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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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Pulse-chase analysis of proPM II–GFP maturation. B7 trophozoites were pulse labeled for 15 min with [35S]methionine and -cysteine and chased for the times indicated (min). (A) Immunoprecipitation of PM II–containing species. To indicate the position of proPM II (without GFP), this species was immunoprecipitated from labeled wild-type 3D7 parasites. In the B7 lanes, proPM II–GFP is partially obscured by high background in this region of the gel. (B) Immunoprecipitation of polypeptides containing GFP. The low intensity of the GFP band relative to proPM II–GFP 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. (C) Immunoprecipitation of pro- and mPM I. Parasites are from the same labeled populations as those in B.
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fig3: Pulse-chase analysis of proPM II–GFP maturation. B7 trophozoites were pulse labeled for 15 min with [35S]methionine and -cysteine and chased for the times indicated (min). (A) Immunoprecipitation of PM II–containing species. To indicate the position of proPM II (without GFP), this species was immunoprecipitated from labeled wild-type 3D7 parasites. In the B7 lanes, proPM II–GFP is partially obscured by high background in this region of the gel. (B) Immunoprecipitation of polypeptides containing GFP. The low intensity of the GFP band relative to proPM II–GFP 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. (C) Immunoprecipitation of pro- and mPM I. Parasites are from the same labeled populations as those in B.

Mentions: The biosynthesis and maturation of proPM II and the closely related proteins proPM I, proPM IV, and proHAP have been shown to proceed relatively rapidly, with a half-time of ∼20 min (Francis et al., 1997a; Banerjee et al., 2003). To determine the effect of the GFP tag on the rate of maturation of proPM II, B7 parasites were pulse labeled with [35S]methionine and -cysteine, chased for various times, and polypeptides containing PM I, PM II, or GFP were immunoprecipitated (Fig. 3). The PM II and GFP immunoprecipitations indicated that proPM II–GFP was processed to mPM II and GFP without significant accumulation of the intermediate forms proPM II or mPM II–GFP, which suggests that PM II maturation and cleavage at the PM II–GFP linker occur essentially simultaneously (Fig. 3, A and B). The rate of disappearance of proPM II–GFP was considerably slower than that of proPM I. After 45 min, a significant amount of proPM II–GFP remained unprocessed (Fig. 3 B), whereas proPM I was completely converted to mPM I (Fig. 3 C). This slower rate of processing may be a factor in the elevation of steady-state levels of proPM II–GFP observed in the immunoblots.


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)

Pulse-chase analysis of proPM II–GFP maturation. B7 trophozoites were pulse labeled for 15 min with [35S]methionine and -cysteine and chased for the times indicated (min). (A) Immunoprecipitation of PM II–containing species. To indicate the position of proPM II (without GFP), this species was immunoprecipitated from labeled wild-type 3D7 parasites. In the B7 lanes, proPM II–GFP is partially obscured by high background in this region of the gel. (B) Immunoprecipitation of polypeptides containing GFP. The low intensity of the GFP band relative to proPM II–GFP 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. (C) Immunoprecipitation of pro- and mPM I. Parasites are from the same labeled populations as those in B.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2171955&req=5

fig3: Pulse-chase analysis of proPM II–GFP maturation. B7 trophozoites were pulse labeled for 15 min with [35S]methionine and -cysteine and chased for the times indicated (min). (A) Immunoprecipitation of PM II–containing species. To indicate the position of proPM II (without GFP), this species was immunoprecipitated from labeled wild-type 3D7 parasites. In the B7 lanes, proPM II–GFP is partially obscured by high background in this region of the gel. (B) Immunoprecipitation of polypeptides containing GFP. The low intensity of the GFP band relative to proPM II–GFP 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. (C) Immunoprecipitation of pro- and mPM I. Parasites are from the same labeled populations as those in B.
Mentions: The biosynthesis and maturation of proPM II and the closely related proteins proPM I, proPM IV, and proHAP have been shown to proceed relatively rapidly, with a half-time of ∼20 min (Francis et al., 1997a; Banerjee et al., 2003). To determine the effect of the GFP tag on the rate of maturation of proPM II, B7 parasites were pulse labeled with [35S]methionine and -cysteine, chased for various times, and polypeptides containing PM I, PM II, or GFP were immunoprecipitated (Fig. 3). The PM II and GFP immunoprecipitations indicated that proPM II–GFP was processed to mPM II and GFP without significant accumulation of the intermediate forms proPM II or mPM II–GFP, which suggests that PM II maturation and cleavage at the PM II–GFP linker occur essentially simultaneously (Fig. 3, A and B). The rate of disappearance of proPM II–GFP was considerably slower than that of proPM I. After 45 min, a significant amount of proPM II–GFP remained unprocessed (Fig. 3 B), whereas proPM I was completely converted to mPM I (Fig. 3 C). This slower rate of processing may be a factor in the elevation of steady-state levels of proPM II–GFP observed in the immunoblots.

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