the GFP label was associated with the nuclear envelope, but some was also observed in elements of the peripheral ER, which consists of tubulovesicular structures extending away from the nucleus toward the parasite plasma membrane (Fig. 6 D). The bulk of the BiP label was associated with the peripheral ER, although it could also be detected in the nuclear envelope (Fig. 6 D and unpublished data). In some sections, continuity between the nuclear envelope and tubulovesicular elements of the peripheral ER could be observed (unpublished data).

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