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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

Creation of a chromosomal PM II–GFP chimera. (A) Schematic diagram of the integration plasmid pPM2GT linearized at the unique XhoI site (X). 1 kb of the 3′ end of the PM II coding sequence (gray box) was fused in frame to a linker sequence followed by the GFPmut2 open reading frame (white box). The amino acid sequence of the linker is shown. A WR99210-resistant variant of human dihydrofolate reductase (Fidock and Wellems, 1997) was incorporated as a selectable marker. The black bar indicates the PM II sequence used for probing Southern blots. Elements are not drawn to scale. (B) Schematic representation of events leading to a chromosomal PM II–GFP chimera. Single-site homologous recombination between the episomal pPM2GT target sequence and the chromosomal PM II locus produces the PM II–GFP chimera. The integration event produces a full-length PM II ORF (designated PM IIa) fused to GFP and a downstream promoterless copy of the PM II target sequence (PM IIb). Some elements of the plasmid, including the drug-resistance cassette, have been omitted for clarity. (C) Southern blot of StuI–NotI digested total DNA from untransfected parasites (3D7), stably transfected parasites before cycling (cycle 0) and after one and two drug cycles, and from three clones (B7, C9, and F4). The identity of the StuI–NotI fragments is indicated at left. The band identified with an asterisk is of unknown origin and probably reflects a rearrangement of pPM2GT after transfection. This figure was assembled from two experiments. wt locus, wild-type PM II locus.
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fig1: Creation of a chromosomal PM II–GFP chimera. (A) Schematic diagram of the integration plasmid pPM2GT linearized at the unique XhoI site (X). 1 kb of the 3′ end of the PM II coding sequence (gray box) was fused in frame to a linker sequence followed by the GFPmut2 open reading frame (white box). The amino acid sequence of the linker is shown. A WR99210-resistant variant of human dihydrofolate reductase (Fidock and Wellems, 1997) was incorporated as a selectable marker. The black bar indicates the PM II sequence used for probing Southern blots. Elements are not drawn to scale. (B) Schematic representation of events leading to a chromosomal PM II–GFP chimera. Single-site homologous recombination between the episomal pPM2GT target sequence and the chromosomal PM II locus produces the PM II–GFP chimera. The integration event produces a full-length PM II ORF (designated PM IIa) fused to GFP and a downstream promoterless copy of the PM II target sequence (PM IIb). Some elements of the plasmid, including the drug-resistance cassette, have been omitted for clarity. (C) Southern blot of StuI–NotI digested total DNA from untransfected parasites (3D7), stably transfected parasites before cycling (cycle 0) and after one and two drug cycles, and from three clones (B7, C9, and F4). The identity of the StuI–NotI fragments is indicated at left. The band identified with an asterisk is of unknown origin and probably reflects a rearrangement of pPM2GT after transfection. This figure was assembled from two experiments. wt locus, wild-type PM II locus.

Mentions: The single-copy PM II gene of P. falciparum strain 3D7 was altered to encode a proPM II–GFP fusion by modifying an established gene disruption procedure (Crabb et al., 1997a). Plasmid pPM2GT was constructed with a targeting sequence of 1 kb of the 3′ end of the PM II coding region fused in-frame to a sequence encoding a linker and the enhanced GFP variant GFPmut2 (Fig. 1 A). Parasites transfected with pPM2GT were selected with the antifolate drug WR99210 and subjected to two rounds of drug cycling to enrich the population for parasites that had integrated pPM2GT into the PM II gene (Fig. 1, B and C). Single-cell cloning of the twice-cycled culture was undertaken to obtain parasites of defined genotype. The clone studied extensively here, designated B7, contains a single copy of pPM2GT integrated into the PM II gene (Fig. 1 C).


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)

Creation of a chromosomal PM II–GFP chimera. (A) Schematic diagram of the integration plasmid pPM2GT linearized at the unique XhoI site (X). 1 kb of the 3′ end of the PM II coding sequence (gray box) was fused in frame to a linker sequence followed by the GFPmut2 open reading frame (white box). The amino acid sequence of the linker is shown. A WR99210-resistant variant of human dihydrofolate reductase (Fidock and Wellems, 1997) was incorporated as a selectable marker. The black bar indicates the PM II sequence used for probing Southern blots. Elements are not drawn to scale. (B) Schematic representation of events leading to a chromosomal PM II–GFP chimera. Single-site homologous recombination between the episomal pPM2GT target sequence and the chromosomal PM II locus produces the PM II–GFP chimera. The integration event produces a full-length PM II ORF (designated PM IIa) fused to GFP and a downstream promoterless copy of the PM II target sequence (PM IIb). Some elements of the plasmid, including the drug-resistance cassette, have been omitted for clarity. (C) Southern blot of StuI–NotI digested total DNA from untransfected parasites (3D7), stably transfected parasites before cycling (cycle 0) and after one and two drug cycles, and from three clones (B7, C9, and F4). The identity of the StuI–NotI fragments is indicated at left. The band identified with an asterisk is of unknown origin and probably reflects a rearrangement of pPM2GT after transfection. This figure was assembled from two experiments. wt locus, wild-type PM II locus.
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Related In: Results  -  Collection

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fig1: Creation of a chromosomal PM II–GFP chimera. (A) Schematic diagram of the integration plasmid pPM2GT linearized at the unique XhoI site (X). 1 kb of the 3′ end of the PM II coding sequence (gray box) was fused in frame to a linker sequence followed by the GFPmut2 open reading frame (white box). The amino acid sequence of the linker is shown. A WR99210-resistant variant of human dihydrofolate reductase (Fidock and Wellems, 1997) was incorporated as a selectable marker. The black bar indicates the PM II sequence used for probing Southern blots. Elements are not drawn to scale. (B) Schematic representation of events leading to a chromosomal PM II–GFP chimera. Single-site homologous recombination between the episomal pPM2GT target sequence and the chromosomal PM II locus produces the PM II–GFP chimera. The integration event produces a full-length PM II ORF (designated PM IIa) fused to GFP and a downstream promoterless copy of the PM II target sequence (PM IIb). Some elements of the plasmid, including the drug-resistance cassette, have been omitted for clarity. (C) Southern blot of StuI–NotI digested total DNA from untransfected parasites (3D7), stably transfected parasites before cycling (cycle 0) and after one and two drug cycles, and from three clones (B7, C9, and F4). The identity of the StuI–NotI fragments is indicated at left. The band identified with an asterisk is of unknown origin and probably reflects a rearrangement of pPM2GT after transfection. This figure was assembled from two experiments. wt locus, wild-type PM II locus.
Mentions: The single-copy PM II gene of P. falciparum strain 3D7 was altered to encode a proPM II–GFP fusion by modifying an established gene disruption procedure (Crabb et al., 1997a). Plasmid pPM2GT was constructed with a targeting sequence of 1 kb of the 3′ end of the PM II coding region fused in-frame to a sequence encoding a linker and the enhanced GFP variant GFPmut2 (Fig. 1 A). Parasites transfected with pPM2GT were selected with the antifolate drug WR99210 and subjected to two rounds of drug cycling to enrich the population for parasites that had integrated pPM2GT into the PM II gene (Fig. 1, B and C). Single-cell cloning of the twice-cycled culture was undertaken to obtain parasites of defined genotype. The clone studied extensively here, designated B7, contains a single copy of pPM2GT integrated into the PM II gene (Fig. 1 C).

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