Anionic sites,fucose residues and class I human leukocyte antigen fate during interaction of Toxoplasma gondii with endothelial cells

Toxoplasma gondii invades and proliferates in human umbilical vein endothelial cells where it resides in a parasitophorous vacuole. In order to analyze which components of the endothelial cell plasma membrane are internalized and become part of the parasitophorous vacuole membrane, the culture of endothelial cells was labeled with cationized ferritin or UEA I lectin or anti Class I human leukocyte antigen (HLA) before or after infection with T. gondii. The results showed no cationized ferritin and UEA I lectin in any parasitophorous vacuole membrane, however, the Class I HLA molecule labeling was observed in some endocytic vacuoles containing parasite until 1 h of interaction with T. gondii. After 24 h parasite-host cell interaction, the labeling was absent on the vacuolar membrane, but presents only in small vesicles near parasitophorous vacuole. These results suggest the anionic site and fucose residues are excluded at the time of parasitophorous vacuole formation while Class I HLA molecules are present only on a minority of Toxoplasma-containing vacuoles.     

Presence of a Na+-stimulated P-type ATPase in the plasma membrane of the alkaliphilic halotolerant cyanobacterium Aphanothece halophytica

Aphanothece cells could take up Na(+) and this uptake was strongly inhibited by the protonophore, carbonyl cyanide m-chlorophenylhydrazone (CCCP). Cells preloaded with Na(+) exhibited Na(+) extrusion ability upon energizing with glucose. Na(+) was also taken up by the plasma membranes supplied with ATP and the uptake was abolished by gramicidin D, monensin or Na(+)-ionophore. Orthovanadate and CCCP strongly inhibited Na(+) uptake, whereas N, N'-dicyclohexylcarbodiimide (DCCD) slightly inhibited the uptake. Plasma membranes could hydrolyse ATP in the presence of Na(+) but not with K(+), Ca(2+) and Li(+). The K(m) values for ATP and Na(+) were 1.66+/-0.12 and 25.0+/-1.8 mM, respectively, whereas the V(max) value was 0.66+/-0.05 mumol min(-1) mg(-1). Mg(2+) was required for ATPase activity whose optimal pH was 7.5. The ATPase was insensitive to N-ethylmaleimide, nitrate, thiocyanate, azide and ouabain, but was substantially inhibited by orthovanadate and DCCD. Amiloride, a Na(+)/H(+) antiporter inhibitor, and CCCP showed little or no effect. Gramicidin D and monensin stimulated ATPase activity. All these results suggest the existence of a P-type Na(+)-stimulated ATPase in Aphanothece halophytica. Plasma membranes from cells grown under salt stress condition showed higher ATPase activity than those from cells grown under nonstress condition.     

Penetration of Toxoplasma gondii into host cells induces changes in the distribution of the mitochondria and the endoplasmic reticulum.

Fluorescence microscopy, using dyes which specifically label mitochondria, endoplasmic reticulum and the Golgi complex, and transmission electron microscopy, were used to analyze the changes which occur in the organization of these structures during interaction of Toxoplasma gondii with host cells. In uninfected cells the mitochondria are long filamentous structures which radiate from the nuclear region toward the cell periphery. After parasite penetration they become shorter and tend to concentrate around the parasite-containing vacuole (parasitophorous vacuole) located in the cytoplasm of the host cell. The mitochondria of extracellular parasites, but not of those located within the parasitophorous vacuole, were also stained by rhodamine 123. Labeling with DiOC6, which binds to elements of the endoplasmic reticulum, in association with transmission electron microscopy, revealed a concentration of this structure around the parasitophorous vacuole. The membrane lining this vacuole was also stained, suggesting that components of the endoplasmic reticulum are also incorporated into this membrane. The Golgi complex, as revealed by staining with NBD-ceramide and electron microscopy, maintains its perinuclear position throughout the evolution of the intracellular parasitism.     

