Endocytosis and secretion in trypanosomatid parasites - Tumultuous traffic in a pocket.

Trypanosomatids are flagellated protozoan parasites of invertebrates, vertebrates and plants. Some species, found in the subtropics and tropics, cause chronic diseases in humans and domestic animals. The surface of the trypanosomatid provides a shield against environmental challenges, ligands for interaction with host cells, as well as receptors and transporters for the uptake of nutrients. Communication between the parasite and its environment is confined to the flagellar pocket, an invagination of the plasma membrane around the base of the flagellum. In this review, the authors discuss endocytosis, secretion and membrane trafficking in Trypanosoma and Leishmania.

Import of a DHFR hybrid protein into glycosomes in vivo is not inhibited by the folate-analogue aminopterin.

Dihydrofolate reductase fusion proteins have been widely used to study conformational properties of polypeptides translocated across membranes. We have studied the import of dihydrofolate reductase fusion proteins into glycosomes and mitochondria of Trypanosoma brucei. As signal sequences we used the last 22 carboxy-terminal amino acids of glycosomal phosphoglycerate kinase for glycosomes, and the cleavable presequences of yeast cytochrome b2 or cytochrome oxidase subunit IV for mitochondria. Upon addition of aminopterin, a folate analogue that stabilizes the dihydrofolate reductase moiety, import of the fusion protein targeted to glycosomes was not inhibited, although the results of protease protection assays showed that the fusion protein could bind the drug. Under the same conditions, import of a DHFR fusion protein targeted to mitochondria was inhibited by aminopterin. When DHFR fusion proteins targeted simultaneously to both glycosomes and mitochondria were expressed, import into mitochondria was inhibited by aminopterin, whereas uptake of the same proteins into glycosomes was either unaffected or slightly increased. These findings suggest that the glycosomes possess either a strong unfolding activity or an unusually large or flexible translocation channel.

The centrosomal protein C-Nap1 is required for cell cycle-regulated centrosome cohesion.

Duplicating centrosomes are paired during interphase, but are separated at the onset of mitosis. Although the mechanisms controlling centrosome cohesion and separation are important for centrosome function throughout the cell cycle, they remain poorly understood. Recently, we have proposed that C-Nap1, a novel centrosomal protein, is part of a structure linking parental centrioles in a cell cycle-regulated manner. To test this model, we have performed a detailed structure-function analysis on C-Nap1. We demonstrate that antibody-mediated interference with C-Nap1 function causes centrosome splitting, regardless of the cell cycle phase. Splitting occurs between parental centrioles and is not dependent on the presence of an intact microtubule or microfilament network. Centrosome splitting can also be induced by overexpression of truncated C-Nap1 mutants, but not full-length protein. Antibodies raised against different domains of C-Nap1 prove that this protein dissociates from spindle poles during mitosis, but reaccumulates at centrosomes at the end of cell division. Use of the same antibodies in immunoelectron microscopy shows that C-Nap1 is confined to the proximal end domains of centrioles, indicating that a putative linker structure must contain additional proteins. We conclude that C-Nap1 is a key component of a dynamic, cell cycle-regulated structure that mediates centriole-centriole cohesion.

Transferrin-binding protein complex is the receptor for transferrin uptake in Trypanosoma brucei.

In Trypanosoma brucei, the products of two genes, ESAG 6 and ESAG 7, located upstream of the variant surface glycoprotein gene in a polycistronic expression site form a glycosylphosphatidylinositol-anchored transferrin-binding protein (TFBP) complex. It is shown by gel filtration and membrane-binding experiments that the TFBP complex is heterodimeric and binds one molecule of transferrin with high affinity (2,300 binding sites per cell; KD = 2.1 nM for the dominant expression site from T. brucei strain 427 and KD = 131 nM for ES1.3A of the EATRO 1125 stock). The ternary transferrin-TFBP complexes with iron-loaded or iron-free ligand are stable between pH 5 and 8. Cellular transferrin uptake can be inhibited by 90% with Fab fragments from anti-TFBP antibodies. After uptake, the TFBP complex and its ligand are routed to lysosomes where transferrin is proteolytically degraded. While the degradation products are released from the cells, iron remains cell associated and the TFBP complex is probably recycled to the membrane of the flagellar pocket, the only site for exo- and endocytosis in this organism. It is concluded that the TFBP complex serves as the receptor for the uptake of transferrin in T. brucei by a mechanism distinct from that in mammalian cells.

