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First published online 13 July 2004
doi: 10.1242/jcs.01237

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The Plasmodium falciparum Vps4 homolog mediates multivesicular body formation

Yale University School of Medicine, Department of Medicine, Section of Infectious Disease, 333 Cedar Street, PO Box 208022, New Haven, CT 06520-8022, USA

View larger version (68K): [in a new window]   Fig. 1. PfVps4 localizes to the cytosol of P. falciparum trophozoites. (A) Immunoblot of P. falciparum trophozoites with rabbit anti-PfVps4 (lanes 2 and 3) and mouse anti-PfVps4 (lanes 5 and 6). Lanes 1 and 4 are pre-immune serum. Lanes 2 and 3 correspond to two different preparations of P. falciparum, as do lanes 5 and 6. (B) Specific labeling with mouse anti-Vsp4 of the cytosol. ER, endoplasmic reticulum; N, nucleus; Pf, Plasmodium falciparum; DV, digestive vacuole. Bar, 0.15 µm.

View larger version (56K): [in a new window]   Fig. 2. Mutant GFP-PfVps4 localizes to punctuate structures when transiently expressed in P. falciparum. (A) Cartoon illustrating the general domain structure of the GFP-PfVps4 chimera and the mutations predicted to block ATP binding or ATP hydrolysis. (B) Pattern of GFP fluorescence in stable line of P. falciparum expressing GFP-PfVps4-WT. Corresponding phase contrast images are illustrated. (C) Pattern of GFP fluorescence in P. falciparum transiently transfected with GFP-PfVps4-E214Q. Arrows indicate the punctuate structures.

View larger version (40K): [in a new window]   Fig. 3. Subcellular fractionation of P. falciparum and T. gondii expressing GFP-PfVps4. (A) Cell homogenates (T, total cell lysate) prepared from a non-transfected control (3D7) and from P. falciparum stably expressing GFP-PfVps4-WT were fractionated into supernatant (S) and pellet (P) as described in Materials and Methods. Fractions were subsequently analyzed by immunoblot using an affinity purified rabbit anti-GFP antibody. Results are described in the text. (B) The same blot from panel A was stripped and re-analyzed using an affinity purified rabbit anti-PfVps4 antibody. (C) Wild-type T. gondii RH strain, and the RH strain stably expressing GFP-PfVps4-E214Q or GFP-PfVps4 WT were subjected to subcellular fractionation, as described in Materials and Methods. The fractions were analyzed by immunoblot using a rabbit anti-GFP antibody. Results are described in the text.

View larger version (63K): [in a new window]   Fig. 4. GFP-PfVps4-E214Q does not localize to secretory organelles in T. gondii but partially co-localizes with the early endosomal compartment and the immature rhoptry compartment. (A) GFP-PfVps4 and GFP-PfVps4-E214Q were expressed in T. gondii, and detected by GFP fluorescence. (B) The punctuate structures observed with the mutant did not co-localize with endogenous markers of mature parasite secretory organelles, including dense granules (GRA3), rhoptries (ROP2,3,4) or micronemes (MIC2). (C,D) T. gondii stably expressing wild type or mutant PfVps4 were transiently transfected with TgRab51 or with ROP2 (YEQL)-HA. Partial overlap between the GFP-PfVps4 mutant and the other compartments was observed.

View larger version (45K): [in a new window]   Fig. 5. Mutant PfVps4 does not co-localize with lipid droplets in T. gondii. (A) Parasite lipid droplets, detected with oil red O (ORO) do not co-localize with mutant PfVps4-positive structures. (B) The distribution of NBD-cholesterol which accumulates in T. gondii is altered by expression of mutant PfVps4, consistent with accumulation of cholesterol in the PfVps4-positive compartments as determined by filipin staining.

View larger version (189K): [in a new window]   Fig. 6. Mutant PfVps4 localizes to large multivesicular bodies in T. gondii. (A-C) EM of T. gondii stably expressing mutant PfVps4 showing multivesicular bodies. (D) Immunocryolabeling of T. gondii stably expressing mutant PfVps4 showing the PfVps4 distribution in multivesicular bodies. Results are described in the text. Rh, rhoptry. Bars, 0.2 µm.

View larger version (69K): [in a new window]   Fig. 7. Mutant GFP-PfVps4 generates enlarged, cholesterol-enriched vesicles when expressed in COS cells. Wild type and mutant PfVps4 were transiently expressed in COS cells. GFP fluorescence is illustrated, as is the distribution of cholesterol, detected by staining with filipin. Importantly, immunofluorescence staining of transgenic COS cells revealed precise co-localization between the GFP fluorescence and PfVps4 detected using rabbit or mouse antiserum (not shown).