Subcellular antigen location influences T-cell activation during acute infection with Toxoplasma gondii

Effective control of the intracellular protozoan parasite Toxoplasma gondii depends on the activation of antigen-specific CD8(+) T-cells that manage acute disease and prevent recrudescence during chronic infection. T-cell activation in turn, requires presentation of parasite antigens by MHC-I molecules on the surface of antigen presenting cells. CD8(+) T-cell epitopes have been defined for several T. gondii proteins, but it is unclear how these antigens enter into the presentation pathway. We have exploited the well-characterized model antigen ovalbumin (OVA) to investigate the ability of parasite proteins to enter the MHC-I presentation pathway, by engineering recombinant expression in various organelles. CD8(+) T-cell activation was assayed using 'B3Z' reporter cells in vitro, or adoptively-transferred OVA-specific 'OT-I' CD8(+) T-cells in vivo. As expected, OVA secreted into the parasitophorous vacuole strongly stimulated antigen-presenting cells. Lower levels of activation were observed using glycophosphatidyl inositol (GPI) anchored OVA associated with (or shed from) the parasite surface. Little CD8(+) T-cell activation was detected using parasites expressing intracellular OVA in the cytosol, mitochondrion, or inner membrane complex (IMC). These results indicate that effective presentation of parasite proteins to CD8(+) T-cells is a consequence of active protein secretion by T. gondii and escape from the parasitophorous vacuole, rather than degradation of phagocytosed parasites or parasite products.     

Ion metabolism in malaria-infected erythrocytes

Malaria parasites of the genus Plasmodium spend much of their asexual life cycle inside the erythrocytes of their vertebrate hosts. Parasites presumably have to exploit metabolic and transport mechanisms to adapt themselves to the host erythrocyte's physicochemical environment. This review surveys the metabolism and transport of Ca2+, alkali cations, and H+ in malaria-infected erythrocytes. The Ca2+ content of Plasmodium-infected erythrocytes increases as the parasite matures. An increase in the influx of extracellular Ca2+ into infected erythrocytes is evident at later stages of parasite development. In infected erythrocytes, Ca2+ is almost exclusively localized in the parasite compartment and changes but little in the cytosol of the host cell. The importance of Ca2+ in supporting the growth of intraerythrocytic parasites and the invasion of erythrocytes by the merozoite has been assessed by depletion of extracellular Ca2+ with chelators, or by disturbance of the metabolism and transport of Ca2+ with a variety of Ca2+ modulators. Membranes of malaria-infected erythrocytes change their permeability to alkali cations. Hence, levels of K+ decrease and levels of Na+ increase in the cytosol of infected erythrocytes. Intraerythrocytic parasites maintain a high K+, low Na+ state, suggesting a mechanism for transporting K+ inward and Na+ outward against concentration gradients of the alkali cations across the parasite plasma membrane and/or the parasitophorous vacuole membrane (PVM). Concomitantly, P. falciparum can grow in Na(+)-enriched human erythrocytes. Experimental evidence suggests that Plasmodium possesses in its plasma membrane a proton pump which is very sensitive to orthovanadate, carbonylcyanide m-chlorophenylhydrazone, a protonophore, and dicyclohexylcarbodiimide, an inhibitor of H(+)-ATPase, but is only slightly sensitive to inhibitors of bacterial and mitochondrial respiration, such as antimycin A, CN-, or N3-, and ouabain, a Na+, K(+)-ATPase inhibitor. By operating this proton pump, parasites extrude H+ and thus generate an electrochemical gradient of protons (an internal negative membrane potential and a concentration gradient of protons) across the parasite plasma membrane. The electrochemical gradient apparently drives inward movement of Ca2+ and, possibly, glucose from the cytosol of infected erythrocytes. Little is known about the transport properties of the PVM. Recent sequence studies suggest that Plasmodium contains a cation-transporting ATPase which exhibits a high homology to the Ca2(+)-ATPase of rabbit skeletal muscle sarcoplasmic reticulum.(ABSTRACT TRUNCATED AT 250 WORDS)     

Molecular partitioning during host cell penetration by Toxoplasma gondii

During invasion by Toxoplasma gondii, host cell transmembrane proteins are excluded from the forming parasitophorous vacuole membrane (PVM) by the tight apposition of host and parasite cellular membranes. Previous studies suggested that the basis for the selective partitioning of membrane constituents may be a preference for membrane microdomains, and this hypothesis was herein tested. The partitioning of a diverse group of molecular reporters for raft and nonraft membrane subdomains was monitored during parasite invasion by time-lapse video or confocal microscopy. Unexpectedly, both raft and nonraft lipid probes, as well as both raft and nonraft cytosolic leaflet proteins, flowed unhindered past the host-parasite junction into the PVM. Moreover, neither a raft-associated type 1 transmembrane protein nor its raft-dissociated counterpart accessed the PVM, while a multispanning membrane raft protein readily did so. Considered together with previous data, these studies demonstrate that selective partitioning at the host-parasite interface is a highly complex process, in which raft association favors, but is neither necessary nor sufficient for, inclusion into the T. gondii PVM.     

