Proc. Natl. Acad. Sci. USA Vol. 91, pp. 509-513, January 1994 Cell Biology The parasitophorous membrane surrounding intracellular functions as a molecular sieve (//channel/microinjection/fluorescence microscopy) J. C. SCHWAB*, C. J. M. BECKERS, AND K. A. JOINERt Department of Internal Medicine, Infectious Diseases Section, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06510-8056 Communicated by Edward A. Adelberg, September 27, 1993

ABSTRACT The obligate intracellular protozoan parasite MATERIALS AND METHODS Toxoplasma gondii creates and enters into a unique membrane- the vac- Fluorophores. Lucifer yellow dipotassium salt was pur- bounded cytoplasmic compartment, parasitophorous chased from Sigma and dissolved as a 1% aqueous solution. uole, when invading mammalian host cells. By microinjecting Chromatography-purified calcein (Molecular Probes) was 3 polar fluorescent molecules into individual T. gondii-infected mg/ml in potassium phosphate buffer (9). Fluo-3 salt (Mo- fibroblasts, we show here that the parasitophorous vacuole lecular Probes) was injected as a 1 mM aqueous solution. membrane (PVM) surrounding the parasite functions as a Texas Red dextran (3000 and 10,000 Da), rhodamine dex- molecular sieve. Lucifer yellow (457 Da) displayed free bidi- tran (70,000 Da), Texas Red ovalbumin, and lactalbumin rectional flux across the PVM and distinctly outlined the were purchased from Molecular Probes. Residual unconju- parasites, which did not take up the dye, within the vacuole. gated fluorophore was removed by size-exclusion chroma- This dye movement was not appreciably delayed by pretreat- tography and SM-2 biobeads (10) or butanol extraction (11). ment of cells with 5 mM probenecid or chilling the monolayer A series of intermediate-sized fluorescein isothiocyanate to 5°C, suggesting that dye movement was due to passive (FITC)-conjugated peptide probes was generated (Table 1). permeation through a membrane pore rather than active KF and RSR were from Sigma; SGALDVLQ is from the N transport. Calcein, fluo-3, and a series of fluorescein isothio- terminus of the 32/67-kDa laminin binding protein and was cyanate-labeled peptides up to 1291 Da crossed the PVM in a synthesized by the Yale Peptide Synthesis facility; size-restricted fashion. A labeled peptide of 1926 Da and SETRTFY is from the fibronectin collagen-binding region labeled dextrans and proteins (.3000 Da) failed to transit the and was synthesized by Lee Maloy (National Institute of PVM. This putative channel in the PVM therefore allows Allergy and Infectious Diseases, National Institutes of exchange ofmolecules up to 1300-1900 Da between the host cell Health, Bethesda, MD). ADSGEGDFLAEGGGVR is fibri- and the parasitophorous vacuolar space. nopeptide A, a cleavage product of thrombin acting on fibrinogen (purchased from Bachem). All peptides gave a Obligate intracellular parasites must exchange nutrients and single predominant absorbance peak at 220 nm when ana- metabolites with their host cell (1). Most intracellular patho- lyzed by C18 reverse-phase HPLC prior to conjugation with gens are surrounded by a vacuolar membrane, which estab- FITC. Conjugated peptides were separated from unreacted lishes a confined vacuolar space external to the pathogen but dye by HPLC and lyophilized. Peptides were dissolved for contained by the vacuolar membrane. Exchange between microinjection in potassium phosphate buffer (9) and stored host cell cytoplasm and the vacuolar space must therefore at -40°C. Molecular mass of conjugated peptides was con- occur across the vacuolar membrane. In principle, this ex- firmed by mass spectroscopy (Yale Department of Biochem- change could occur by diffusion, by transport, or by vesicular istry and Biophysics). traffic. In practice, the mechanisms for the exchange are Cells and Parasites. Human fibroblasts and the RH strain of largely unexplored. T. gondii were maintained in culture as described (2). The obligate intracellular protozoan Toxoplasma gondii Fibroblasts (-5 x 104) in aMEM/10% fetal calf serum were actively invades all nucleated cells and resides in a special- plated and incubated (37°C in 5% C02/95% air) in modified ized intracellular vacuole, which is isolated from vesicular 6-cm round tissue culture dishes. Subconfluent fibroblast traffic in the host cell within this monolayers were infected with -5 x 105 tachyzoites and (2-4). Sequestered para- incubated for an additional 18-24 h. sitophorous vacuole, the parasite salvages essential nutrients Micro'ljection. Before microinjection, the medium in the from the host cell (5, 6) and multiplies rapidly. Since plasma dishes was replaced with fresh medium containing 20 mM membrane proteins ofthe host cell that might form membrane Hepes (pH 7.2). Microinjection needles (0.6-gm tip) (12) were channels or transporters are excluded from the parasitopho- formed (P-80/PC micropipette puller; Sutter Instruments, rous vacuole membrane (PVM) (2, 7, 8), the pathway for Novata, CA) from 1-mm glass capillary tubing (World Pre- nutrient acquisition by the parasite is enigmatic. cision Instruments, Sarasota, FL) and loaded retrograde with To survey for molecular exchange occurring across the dye prepared as described above. Individual fibroblasts with PVM, we microinjected polar fluorescent molecules into the one or more discrete parasitophorous containing cytoplasm of the T. gondii-infected fibroblasts. The PVM 2-16 parasites (1-4 replications of a single invading parasite) surrounding T. gondii permits free bidirectional diffusion of were identified by phase-contrast microscopy (Zeiss Axio- small charged and uncharged molecules between the host cell vert; x40 or x 100 objective) and fluorophore microinjected cytoplasm and vacuolar space, a result most consistent with into the host cell cytoplasm using a micromanipulator and a pore of protein origin across the PVM. Abbreviations: PVM, parasitophorous vacuole membrane; FITC, fluorescein isothiocyanate. The publication costs of this article were defrayed in part by page charge *Present address: Department of Medicine, State University of New payment. This article must therefore be hereby marked "advertisement" York Health Science Center, Syracuse, NY 13210. in accordance with 18 U.S.C. §1734 solely to indicate this fact. tTo whom reprint requests should be addressed. 509 Downloaded by guest on September 28, 2021 510 Cell Biology: Schwab et al. Proc. Natl. Acad. Sci. USA 91 (1994) Table 1. Fluorescent probes used in evaluating low entered the parasitophorous vacuole space within sec- PVM permeability onds after microinjection into the cytoplasm of T. gondii- Flux across infected fibroblasts. Equilibration of fluorescence across the Polar fluorescent probe n* Da PVMt PVM was rapid and stable over 20 min of observation (Table 1; Fig. 1 A and B). In contrast, the parasite plasma membrane Small fluorophores represented a true diffusion barrier, since parasites within the Lucifer yellowt 14 457 + + vacuole did not take up dye. Probenecid (5 mM) 4 + + Vacuolar uptake of Lucifer yellow was a passive process. Temperature 5°C 5 + + Cooling of cells to 5°C prior to microinjection did not appre- Intravacuolar injection 4 + + ciably delay Lucifer yellow entry (Table 1). Pretreatment of Octanol (500 ,uM) 7 + + cells with the organic anion transport inhibitor probenecid, Calcein 3 623 + + which blocks Lucifer yellow transport from the cytosol into Fluo-3 14 781 + cellular endosomal compartments (15), likewise did not delay Peptides (charge)§ Lucifer yellow entry into the vacuolar space. This rapid, KF FITC (+1) 2 680 ++ non-carrier-mediated transfer of Lucifer yellow across the RSR FITC (+2) 6 806 ++ PVM is analogous to the dye coupling described between KF (FITC)2 (+1) 3 1,070 + cells connected by intercellular gap junctions (16, 17). Dye SGALDVLQ FITC (-1) 4 1,191 + coupling of the parasitophorous vacuole space and host cell SETRTFY FITC (0) 5 1,291 + cytoplasm is in striking contrast to the exclusion of Lucifer ADSGEGDFLAEGGGVR FITC (-3) 3 1,926 - yellow from the parasitophorous vacuole found when dye Protein and dextrans was loaded into host cell endosomal compartments (2). 3-kDa Texas Red dextran 3 3,000 - Size-Restricted Entry into the Parasitophorous Vacuole. 10-kDa Texas Red dextran 2 10,000 - Basic, neutral, and acidic peptides with a molecular mass of Texas Red lactalbumin 4 14,500 - <1300 Da crossed the PVM (Table 1). In contrast, no entry 70-kDa rhodamine dextran 5 70,000 - of a peptide of 1926 Da (Fig. 2 E and F) or Texas Red Intravacuolar injection >5 - lactalbumin (14,500 Da) (Fig. 2 G and H) was detected. *Number of T. gondii-infected cells observed. Neutral fluorescent dextrans with molecular masses of tMovement offluorescent tracers across the PVM was monitored for 70,000 (Fig. 1 C and D), 10,000 and 3000 Da were also up to 20 min after injection. + +, Extremely rapid entry, with excluded from the parasitophorous vacuole (Table 1). When intravacuolar dye silhouetting the parasites and of equal fluores- Texas Red dextran (3000 or 10,000 Da) and Lucifer yellow cence intensity to cytosolic dye at the first fluorescent observation were the same Lucifer yellow entry (15-30 s) after cytoplasmic microinjection; +, rapidly increasing injected into cell, rapid intravacuolar dye fluorescence over the observation period, with and complete exclusion of the fluorescent dextran were parasites clearly silhouetted, and fluorescence intensity equili- observed at all times. brated across the PVM before the end ofthe observation period; -, These results indicate that microinjection does not disrupt no detectable dye entry into the PVM, resulting in persistent the integrity of the PVM and that the rate of diffusion across