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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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PM II–GFP expression in B7 parasites. (A) Schematic representation of the proPM II–GFP fusion. The propiece is shown with the single transmembrane domain (TM) in black. proPM II–GFP is proteolytically processed twice (arrowheads) to generate mPM II (gray box). (B) Immunoblot analysis of PM II and GFP expression in synchronized trophozoites before and after integration of pPM2GT. SDS-solubilized protein from 107 saponin-treated trophozoites was loaded in each lane. To assess relative amounts of protein loaded, blots were stripped and reprobed with anti-PfBiP antibody (bottom). Recombinant mature PM II (rmPM II) was used as a marker in the PM II blot. The species labeled “GFP” comigrated with recombinant GFPmut2 (not depicted). The band denoted with an asterisk reacted with secondary anti–rabbit Ig antibody alone (not depicted). Sizes of molecular mass markers are indicated in kD. (C) Immunoelectron micrograph illustrating labeling of the food vacuole of a B7 trophozoite with affinity-purified anti–PM II. The food vacuole membrane is indicated with arrowheads, and the closely apposed parasitophorous vacuole and parasite plasma membranes are indicated with an arrow. A low magnification image of this parasite is provided in Fig. S2, http://www.jcb.org/cgi/content/full/jcb.200307147/DC1. fv, food vacuole. Bar, 200 nm.
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fig2: PM II–GFP expression in B7 parasites. (A) Schematic representation of the proPM II–GFP fusion. The propiece is shown with the single transmembrane domain (TM) in black. proPM II–GFP is proteolytically processed twice (arrowheads) to generate mPM II (gray box). (B) Immunoblot analysis of PM II and GFP expression in synchronized trophozoites before and after integration of pPM2GT. SDS-solubilized protein from 107 saponin-treated trophozoites was loaded in each lane. To assess relative amounts of protein loaded, blots were stripped and reprobed with anti-PfBiP antibody (bottom). Recombinant mature PM II (rmPM II) was used as a marker in the PM II blot. The species labeled “GFP” comigrated with recombinant GFPmut2 (not depicted). The band denoted with an asterisk reacted with secondary anti–rabbit Ig antibody alone (not depicted). Sizes of molecular mass markers are indicated in kD. (C) Immunoelectron micrograph illustrating labeling of the food vacuole of a B7 trophozoite with affinity-purified anti–PM II. The food vacuole membrane is indicated with arrowheads, and the closely apposed parasitophorous vacuole and parasite plasma membranes are indicated with an arrow. A low magnification image of this parasite is provided in Fig. S2, http://www.jcb.org/cgi/content/full/jcb.200307147/DC1. fv, food vacuole. Bar, 200 nm.

Mentions: Before examining the trafficking of the proPM II–GFP fusion, it was necessary to demonstrate that the addition of the GFP tag did not interfere with transport to and maturation in the food vacuole. Like untagged proPM II, proPM II–GFP is synthesized as an integral membrane proenzyme (Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200307147/DC1). To assess whether proPM II–GFP is appropriately processed to the mature protease (Fig. 2 A), trophozoite extracts were examined by immunoblotting with anti–PM II and anti-GFP antibodies (Fig. 2 B). Only mPM II was observed in parasites containing a wild-type copy of the PM II gene. mPM II was also the predominant form of the protein in B7 parasites. Along with the proregion, GFP appeared to be proteolytically cleaved from mPM II, as the mobility of mPM II in SDS-PAGE was only slightly slower than that from 3D7 parasites. This slight shift in mobility presumably derives from retention of some of the linker sequence at the COOH terminus of mPM II. Significantly, addition of the GFP tag had a relatively small effect on the steady-state levels of mPM II: densitometric quantitation of Fig. 2 B using BiP as a normalization reference indicated that the amount of mPM II in B7 extract was only 1.2-fold greater than that in transfected parasites before cycling (cycle 0) and 1.6-fold greater than that in 3D7 extract. In addition to mPM II, a small but significant amount of proPM II–GFP was observed in extracts of B7 parasites; in contrast, proPM II was not detected in 3D7 extracts. Immunoblotting with anti-GFP antibodies indicated that GFP is undetectable in stably transfected parasites before drug cycling, which demonstrates that integration of pPM2GT into the PM II gene is a prerequisite for GFP expression.