The Toxoplasma gondii rhoptry protein ROP 2 is inserted into the parasitophorous vacuole membrane, surrounding the intracellular parasite, and is exposed to the host cell cytoplasm

The origin of the vacuole membrane surrounding the intracellular protozoan parasite Toxoplasma gondii is not known. Although unique secretory organelles, the rhoptries, discharge during invasion of the host cell and may contribute to the formation of this parasitophorous vacuole membrane (PVM), no direct evidence for this hypothesis exists. Using a novel approach we have determined that parasite-encoded proteins are present in the PVM, exposed to the host cell cytoplasm. In infected cells incubated with streptolysin-O or low concentrations of digitonin, the host cell plasma membrane was selectively permeabilized without significantly affecting the integrity of the PVM. Antisera prepared against whole parasites or a parasite fraction enriched in rhoptries and dense granules reacted with the PVM in these permeabilized cells, indicating that parasite-encoded antigens were exposed on the cytoplasmic side of the PVM. Parasite antigens responsible for this staining of the PVM were identified by fractionating total parasite proteins by SDS-PAGE and velocity sedimentation, and then affinity purifying "fraction-specific" antibodies from the crude antisera. Proteins responsible for the PVM- staining, identified with fraction-specific antibodies, cofractionated with known rhoptry proteins. The gene encoding one of the rhoptry proteins, ROP 2, was cloned and sequenced, predicting and integral membrane protein. Antibodies specific for ROP 2 reacted with the intact PVM. These results provide the first direct evidence that rhoptry contents participate in the formation of the PVM of T. gondii and suggest a possible role of ROP 2 in parasite-host cell interactions.     

Abolishment of proton pumping and accumulation in the E1P conformational state of a plant plasma membrane H+-ATPase by substitution of a conserved aspartyl residue in transmembrane segment 6

The plasma membrane H(+)-ATPase AHA2 of Arabidopsis thaliana, which belongs to the P-type ATPase superfamily of cation-transporting ATPases, pumps protons out of the cell. To investigate the mechanism of ion transport by P-type ATPases we have mutagenized Asp(684), a residue in transmembrane segment M6 of AHA2 that is conserved in Ca(2+)-, Na(+)/K(+)-, H(+)/K(+)-, and H(+)-ATPases and which coordinates Ca(2+) ions in the SERCA1 Ca(2+)-ATPase. We describe the expression, purification, and biochemical analysis of the Asp(684) --> Asn mutant, and provide evidence that Asp(684) in the plasma membrane H(+)-ATPase is required for any coupling between ATP hydrolysis, enzyme conformational changes, and H(+)-transport. Proton pumping by the reconstituted mutant enzyme was completely abolished, whereas ATP was still hydrolyzed. The mutant was insensitive to the inhibitor vanadate, which preferentially binds to P-type ATPases in the E(2) conformation. During catalysis the Asp(684) --> Asn enzyme accumulated a phosphorylated intermediate whose stability was sensitive to addition of ADP. We conclude that the mutant enzyme is locked in the E(1) conformation and is unable to proceed through the E(1)P-E(2)P transition.     

Anticipating antiport in P-type ATPases

Cation-transporting P-type ATPases show a high degree of structural and functional homology. Nevertheless, for many members of this large family, the molecular mechanism of transport is unclear; namely, whether transport is electrogenic or not and if countertransport is involved remains to be established. In a few well-studied cases such as the Na(+)-K(+)-ATPase, plasma membrane Ca(2+) ATPase (PMCA) and sarcoplasmic reticulum Ca(2+) ATPase (SERCA) countertransport has been clearly demonstrated. New data based on the crystal structure of SERCA now strongly indicate that countertransport could be mandatory for all P-type ATPases. This concept should be verified for other known and for all newly characterized P-type ATPases.     

Trafficking of PfExp1 to the parasitophorous vacuolar membrane of Plasmodium falciparum is independent of protein folding and the PTEX translocon

Having entered the mature human erythrocyte, the malaria parasite survives and propagates within a parasitophorous vacuole, a membrane‐bound compartment separating the parasite from the host cell cytosol. The bounding membrane of this vacuole, referred to as the parasitophorous vacuolar membrane (PVM), contains parasite‐encoded proteins, but how these membrane proteins are trafficked to the PVM remains unknown. Here, we have studied the trafficking of PfExp1 to the PVM. We find that trafficking of PfExp1 to the PVM is independent of the folding state of the protein and also continues unabated upon inactivation of the PVM translocon Plasmodium Translocon of Exported proteins (PTEX). Our data strongly suggest that the trafficking of membrane proteins to the PVM occurs by as yet unknown mechanism, potentially unique to Plasmodium.     