Characterization of polymer release from the flagellar pocket of Leishmania mexicana promastigotes.

Trypanosomatids contain a unique compartment, the flagellar pocket, formed by an invagination of the plasma membrane at the base of the flagellum, which is considered to be the sole cellular site for endocytosis and exocytosis of macromolecules. The culture supernatant of Leishmania mexicana promastigotes, the insect stage of this protozoan parasite, contains two types of polymers: a filamentous acid phosphatase (sAP) composed of a 100-kD phosphoglycoprotein with non-covalently associated proteo high molecular weight phosphoglycan (proteo-HMWPG) and fibrous material termed network consisting of complex phosphoglycans. Secretion of both polymers is investigated using mAbs and a combination of light and electron microscopic techniques. Long filaments of sAP are detectable in the lumen of the flagellar pocket. Both sAP filaments and network material emerge from the ostium of the flagellar pocket. While sAP filaments detach from the cells, the fibrous network frequently remains associated with the anterior end of the parasites and can be found in the center of cell aggregates. The related species L. major forms similar networks. Since polymeric structures cannot be detected in intracellular compartments, it is proposed that monomeric or, possibly, oligomeric subunits synthesized in the cells are secreted into the flagellar pocket. Polymer formation from subunits is suggested to occur in the lumen of the pocket before release into the culture medium or, naturally, into the gut of infected sandflies.

C-Nap1, a novel centrosomal coiled-coil protein and candidate substrate of the cell cycle-regulated protein kinase Nek2.

Nek2 (for NIMA-related kinase 2) is a mammalian cell cycle-regulated kinase structurally related to the mitotic regulator NIMA of Aspergillus nidulans. In human cells, Nek2 associates with centrosomes, and overexpression of active Nek2 has drastic consequences for centrosome structure. Here, we describe the molecular characterization of a novel human centrosomal protein, C-Nap1 (for centrosomal Nek2-associated protein 1), first identified as a Nek2-interacting protein in a yeast two-hybrid screen. Antibodies raised against recombinant C-Nap1 produced strong labeling of centrosomes by immunofluorescence, and immunoelectron microscopy revealed that C-Nap1 is associated specifically with the proximal ends of both mother and daughter centrioles. On Western blots, anti-C-Nap1 antibodies recognized a large protein (>250 kD) that was highly enriched in centrosome preparations. Sequencing of overlapping cDNAs showed that C-Nap1 has a calculated molecular mass of 281 kD and comprises extended domains of predicted coiled-coil structure. Whereas C-Nap1 was concentrated at centrosomes in all interphase cells, immunoreactivity at mitotic spindle poles was strongly diminished. Finally, the COOH-terminal domain of C-Nap1 could readily be phosphorylated by Nek2 in vitro, as well as after coexpression of the two proteins in vivo. Based on these findings, we propose a model implicating both Nek2 and C-Nap1 in the regulation of centriole-centriole cohesion during the cell cycle.

Topologically restricted appearance in the developing chick retinotectal system of Bravo, a neural surface protein: experimental modulation by environmental cues.

A novel neural surface protein, Bravo, shows a pattern of topological restriction in the embryonic chick retinotectal system. Bravo is present on the developing optic fibers in the retina; however, retinal axons in the tectum do not display Bravo. The appearance of Bravo in vitro is modulated by environmental cues. Axons growing out from retinal explants on retinal basal lamina, their natural substrate, express Bravo, whereas such axons growing on collagen do not. Retinal explants provide a valuable system to characterize the mechanism of Bravo restriction, as well as the cellular signals controlling it. Bravo was identified with monoclonal antibodies from a collection generated against exposed molecules isolated by using a selective cell surface biotinylation procedure. The NH2-terminal sequence of Bravo shows similarity with L1, a neural surface molecule which is a member of the immunoglobulin superfamily. This possible relationship to L1, together with its restricted appearance, suggests an involvement of Bravo in axonal growth and guidance.

Structure of a filamentous phosphoglycoprotein polymer: the secreted acid phosphatase of Leishmania mexicana.