silhouetting of the vacuole rather than the contained parasites. the PVM is influenced by solute size, an essential feature of tIn 3 of the 14 cells, Lucifer yellow was microinjected into cells channel-mediated permeation (13, 14, 18). previously microinjected with Texas Red dextran (3000 or 10,000 Calcium Equilibrates Across the PVM. The fluorescent Da). probes calcein (Fig. 2 C and D) and fluo-3, which do not cross §Charge is that of the peptide before FITC conjugation. Coupling of FITC to either the a or the e amino group will remove 1 positive unmodified biological membranes, also crossed the PVM charge from the peptide. Contribution of negatively charged FITC (Table 1). Fluo-3 (781 Da) equilibration was delayed (Fig. 3) molecule to overall charge of conjugated peptides is not defined. in comparison to cells injected with Lucifer yellow (457 Da) or calcein (623 Da), requiring several minutes for equilibra- pressure microinjector (Narishige, Greenvale, NY). Dye tion, again consistent with a size-dependent rate of entry. redistribution across the parasitophorous vacuole membrane Fluo-3 fluorescence equilibration suggests that ionized was observed for up to 20 min after microinjection and images calcium also equilibrates across the PVM. The fluorescence were acquired on film (Tri X Pan 400ASA) or on optical intensity offluo-3 depends not only on cell thickness and dye memory disk (Panasonic) using a SIT camera (Dage-MTI, concentration but also on the local ionized calcium concen- Michigan City, IN). Cells damaged by microinjection (9) or in tration (19). To confirm that the fluo-3 fluorescence within which parasites subsequently reoriented within or exited the vacuole was calcium sensitive, two cells that had been from the vacuole were excluded from further study. Prelim- microinjected with fluo-3 were subsequently injected with 1 inary experiments demonstrated that Lucifer yellow micro- mM CaCl2 in 150 mM KCI solution. This resulted in an injection did not cause parasites to exit the host cell upon immediate and simultaneous increase in fluo-3 fluorescence reincubation at 37°C or inhibit the next cycle of parasite in both cytoplasm and vacuolar space. Of interest, this was division. followed within seconds by initiation ofactive parasite move- Experiments were performed to evaluate the effect of ment and breakout from the vacuole, extending earlier ob- potential inhibitors on Lucifer yellow flux across the PVM. servations that the calcium ionophore A23187 triggers rapid The organic anion transport inhibitor probenecid (5 mM; parasite exit from infected cells (20). Sigma) was added to monolayers in aMEM/20 mM Hepes Dye Movement Across the PVM Is Bidirectional with the 15-30 min before microinjection. Octanol (500 ,uM; Sigma) Host Cell Cytoplasm. Microinjection of Lucifer yellow di- was added to the monolayer 1-15 min before microinjection. rectly into the parasitophorous vacuole space showed rapid Injection at 5°C was performed by cooling the monolayer on spread of dye to the host cell cytoplasm with equilibration the microscope stage with - 17°C saline pumped through across the PVM. In contrast, when rhodamine dextran of 70 coiled HPLC tubing immersed in the medium. Microinjection kDa was injected into parasitophorous vacuoles (Table 1), was otherwise performed at ambient (24°C) temperature. fluorescence was stably restricted to the vacuolar space over 20 min of observation. Similar stable retention of large polar fluorescent markers within the parasitophorous vacuole has RESULTS been demonstrated previously (4, 21). These results indicate Dye Coupling Between the Host Cell Cytoplasm and Para- that controlled injection ofthe vacuole does not disrupt PVM sitophorous Vacuole. The polar fluorescent dye Lucifer yel- integrity, that passage of small molecules and retention of Downloaded by guest on September 28, 2021 Cell Biology: Schwab et al. Proc. Natl. Acad. Sci. USA 91 (1994) 511

N

A I

* C Ir

FIG. 1. Parasitophorous vacuole entry versus exclusion of polar fluorescent molecules microinjected into T. gondii-infected fibroblasts. (A) Four parasites (shaded) resulting from two replications of a single invading parasite and surrounded by a single PVM (arrow) are located in the cytoplasm of a host fibroblast (N; nucleus). (B) Fluorescence image. Following microinjection of Lucifer yellow, dye enters the vacuole and silhouettes the parasites, which do not take up the dye. The nucleus displays increased tracer relative to the cytoplasm, a finding also observed by others (13, 14). (C) A different T. gondii-infected fibroblast, with vacuoles containing either two or four (arrow) parasites. (D) Corresponding fluorescence image 20 min after microinjection ofneutral rhodamine dextran (70 kDa) reveals that this dye is excluded from each parasitophorous vacuole and from the host cell nucleus. (Bar = 10 ,um.)

