Limits...
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.

Show MeSH

Related in: MedlinePlus

ALLN treatment results in accumulation of proPM II–GFP in the food vacuole membrane. (A) GFP fluorescence in a live B7 trophozoite treated with BFA for 2 h followed by replacement of BFA with 100 μM ALLN for a further 2 h. A bright rim of fluorescence circumscribes the food vacuole (arrow). A local concentration of fluorescence on the food vacuole membrane is indicated with an arrowhead. Two cytostomal vacuoles above the food vacuole are also visible. Bar, 2 μm. (B) A trophozoite treated as in A in which the Hoechst 33342–stained nucleus is pseudocolored red. (C and D) Trophozoites treated as in A and labeled with either (C) anti-GFP or (D) anti–PM II antibody. Low magnification images of these parasites are provided in Fig. S2. Abbreviations are given in the legend to Fig. 5. Bars, 200 nm.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2171955&req=5

fig7: ALLN treatment results in accumulation of proPM II–GFP in the food vacuole membrane. (A) GFP fluorescence in a live B7 trophozoite treated with BFA for 2 h followed by replacement of BFA with 100 μM ALLN for a further 2 h. A bright rim of fluorescence circumscribes the food vacuole (arrow). A local concentration of fluorescence on the food vacuole membrane is indicated with an arrowhead. Two cytostomal vacuoles above the food vacuole are also visible. Bar, 2 μm. (B) A trophozoite treated as in A in which the Hoechst 33342–stained nucleus is pseudocolored red. (C and D) Trophozoites treated as in A and labeled with either (C) anti-GFP or (D) anti–PM II antibody. Low magnification images of these parasites are provided in Fig. S2. Abbreviations are given in the legend to Fig. 5. Bars, 200 nm.

Mentions: To better visualize proPM II–GFP fluorescence against a background of GFP in the food vacuole, B7 parasites were first treated with BFA for 2 h to accumulate proPM II–GFP, and then the BFA was washed out in the presence of 100 μM ALLN. A marked redistribution of proPM II–GFP was observed in live parasites upon replacement of BFA with ALLN. The perinuclear ring of fluorescence observed upon BFA treatment disappeared and was replaced by a fluorescent rim around the food vacuole (Fig. 7, A and B). In many parasites, regions of more intense fluorescence could be observed within this rim (Fig. 7 A). These may reflect a local concentration of proPM II–GFP in the food vacuole membrane, or may arise from the docking of transport vesicles to the food vacuole; an explanation for this phenomenon was not obvious from immunoEM experiments. In addition, peripheral fluorescent spots indicative of cytostomal vacuoles were seen (Fig. 7 A). The observation of fluorescence circumscribing the food vacuole suggested that the integral membrane protein proPM II–GFP had been transported to the food vacuole and, in the absence of cleavage of proPM II or GFP, remained anchored in the food vacuole membrane. To confirm this idea, B7 parasites treated sequentially with BFA and ALLN were analyzed by immunoEM with either anti-GFP or anti–PM II antibody. In contrast to the lumenal labeling observed with untreated parasites (Fig. 2 C and Fig. 5 A), BFA/ALLN-treated parasites exhibited accumulation of gold label primarily at the food vacuole membrane (Fig. 7, C and D). Control experiments confirmed that parasites remained viable throughout the BFA and ALLN treatments.


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)

ALLN treatment results in accumulation of proPM II–GFP in the food vacuole membrane. (A) GFP fluorescence in a live B7 trophozoite treated with BFA for 2 h followed by replacement of BFA with 100 μM ALLN for a further 2 h. A bright rim of fluorescence circumscribes the food vacuole (arrow). A local concentration of fluorescence on the food vacuole membrane is indicated with an arrowhead. Two cytostomal vacuoles above the food vacuole are also visible. Bar, 2 μm. (B) A trophozoite treated as in A in which the Hoechst 33342–stained nucleus is pseudocolored red. (C and D) Trophozoites treated as in A and labeled with either (C) anti-GFP or (D) anti–PM II antibody. Low magnification images of these parasites are provided in Fig. S2. Abbreviations are given in the legend to Fig. 5. Bars, 200 nm.
© Copyright Policy
Related In: Results  -  Collection

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

fig7: ALLN treatment results in accumulation of proPM II–GFP in the food vacuole membrane. (A) GFP fluorescence in a live B7 trophozoite treated with BFA for 2 h followed by replacement of BFA with 100 μM ALLN for a further 2 h. A bright rim of fluorescence circumscribes the food vacuole (arrow). A local concentration of fluorescence on the food vacuole membrane is indicated with an arrowhead. Two cytostomal vacuoles above the food vacuole are also visible. Bar, 2 μm. (B) A trophozoite treated as in A in which the Hoechst 33342–stained nucleus is pseudocolored red. (C and D) Trophozoites treated as in A and labeled with either (C) anti-GFP or (D) anti–PM II antibody. Low magnification images of these parasites are provided in Fig. S2. Abbreviations are given in the legend to Fig. 5. Bars, 200 nm.
Mentions: To better visualize proPM II–GFP fluorescence against a background of GFP in the food vacuole, B7 parasites were first treated with BFA for 2 h to accumulate proPM II–GFP, and then the BFA was washed out in the presence of 100 μM ALLN. A marked redistribution of proPM II–GFP was observed in live parasites upon replacement of BFA with ALLN. The perinuclear ring of fluorescence observed upon BFA treatment disappeared and was replaced by a fluorescent rim around the food vacuole (Fig. 7, A and B). In many parasites, regions of more intense fluorescence could be observed within this rim (Fig. 7 A). These may reflect a local concentration of proPM II–GFP in the food vacuole membrane, or may arise from the docking of transport vesicles to the food vacuole; an explanation for this phenomenon was not obvious from immunoEM experiments. In addition, peripheral fluorescent spots indicative of cytostomal vacuoles were seen (Fig. 7 A). The observation of fluorescence circumscribing the food vacuole suggested that the integral membrane protein proPM II–GFP had been transported to the food vacuole and, in the absence of cleavage of proPM II or GFP, remained anchored in the food vacuole membrane. To confirm this idea, B7 parasites treated sequentially with BFA and ALLN were analyzed by immunoEM with either anti-GFP or anti–PM II antibody. In contrast to the lumenal labeling observed with untreated parasites (Fig. 2 C and Fig. 5 A), BFA/ALLN-treated parasites exhibited accumulation of gold label primarily at the food vacuole membrane (Fig. 7, C and D). Control experiments confirmed that parasites remained viable throughout the BFA and ALLN treatments.

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