The insect stage of the protozoan parasite Leishmania mexicana secretes a filamentous acid phosphatase (secreted acid phosphatase, SAP), a polymeric phosphoglycoprotein. The wild-type (wt) SAP filament is a copolymer composed of two related gene products SAP1 and SAP2, which are identical in the enzymatically active NH2-terminal domain and the COOH-terminal domain, but differ in the length of a highly glycosylated Ser/Thr-rich repeat region (32 amino acids and 383 amino acids, respectively) which is located between these domains. When expressed separately, full length SAP1, SAP2, or the NH2-terminal domain alone, are able to assemble into filaments. The Ser/Thr-rich region is the exclusive target for a novel type of O-glycosylation via phosphoserines. By using glycerol spraying/low-angle rotary metal shadowing and labelling with monoclonal antibodies it is demonstrated that the repetitive region adopts an extended conformation forming side arms which project radially from the filament core and terminate with the COOH-terminal domain. The length of the side arms of SAP1 and SAP2 (20 nm and 90 nm, respectively) corresponds to the predicted length of the Ser/Thr-rich repeat region of SAP1 and SAP2. Mass determination by scanning electron microscopy (STEM) shows that one morphologically defined globular particle of the filament core is a polypeptide dimer. We propose a model for the filament core, in which the globular NH2-terminal SAP domains form one strand composed of polypeptide dimers or two tightly associated strands of monomers which may twist into a double helix, similar to actin filaments. The highly O-glycosylated side arms project from the filament core conferring an overall bottle-brush-like appearance. The L. mexicana SAP is compared to SAPs secreted by the closely related species L. amazonensis and L. donovani.

Purification, partial characterization and immunolocalization of a proteophosphoglycan secreted by Leishmania mexicana amastigotes.

The intracellular amastigote form of the parasitic protozoon Leishmania mexicana expresses a high-molecular weight phosphoglycan, which is antigenically related to the surface glycolipid lipophosphoglycan and the secreted enzyme acid phosphatase of Leishmania promastigotes. This antigen was purified from a cell-free homogenate of infected mouse tissue and from amastigotes. Compositional and immunological analysis of the purified components indicate a proteophosphoglycan structure consisting of serine-rich polypeptide chains and mild acid-labile phosphooligosaccharides capped by mannooligosaccharides. Immunofluorescence and immunoelectron microscopy of parasitized mouse peritoneal macrophages and infected mouse tissue suggest that the proteophosphoglycan is secreted in large amounts by amastigotes via their flagellar pockets into the parasitophorous vacuoles of host cells. In some infected macrophages proteophosphoglycan is also located in vesicles apparently originating from the parasitophorous vacuole, which demonstrates redistribution of a secreted amastigote antigen in parasitized host cells.

Conservation of mitochondrial targeting sequence function in mitochondrial and hydrogenosomal proteins from the early-branching eukaryotes Crithidia, Trypanosoma and Trichomonas.

Kinetoplastid protozoa are the earliest-branching eukaryotes to possess a true mitochondrion. This organelle is host to a variety of intriguing and unique features, including RNA editing. We examined the characteristics of protein import into mitochondria of Trypanosoma brucei. Dihydrofolate reductase (DHFR) carrying a yeast mitochondrial targeting signal was correctly translocated into trypanosome mitochondria in vivo, as were DHFR fusion proteins bearing two unusually short (7-9 amino acids) presequences from trypanosomatids. The short trypanosomal targeting signals were functional in Saccharomyces cerevisiae as well, but their targeting efficiency was lower and processing was absent. Trichomonads branched even earlier than kinetoplastids in eukaryotic evolution and contain energy-generating organelles called hydrogenosomes. The origin of hydrogenosomes has been controversial, but most evidence suggests that they are related to mitochondria. Putative hydrogenosomal targeting signals from Trichomonas vaginalis are short (5-12 amino acids). Three such sequences were capable of targeting a passenger protein to mitochondria both in yeast and in trypanosomes, and one of the hydrogenosomal presequences was efficiently processed in both organisms. These findings suggest a resemblance between the import machineries of mitochondria and hydrogenosomes.

ESAG 6 and 7 products of Trypanosoma brucei form a transferrin binding protein complex.