large molecules occur on both faces of the PVM, and that a infected fibroblasts with the gap junction uncoupler octanol functional connection between the parasitophorous vacuole (17, 31) (500 ,M) did not inhibit Lucifer yellow entry into the and the extracellular space is unlikely given the stability of vacuole (Fig. 2 A and B). Therefore, gap junction proteins of the dye fluorescence. host cell origin seem less likely to form the pore across the PVM. Using patch clamping, Desai et al. (32) recently DISCUSSION reported the presence within the PVM surrounding Plasmo- diumfalciparum of 140-pS channels, which were open >98% The PVM of T. gondii functions as a molecular sieve. Small of the time, and were permeable to small charged molecules molecules and ions cross the PVM at a rate inversely pro- (i.e., lysine, glucuronate). The pores in the T. gondii PVM portional to their size. This sieving effect is present in permitted passage of much larger molecules than those vacuoles of different ages, containing 2-16 parasites (Figs. 1 studied by Desai. In addition, our approach (unlike patch and 2), indicating that putative channels or pores are stably clamping) can evaluate flux of compounds independent of retained as and/or constantly replenished the vacuole en- charge. Nonetheless, the functional pores we in larges. We favor the notion that form the observe the parasite proteins PVM surrounding T. gondii may be analogous to the channels pore across the PVM. Parasite proteins derived from two in different secretory , the and dense gran- the P. falciparum PVM. ules, are associated with the PVM and could contribute to Membranous tubules and vesicles of parasite origin are pore formation (refs. 22-26 and t). Association of parasite observed in the cytoplasm of erythrocytes infected with the proteins with the PVM has also been reported in related sporozoan parasite Plasmodium sp. (33-35). Al- sp. (ref. 27; reviewed in ref. 28). though these structures may function in phospholipid transfer The permeability characteristics of the T. gondii PVM are between host and parasite (35), they do not appear to mediate similar to those described for gap junctions between cells transfer of fluid-phase markers between the vacuole and (29). Infection with another intracellular protozoan parasite, cytoplasm. Pouvelle et al. (36) reported the presence of Trypanosoma cruzi, alters gap junction distribution in in- membranous tubules, termed parasitophorous ducts, con- fected cells (30). In our experiments, however, treatment of necting the P. falciparum parasitophorous vacuole with the plasma membrane of the infected erythrocyte. These struc- tBeckers, C. J. M., Dubremetz, J. F. & Joiner, K. A., Molecular tures admitted probes as large as 30-nm fluorescent latex Parasitology Meeting, Marine Biological Laboratories, September beads. Earlier studies with T. gondii (4, 21), combined with 13-16, 1992, Woods Hole, MA, abstr. 130. our results showing retention of dextran (70 kDa) within the Downloaded by guest on September 28, 2021 512 Cell Biology: Schwab et al. Proc. Natl. Acad. Sci. USA 91 (1994)