Evidence for host cells as the major contributor of lipids in the intravacuolar network of Toxoplasma-infected cells

The intracellular parasite Toxoplasma gondii develops inside a parasitophorous vacuole (PV) that derives from the host cell plasma membrane during invasion. Previous electron micrograph images have shown that the membrane of this vacuole undergoes an extraordinary remodeling with an extensive network of thin tubules and vesicles, the intravacuolar network (IVN), which fills the lumen of the PV. While dense granule proteins, secreted during and after invasion, are the main factors for the organization and tubulation of the network, little is known about the source of lipids used for this remodeling. By selectively labeling host cell or parasite membranes, we uncovered evidence that strongly supports the host cell as the primary, if not exclusive, source of lipids for parasite IVN remodeling. Fluorescence recovery after photobleaching (FRAP) microscopy experiments revealed that lipids are surprisingly dynamic within the parasitophorous vacuole and are continuously exchanged or replenished by the host cell. The results presented here suggest a new model for development of the parasitophorous vacuole whereby the host provides a continuous stream of lipids to support the growth and maturation of the PVM and IVN.     

The Toxoplasma gondii parasitophorous vacuole membrane: transactions across the border

The obligate intracellular protozoan Toxoplasma gondii establishes its replication permissive niche within the infected host cell. This niche, the parasitophorous vacuole (PV), is delimited from the host cell cytoplasm by the PV membrane (PVM). In this chapter we highlight the roles of the PVM in the remodeling of host cell architecture, nutrient acquisition, the manipulation of signaling, and touch upon the potential roles in the parasite developmental cycle. We further present the PVM as a unique and dynamic "organelle" found only within the infected cell where it is established outside the parent organism. Despite its importance little is known about the biology of the PVM. There has, however, been a recent renewal of interest in the PVM, the study of which has become more tractable with the application of both classical approaches as well as genomic and proteomic analyses. In this review we discuss the diverse activities associated with the PVM and present pressing questions that remain to be elucidated regarding this enigmatic organelle.     

Calcium and Hydrogen Ion Concentrations in the Parasitophorous Vacuoles of Epithelial Cells Infected with the Microsporidian Encephalitozoon hellem

ABSTRACT. Microsporidia of the genus Encephalitozoon undergo merogony and sporogony in a parasitophorous vacuole within the host cell. Cultured green monkey kidney cells infected with Encephalitozoon hellem were loaded with the fluorescent dyes fura-2 or BCECF in order to measure intracellular concentrations of calcium and hydrogen ions respectively. Both the parasitophorous vacuole calcium concentration and pH values resembled those of the host cell cytoplasm in infected cells. Calcein entered the parasitophorous vacuole but not other host cell vacuoles or parasite stages within the parasitophorous vacuole. The lack of a pH or calcium concentration gradient across the parasitophorous vacuole membrane and the permeability of this membrane to a large anion such as calcein suggest that the vacuole membrane surrounding E. hellem resembles that surrounding some other intracellular parasites such as Toxoplasma gondii. A potential role is discussed for the parasitophorous vacuole calcium concentration in germination in situ.     