In Trypanosoma brucei, the gene for the expressed variant surface glycoprotein (VSG) is preceded by a series of open reading frames designated expression site associated genes (ESAGs), which together with the VSG gene form a polycistronic transcription unit. It is shown that the products derived from two ESAGs (ESAG 6 and 7 in the nomenclature of Pays, E., et al. Cell 57, 835-845 (1989)) form a complex, which binds transferrin with high affinity. Transferrin affinity chromatography yields heterodimers or higher order heteroligomers composed of the products of ESAG 6 and ESAG 7. The former is a heterogeneously glycosylated protein of 50 to 60 kDa modified by a glycosylphosphatidylinositol membrane anchor at the COOH-terminus, while the latter is the previously identified 42 kDa glycoprotein carrying an unmodified COOH-terminus (Schell, D., et al. EMBO J. 10, 1061-1066 (1991) and Schell, D., et al. EMBO J. 12, 2990 (1993)). When isolated from trypanosomes grown in rodents, the complex is in part free and in part associated with transferrin. Also, the complex is present both in the membrane fraction and the soluble fraction of cell lysates. As shown by immunoelectron microscopy, both transferrin and ESAG 6/7-derived proteins can be demonstrated in the lumen of the flagellar pocket, an invagination of the plasma membrane serving as the sole site for endocytotic uptake of macromolecular nutrients. Weak labeling is also obtained on the flagellar pocket membrane and in intracellular vesicles. The possibility that the binding protein complex serves as a receptor for the uptake of transferrin in T. brucei is discussed.

Filamentous proteophosphoglycan secreted by Leishmania promastigotes forms gel-like three-dimensional networks that obstruct the digestive tract of infected sandfly vectors.

Development of Leishmania parasites in the digestive tract of their sandfly vectors involves several morphological transformations from the intracellular mammalian amastigote via a succession of free and gut wall-attached promastigote stages to the infective metacyclic promastigotes. At the foregut midgut transition of Leishmania-infected sandflies a gel-like plug of unknown origin and composition is formed, which contains high numbers of parasites, that occludes the gut lumen and which may be responsible for the often observed inability of infected sandflies to draw blood. This "blocked fly" phenotype has been linked to efficient transmission of infectious metacyclic promastigotes from the vector to the mammalian host. We show by immunofluorescence and immunoelectron microscopy on two Leishmania/sandfly vector combinations (Leishmania mexicana/Lutzomyia longipalpis and L. major/Phlebotomus papatasi) that the gel-like mass is formed mainly by a parasite-derived mucin-like filamentous proteophosphoglycan (fPPG) whereas the Leishmania polymeric secreted acid phosphatase (SAP) is not a major component of this plug. fPPG forms a dense three-dimensional network of filaments which engulf the promastigote cell bodies in a gel-like mass. We propose that the continuous secretion of fPPG by promastigotes in the sandfly gut, that causes plug formation, is an important factor for the efficient transmission to the mammalian host.

Purification and characterization of a tartrate-sensitive acid phosphatase of Trypanosoma brucei.

In search for invariant surface proteins in Trypanosoma brucei bloodstream forms, acid phosphatase was investigated. Earlier work had shown that part of the cellular phosphatase activity is associated with the flagellar pocket of the parasite. It is demonstrated that T. brucei contains at least two membrane-bound enzymes, one is sensitive to the inhibitor L-(+)-tartrate while the other is resistant. The tartrate-sensitive phosphatase was purified to homogeneity by monoclonal antibody affinity chromatography and shown to be a glycoprotein of low abundance (13,000 molecules/cell). It has an apparent molecular weight of 70,000 Da. The usefulness of acid phosphatase as a marker for characterizing the membrane lining the flagellar pocket is discussed.

Protein kinases in control of the centrosome cycle.

The centrosome is the major microtubule nucleating center of the animal cell and forms the two poles of the mitotic spindle upon which chromosomes are segregated. During the cell division cycle, the centrosome undergoes a series of major structural and functional transitions that are essential for both interphase centrosome function and mitotic spindle formation. The localization of an increasing number of protein kinases to the centrosome has revealed the importance of protein phosphorylation in controlling many of these transitions. Here, we focus on two protein kinases, the polo-like kinase 1 and the NIMA-related kinase 2, for which recent data indicate key roles during the centrosome cycle.