FIG. 2. Size-related entry of polar fluorescent probes into the parasitophorous vacuole. Phase- contrast (A, C, E, and G) and corresponding fluorescence (B, D, X | | 11 -_F, and H) images are shown for Ipi cells 2 (B and D), 10 (F), or 20 (H) min after cytoplasmic injection of the fluorescent probes. (A and B) Lucifer yellow in a fibroblast pre- treated with 500 ,uM octanol. (C and D) Calcein. (E and F) ADS- GEGDFLAEGGGVR FITC. (G and H) Texas Red lactalbumin. All probes enter the host cell nu- cleus. Lucifer yellow (B) and cal- cein (D) permeate and equilibrate across the PVM (arrow), silhou- etting the parasites. The larger peptide (F) and lactalbumin (H) are excluded by the PVM (arrow). Each fibroblast in this panel con- tained only a single parasitopho- vacuole. (Bars = 10 ,um.) ._ .....rous vacuolar space, preclude a direct connection between the By acting as a molecular sieve, the PVM is functionally parasitophorous vacuole and the extracellular space. analogous to the outer membrane of prokaryotic Gram-

F 3. F. ..eq ilbrte

3D _i_ ~~~~~~~~~~~~~~~~~~~~FIG.3. Fluo-3 equilibrates across the PVM over minutes. (A) Phase-contrast image of parasites within a parasitophorous vacuole near the host fibroblast nucleus. (B-D) Fluorescence images 0.5, 2, and 5 min after microinjection of fluo-3 into the fibroblast cyto- plasm. In B, the parasitophorous vacuole is silhouetted (arrow). C and D reveal progressive entry of dye into the vacuole with silhou- etting of the contained parasites and equilibration of fluorescence intensity between cytosol and vac- uole. (Bar = 10 ,um.) Downloaded by guest on September 28, 2021 Cell Biology: Schwab et al. Proc. Natl. Acad. Sci. USA 91 (1994) 513 negative bacteria and eukaryotic mitochondrial and chloro- associated with free-living prokaryotic organisms and eu- plast organelles, in which transmembrane porin proteins form karyotic organelles of putative endosymbiotic origin. nonselective membrane channels (37). In this paradigm, the parasitophorous vacuole space would be analogous to the The authors wish to thank Jonathan Izant and Alan Fanning (Yale University) for use of the microinjection equipment and helpful bacterial periplasmic space or the mitochondrial intermem- advice. The authors also acknowledge the generous assistance of branous space. The impermeable parasite plasma membrane Eric Pamer (Yale University) in purification of FITC-conjugated would be the analog of the bacterial or mitochondrial inner peptides and thank Michael Caplan, Ira Mellman, and Michael membrane and provide specificity for transport of nutrients Nathanson (Yale University) for critical reading of the manuscript. and metabolites (Fig. 4). This work was supported by Public Health Service Grants ROlAI 30060 and U01 Al 31808 from the National Institute of Allergy and The intracellular metabolic requirements of T. gondii are Infectious Diseases to K.A.J. and fellowships to J.C.S. from Miles incompletely characterized. Nonetheless, our data suggest Pharmaceuticals and the American Philosophical Society (Daland that amino acids and monosaccharides should readily transit Fellowship). across the PVM without the requirement for specific trans- porters. Similarly, purines, which the parasite must salvage 1. Moulder, J. W. (1985) Microbiol. Rev. 49, 298-337. 2. Joiner, K. A., Fuhrman, S. A., Miettinen, H. M., Kasper, L. H. & from the host cell (5, 6, 38), should freely permeate the Mellman, I. (1990) Science 249, 641-646. parasitophorous vacuole. ATP, present in cells at high con- 3. Jones, T. C., Yeh, S. & Hirsch, J. G. (1972)J. Exp. Med. 136, 1157-1172. alternative 4. Sibley, L. D., Weidner, E. & Krahenbuhl, J. L. 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(1983) Methods Enzymol. 101, products could freely exit the vacuole. The electrolyte com- 482-492. 10. Spack, E. G., Packard, B., Wier, M. L. & Edidin, M. (1986) Anal. position of the vacuolar space should reflect that of the Biochem. 158, 233-237. infected host cell cytoplasm. 