The p-type ATPase superfamily

P-type ATPases function to provide homeostasis in higher eukaryotes, but they are essentially ubiquitous, being found in all domains of life. Thever and Saier [J Memb Biol 2009;229:115-130] recently reported analyses of eukaryotic P-type ATPases, dividing them into nine functionally characterized and 13 functionally uncharacterized (FUPA) families. In this report, we analyze P-type ATPases in all major prokaryotic phyla for which complete genome sequence data are available, and we compare the results with those for eukaryotic P-type ATPases. Topological type I (heavy metal) P-type ATPases predominate in prokaryotes (approx. tenfold) while type II ATPases (specific for Na(+),K(+), H(+) Ca(2+), Mg(2+) and phospholipids) predominate in eukaryotes (approx. twofold). Many P-type ATPase families are found exclusively in prokaryotes (e.g. Kdp-type K(+) uptake ATPases (type III) and all ten prokaryotic FUPA familes), while others are restricted to eukaryotes (e.g. phospholipid flippases and all 13 eukaryotic FUPA families). Horizontal gene transfer has occurred frequently among bacteria and archaea, which have similar distributions of these enzymes, but rarely between most eukaryotic kingdoms, and even more rarely between eukaryotes and prokaryotes. In some bacterial phyla (e.g. Bacteroidetes, Flavobacteria and Fusobacteria), ATPase gene gain and loss as well as horizontal transfer occurred seldom in contrast to most other bacterial phyla. Some families (i.e. Kdp-type ATPases) underwent far less horizontal gene transfer than other prokaryotic families, possibly due to their multisubunit characteristics. Functional motifs are better conserved across family lines than across organismal lines, and these motifs can be family specific, facilitating functional predictions. In some cases, gene fusion events created P-type ATPases covalently linked to regulatory catalytic enzymes. In one family (FUPA Family 24), a type I ATPase gene (N-terminal) is fused to a type II ATPase gene (C-terminal) with retention of function only for the latter. Several pseudogene-encoded nonfunctional ATPases were identified. Genome minimalization led to preferential loss of P-type ATPase genes. We suggest that in prokaryotes and some unicellular eukaryotes, the primary function of P-type ATPases is protection from extreme environmental stress conditions. The classification of P-type ATPases of unknown function into phylogenetic families provides guides for future molecular biological studies.     

Inverted topology of the Toxoplasma gondii ROP5 rhoptry protein provides new insights into the association of the ROP2 protein family with the parasitophorous vacuole membrane

Toxoplasma gondii, as many intracellular parasites, is separated from the cytosol of its host cell by a parasitophorous vacuole membrane (PVM). This vacuole forms during host cell invasion and parasite apical organelles named rhoptries discharge proteins that associate with its membrane during this process. We report here the characterization of the rhoptry protein ROP5, which is a new member of the ROP2 family. Contrasting with what is known for other ROP2 family proteins, ROP5 is not processed during trafficking to rhoptries. We show here that ROP5 is secreted during invasion and associates with the PVM. Using differential permeabilization of infected cells, we have shown that ROP5 exposes its C-terminus towards the host cell cytoplasm, which corresponds to a reverse topology compared with ROP2 and ROP4. Taken together with recent modelling data suggesting that the C-terminal hydrophobic domain hitherto described as transmembrane may correspond to a hydrophobic helix buried in the catalytic domain of kinase-related proteins, these findings call for a reappraisal of the current view of ROP2 family proteins association with the PVM.     

Host but not parasite cholesterol controls Toxoplasma cell entry by modulating organelle discharge

Host cell cholesterol is implicated in the entry and replication of an increasing number of intracellular microbial pathogens. Although uptake of viral particles via cholesterol-enriched caveolae is increasingly well described, the requirement of cholesterol for internalization of eukaryotic pathogens is poorly understood and is likely to be partly organism specific. We examined the role of cholesterol in active host cell invasion by the protozoan parasite Toxoplasma gondii. The parasitophorous vacuole membrane (PVM) surrounding T. gondii contains cholesterol at the time of invasion. Although cholesterol-enriched parasite apical organelles termed rhoptries discharge at the time of cell entry and contribute to PVM formation, surprisingly, rhoptry cholesterol is not necessary for this process. In contrast, host plasma membrane cholesterol is incorporated into the forming PVM during invasion, through a caveolae-independent mechanism. Unexpectedly, depleting host cell plasma membrane cholesterol blocks parasite internalization by reducing the release of rhoptry proteins that are necessary for invasion. Cholesterol back-addition into host plasma membrane reverses this inhibitory effect of depletion on parasite secretion. These data define a new mechanism by which host cholesterol specifically controls entry of an intracellular pathogen.     

A Helical Membrane-Binding Domain Targets the Toxoplasma ROP2 Family to the Parasitophorous Vacuole

During invasion, the obligate intracellular pathogen, Toxoplasma gondii , secretes into its host cell a variety of effector molecules, several of which have been implicated in strain-specific variation in disease. The largest family of these effectors, defined by the canonical member ROP2, quickly associates with the nascent parasitophorous vacuole membrane (PVM) after secretion. Here we demonstrate that the NH₂-terminal domain of the ROP2 family contains a series of amphipathic helices that are necessary and sufficient for membrane association. While each of the amphipathic helices is individually competent to bind cellular membranes, together they act to bind the PVM preferentially, possibly through sensing its strong negative curvature. This previously uncharacterized helical domain is an evolutionarily robust and energetically efficient design for membrane association.     