Isocitrate lyase of Ashbya gossypii--transcriptional regulation and peroxisomal localization.

The isocitrate lyase-encoding gene AgICL1 from the filamentous hemiascomycete Ashbya gossypii was isolated by heterologous complementation of a Saccharomyces cerevisiae icl1d mutant. The open reading frame of 1680 bp encoded a protein of 560 amino acids with a calculated molecular weight of 62584. Disruption of the AgICL1 gene led to complete loss of AgIcl1p activity and inability to grow on oleic acid as sole carbon source. Compartmentation of AgIcl1p in peroxisomes was demonstrated both by Percoll density gradient centrifugation and by immunogold labeling of ultrathin sections using specific antibodies. This fitted with the peroxisomal targeting signal AKL predicted from the C-terminal DNA sequence. Northern blot analysis with mycelium grown on different carbon sources as well as AgICL1 promoter replacement with the constitutive AgTEF promoter revealed a regulation at the transcriptional level. AgICL1 was subject to glucose repression, derepressed by glycerol, partially induced by the C2 compounds ethanol and acetate, and fully induced by soybean oil.

Expression of lipophosphoglycan, high-molecular weight phosphoglycan and glycoprotein 63 in promastigotes and amastigotes of Leishmania mexicana.

The abundant surface glycoconjugate of Leishmania promastigotes, lipophosphoglycan (LPG), forms a blue-colored complex (lambda max = 649 nm) with the cationic dye Stains-all, which can be quantitated densitometrically on polyacrylamide gels of cell lysates. Promastigotes of Leishmania mexicana, Leishmania major and Leishmania donovani yield values of 1-3 x 10(6) LPG molecules cell-1. In amastigotes the LPG content is down-regulated below the detection limit (< 10(3) molecules cell-1) in L. mexicana and L. donovani, but remains significant in L. major (2 x 10(3) molecules cell-1). In the case of L. mexicana, these results are supported by immunological studies. Using several monoclonal and polyclonal antibodies, LPG is undetectable by immunoblotting in lysates of either amastigotes or infected macrophages and the amastigote surface is devoid of LPG as judged by immunofluorescence and immunoelectron microscopy. Immunoblotting experiments demonstrate that amastigotes synthesize hydrophilic high-molecular weight compounds which stain blue with Stains-all and cross-react with the monoclonal and polyvalent antibodies suggesting the presence of similar phosphoglycan structures as in LPG. The high-molecular weight phosphoglycan appears to be located in the lumen of the flagellar pocket of mouse lesion amastigotes and may be secreted from there into the lumen of the parasitophorous vacuole of parasitized macrophages. In L. mexicana promastigotes the surface protease gp63 is amphiphilic and comprises about 1% of the cellular proteins. In contrast, in amastigotes gp63-related proteins are predominantly hydrophilic; they amount to only about 0.1% of the cellular proteins and are mainly located in the lumen of the extended lysosomes (megasomes) characteristic for this species.

Gene cloning and cellular localization of a membrane-bound acid phosphatase of Leishmania mexicana.

In a previous publication, we described the purification of a membrane-bound acid phosphatase of Leishmania mexicana as a heterogeneously N-glycosylated protein of an apparent molecular mass of 70000-72000 expressed in both the promastigote and the amastigote stage of the parasite [19]. Screening of a genomic DNA library of L. mexicana with degenerate oligonucleotides designed according to the NH2-terminus of the protein led to the cloning of the lmmbap gene, which is present in one copy per haploid genome. The open reading frame predicts a protein of 516 amino acids composed of a signal sequence, a large hydrophilic region, a trans-membrane alpha-helix and a short cytoplasmic tail. The sequence of the hydrophilic region is homologous to acid phosphatases from other organisms. While in wild-type promastigotes, the acid phosphatase is located in the endosomal/lysosomal compartment between the flagellar pocket and the nucleus, overexpression leads to its abundant exposure on the cell surface. In cells transfected with a construct lacking the region corresponding to the trans-membrane and the cytoplasmic parts, the resulting altered acid phosphatase is efficiently secreted into the culture medium. The potential of this system for studies on membrane trafficking in kinetoplastid organisms is discussed.

Active site mapping, biochemical properties and subcellular localization of rhodesain, the major cysteine protease of Trypanosoma brucei rhodesiense.