11. Hosoi, K., Shioda, Y., Kodama, A., Sugita, K., Kurihara, K., Murai, T., The obligate intracellular protozoan parasite T. gondii has Nemoto, A., Atsumi, T. & Ueha, T. (1989) Jpn. J. Physiol. 39, 317-324. 12. Mittman, S., Flaming, D. G., Copenhagen, D. R. & Belgum, J. H. (1987) free access to small cytoplasmic molecules ofthe host cell but J. Neurosci. Methods 22, 161-166. is isolated from the host cell vesicular transport system. 13. Paine, P. L., Moore, L. C. & Horowitz, S. B. (1975) Nature (London) Formation of the specialized PVM is a sophisticated exten- 254, 109-114. 14. Peters, P. (1986) Biochem. Biophys. Acta 864, 305-359. sion of a scheme of membrane organization more commonly 15. Steinberg, T. H., Newman, A. S., Swanson, J. A. & Silverstein, S. C. (1987) J. Cell Biol. 105, 2695-2702. 16. Stewart, W. W. (1978) Cell 14, 741-759. 17. Saez, J. C., Connor, J. A., Spray, D. C. & Bennett, M. V. L. (1989) Proc. Natl. Acad. Sci. USA 86, 2708-2712. 18. Stein, W. D. (1986) Transport and Diffusion Across Cell Membranes (Academic, Orlando, FL), pp. 114-230. 19. Minta, A., Kao, J. P. Y. & Tsien, R. Y. (1989) J. Biol. Chem. 264, 8171-8178. 20. Endo, T., Sethi, K. K. & Piekarski, G. (1982) Exp. Parasitol. 53, 179-188. 21. Tanabe, K. & Murakami, K. (1984) J. Cell Sci. 70, 73-81. 22. Achbarou, A., Mercereau-Puijalon, O., Sadak, A., Fortier, B., Leriche, M. A., Camus, D. & Dubremetz, J. F. (1991) Parasitology 103, 321-329. 23. Linder, E., Thors, C., Edberg, F., Haglund, S. & von Bonsdorff, C.-H. (1992) Parasitol. Res. 78, 175-178. 24. Nichols, B. A., Chiappino, M. L. & O'Connor, G. R. (1983) J. Ultra- struct. Res. 83, 85-98. 25. Kimata, I. & Tanabe, K. (1987) J. Cell Sci. 88, 231-239. 26. Saffer, L., Mercereau-Puijalon, O., Dubremetz, J.-F. & Schwartzman, J. D. (1992) J. Protozool. 39, 526-530. 27. Sam-Yellowe, T. Y., Shio, H. & Perkins, M. E. (1988) J. Cell Biol. 106, 1507-1513. 28. Bannister, L. H. & Dluzewski, A. R. (1990) Blood Cells 16, 257-292. 29. Simpson, I., Rose, B. & Loewenstein, W. R. (1977) Science 195, 294-296. 30. de Carvalho, A. C. C., Tanowitz, H. B., Wittner, M., Dermietzel, R., Roy, C., Hertzberg, E. L. & Spray, D. C. (1992) Circ. Res. 70,733-742. 31. Nathanson, M. H. & Burgstahler, A. D. (1992) Mol. Biol. Cell 3, 113- 121. 32. Desai, S. A., Krogstad, D. J. & McCleskey, E. W. (1993) Nature (Lon- FIG. 4. Schematic of potential nutrient salvage pathways of don) 362, 643-646. intracellular T. gondii. Parasite (Tg) resides in a nucleated host cell 33. Bamwell, J. W. (1990) Blood Cells 16, 379-395. surrounded by a PVM. Nonspecific PVM pores (1) allow rapid 34. Haldar, K. & Uyetake, L. (1992) Mol. Biochem. Parasitol. 50, 161-178. exchange of small molecules of <1300-1900 Da (substrate S) be- 35. Haldar, K. (1992) Infect. Agents Dis. 1, 254-262. tween the host cell cytoplasm and parasitophorous vacuole (PV). 36. Pouvelle, B., Spiegel, R., Hsiao, L., Howard, R. J., Morris, R. L., Thomas, A. P. & Taraschi, T. F. (1991) Nature (London) 353, 73-75. Upon entering the PV, S may be directly salvaged across the parasite 37. Benz, R. (1985) CRC Crit. Rev. Biochem. 19, 145-190. plasma membrane (2) or potentially modified to S' either by parasite 38. Krug, E. C., Marr, J. J. & Berens, R. L. (1989) J. Biol. Chem. 264, proteins secreted into the PV (3) or by enzymes present on the 10601-10607. parasite plasma membrane (4) prior to transport across the parasite 39. Wallach, D. F. H., Surgenor, D. M., Soderberg, J. & Delano, E. (1959) plasma membrane (5). For clarity, only a single parasite is shown. Anal. Chem. 31, 456-460. Downloaded by guest on September 28, 2021