Detection of a novel parasite kinase activity at the Toxoplasma gondii parasitophorous vacuole membrane capable of phosphorylating host IkappaBalpha

Toxoplasma gondii activates the NF-kappaB pathway in the infected host cell resulting in upregulation of pro-survival genes and prevention of apoptosis. Manipulation of the NF-kappaB cascade by T. gondii correlates with the localization of phosphorylated IkappaB at the parasitophorous vacuole membrane (PVM). This suggests a parasite-mediated event, involving the recruitment and activation of the host IkappaB kinase (IKK) complex, as has been observed with the related protozoan Theileria parva. In contrast to Theileria, confocal microscopy studies showed no apparent hijacking of IKKalpha, IKKbeta, or their activated phosphorylated forms at the T. gondii PVM. Remarkably, phosphorylation of IkappaBalpha at Ser 32/36 was observed at the PVM of T. gondii-infected IKKalpha-/-, IKKbeta-/- and IKKalpha/beta double-knockout (IKKalpha/beta-/-) fibroblasts, suggesting the involvement of a parasite kinase activity independent of host IKK. The presence of a putative T. gondii IkappaB kinase was examined by in vitro kinase assays using GST-IkappaBalpha constructs and protein extracts from both extracellular parasites and PVM fractions. Interestingly, an activity capable of phosphorylating IkappaBalpha at the critical Ser 32/36 sites was identified in parasite extracts, a property restricted to the IKK signalosome. Taken together, our data support the role for a T. gondii kinase involved in phosphorylation of host cell IkappaBalpha and suggest an unusual mechanism utilized by an intracellular pathogen capable of manipulating the NF-kappaB pathway.     

Transmembrane insertion of the Toxoplasma gondii GRA5 protein occurs after soluble secretion into the host cell

The intracellular parasite Toxoplasma gondii resides within a specialized compartment, the parasitophorous vacuole (PV), that resists fusion with host cell endocytic and lysosomal compartments. The PV is extensively modified by secretion of parasite proteins, including the dense granule protein GRA5 that is specifically targeted to the delimiting membrane of the PV (PVM). We show here that GRA5 is present both in a soluble form and in hydrophobic aggregates. GRA5 is secreted as a soluble form into the PV after which it becomes stably associated with the PVM. Topological studies demonstrated that GRA5 was inserted into the PVM as a transmembrane protein with its N-terminal domain extending into the cytoplasm and its C terminus in the vacuole lumen. Deletion of 8 of the 18 hydrophobic amino acids of the single predicted transmembrane domain resulted in the failure of GRA5 to associate with the PVM; yet it remained correctly packaged in the dense granules and was secreted as a soluble protein into the PV. Collectively, these studies demonstrate that the secretory pathway in Toxoplasma is unusual in two regards; it allows soluble export of proteins containing typical transmembrane domains and provides a mechanism for their insertion into a host cell membrane after secretion from the parasite.     

The Toxoplasma gondii protein ROP2 mediates host organelle association with the parasitophorous vacuole membrane.

Toxoplasma gondii replicates within a specialized vacuole surrounded by the parasitophorous vacuole membrane (PVM). The PVM forms intimate interactions with host mitochondria and endoplasmic reticulum (ER) in a process termed PVM-organelle association. In this study we identify a likely mediator of this process, the parasite protein ROP2. ROP2, which is localized to the PVM, is secreted from anterior organelles termed rhoptries during parasite invasion into host cells. The NH(2)-terminal domain of ROP2 (ROP2hc) within the PVM is exposed to the host cell cytosol, and has characteristics of a mitochondrial targeting signal. In in vitro assays, ROP2hc is partially translocated into the mitochondrial outer membrane and behaves like an integral membrane protein. Although ROP2hc does not translocate across the ER membrane, it does exhibit carbonate-resistant binding to this organelle. In vivo, ROP2hc expressed as a soluble fragment in the cytosol of uninfected cells associates with both mitochondria and ER. The 30-amino acid (aa) NH(2)-terminal sequence of ROP2hc, when fused to green fluorescent protein (GFP), is sufficient for mitochondrial targeting. Deletion of the 30-aa NH(2)-terminal signal from ROP2hc results in robust localization of the truncated protein to the ER. These results demonstrate a new mechanism for tight association of different membrane-bound organelles within the cell cytoplasm.