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)

PM II–GFP expression in B7 parasites. (A) Schematic representation of the proPM II–GFP fusion. The propiece is shown with the single transmembrane domain (TM) in black. proPM II–GFP is proteolytically processed twice (arrowheads) to generate mPM II (gray box). (B) Immunoblot analysis of PM II and GFP expression in synchronized trophozoites before and after integration of pPM2GT. SDS-solubilized protein from 107 saponin-treated trophozoites was loaded in each lane. To assess relative amounts of protein loaded, blots were stripped and reprobed with anti-PfBiP antibody (bottom). Recombinant mature PM II (rmPM II) was used as a marker in the PM II blot. The species labeled “GFP” comigrated with recombinant GFPmut2 (not depicted). The band denoted with an asterisk reacted with secondary anti–rabbit Ig antibody alone (not depicted). Sizes of molecular mass markers are indicated in kD. (C) Immunoelectron micrograph illustrating labeling of the food vacuole of a B7 trophozoite with affinity-purified anti–PM II. The food vacuole membrane is indicated with arrowheads, and the closely apposed parasitophorous vacuole and parasite plasma membranes are indicated with an arrow. A low magnification image of this parasite is provided in Fig. S2, http://www.jcb.org/cgi/content/full/jcb.200307147/DC1. fv, food vacuole. Bar, 200 nm.
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Related In: Results  -  Collection

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fig2: PM II–GFP expression in B7 parasites. (A) Schematic representation of the proPM II–GFP fusion. The propiece is shown with the single transmembrane domain (TM) in black. proPM II–GFP is proteolytically processed twice (arrowheads) to generate mPM II (gray box). (B) Immunoblot analysis of PM II and GFP expression in synchronized trophozoites before and after integration of pPM2GT. SDS-solubilized protein from 107 saponin-treated trophozoites was loaded in each lane. To assess relative amounts of protein loaded, blots were stripped and reprobed with anti-PfBiP antibody (bottom). Recombinant mature PM II (rmPM II) was used as a marker in the PM II blot. The species labeled “GFP” comigrated with recombinant GFPmut2 (not depicted). The band denoted with an asterisk reacted with secondary anti–rabbit Ig antibody alone (not depicted). Sizes of molecular mass markers are indicated in kD. (C) Immunoelectron micrograph illustrating labeling of the food vacuole of a B7 trophozoite with affinity-purified anti–PM II. The food vacuole membrane is indicated with arrowheads, and the closely apposed parasitophorous vacuole and parasite plasma membranes are indicated with an arrow. A low magnification image of this parasite is provided in Fig. S2, http://www.jcb.org/cgi/content/full/jcb.200307147/DC1. fv, food vacuole. Bar, 200 nm.
Mentions: Before examining the trafficking of the proPM II–GFP fusion, it was necessary to demonstrate that the addition of the GFP tag did not interfere with transport to and maturation in the food vacuole. Like untagged proPM II, proPM II–GFP is synthesized as an integral membrane proenzyme (Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200307147/DC1). To assess whether proPM II–GFP is appropriately processed to the mature protease (Fig. 2 A), trophozoite extracts were examined by immunoblotting with anti–PM II and anti-GFP antibodies (Fig. 2 B). Only mPM II was observed in parasites containing a wild-type copy of the PM II gene. mPM II was also the predominant form of the protein in B7 parasites. Along with the proregion, GFP appeared to be proteolytically cleaved from mPM II, as the mobility of mPM II in SDS-PAGE was only slightly slower than that from 3D7 parasites. This slight shift in mobility presumably derives from retention of some of the linker sequence at the COOH terminus of mPM II. Significantly, addition of the GFP tag had a relatively small effect on the steady-state levels of mPM II: densitometric quantitation of Fig. 2 B using BiP as a normalization reference indicated that the amount of mPM II in B7 extract was only 1.2-fold greater than that in transfected parasites before cycling (cycle 0) and 1.6-fold greater than that in 3D7 extract. In addition to mPM II, a small but significant amount of proPM II–GFP was observed in extracts of B7 parasites; in contrast, proPM II was not detected in 3D7 extracts. Immunoblotting with anti-GFP antibodies indicated that GFP is undetectable in stably transfected parasites before drug cycling, which demonstrates that integration of pPM2GT into the PM II gene is a prerequisite for GFP expression.

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