Cysteine protease activity of African trypanosome parasites is a target for new chemotherapy using synthetic protease inhibitors. To support this effort and further characterize the enzyme, we expressed and purified rhodesain, the target protease of Trypanosoma brucei rhodesiense (MVAT4 strain), in reagent quantities from Pichia pastoris. Rhodesain was secreted as an active, mature protease. Site-directed mutagenesis of a cryptic glycosylation motif not previously identified allowed production of rhodesain suitable for crystallization. An invariable ER(A/V)FNAA motif in the pro-peptide sequence of rhodesain was identified as being unique to the genus Trypanosoma. Antibodies to rhodesain localized the protease in the lysosome and identified a 40-kDa protein in long slender forms of T. b. rhodesiense and all life-cycle stages of T. b. brucei. With the latter parasite, protease expression was five times greater in short stumpy trypanosomes than in the other stages. Radiolabeled active site-directed inhibitors identified brucipain as the major cysteine protease in T. b. brucei. Peptidomimetic vinyl sulfone and epoxide inhibitors designed to interact with the S2, S1 and S' subsites of the active site cleft revealed differences between rhodesain and the related trypanosome protease cruzain. Using fluorogenic dipeptidyl substrates, rhodesain and cruzain had acid pH optima, but unlike some mammalian cathepsins retained significant activity and stability up to pH 8.0, consistent with a possible extracellular function. S2 subsite mapping of rhodesain and cruzain with fluorogenic peptidyl substrates demonstrates that the presence of alanine rather than glutamate at S2 prevents rhodesain from cleaving substrates in which P2 is arginine.

Chemical composition of the attachment pad secretion of the locust Locusta migratoria.

This study is the first attempt to characterise the chemical composition of the secretion of the smooth pads of the locust Locusta migratoria and to relate this to the composition of the cuticle coverage of the pads and the wings. Gas-chromatography and mass-spectrometry (GC-MS) were the principal techniques used for the characterization of these materials. Secretion droplets were visualised and quantified with the aid of diverse microscopic techniques. The chemical composition of prints is shown to differ from the cuticle coverage, in particular, with respect to the fatty acid distribution: in the secretion, saturated and unsaturated fatty acids with chain lengths between C(16) and C(20) in both the free form and as glycerides predominate, whereas cuticle coverage contains waxes of long-chained fatty-acids bound to long-chain primary alcohols. The second important difference is the significant amount of glucose and other saccharides found in methanolyzates of the pad fluid. A considerable amount of the amino acids (up to 53%) was detected in the non-volatile portion of the fluid. Data obtained from the shock-freezing, carbon-platinum coating and replica preparation show that the secretory droplets contain nano-droplets on their surfaces. The results lead us to suggest that the pad secretion is an emulsion consisting of lipidic nano-droplets dispersed in an aqueous liquid. According to the chemical composition of the secretion, a high-viscosity of the fluid may be suggested. Presumably, the fluid is a kind of a coupling agent, promoting and strengthening adhesion between otherwise incompatible materials by providing the proximity of contact for intermolecular forces.

Action of gold chloride ("gold toning") on silver-enhanced 1 nm gold markers.

In conventional immunoelectron microscopy (IEM), very small colloidal gold particles (0.8-3 nm), or the gold compound Nanogold (1.4 nm) are silver-enhanced for easy detection. However, silver enhancement has drawbacks. First, the silver layer is dissolved during fixation with osmium tetroxide, even if the concentration and incubation time are strongly reduced during pre-embedding labeling experiments in transmission electron microscopic (TEM) and scanning electron microscopic (SEM) studies. Second, after exposure to the electron beam the silver layer may migrate on the section or the whole particles may disappear. Sometimes silver migration can be observed even without irradiation. This effect strongly hampers reinvestigation of previously inspected areas, after some time of storage. In both cases, gold chloride treatment after silver enhancement is sufficient to completely protect the silver-enhanced 1 nm gold markers. Gold chloride treatment is part of the so-called "gold toning" procedure, which is a method used to substitute and/or cover the silver by a layer of gold. It can be applied in TEM and SEM experiments. As a serious drawback, gold chloride treatment slightly reduces the size of both unenhanced and silver-enhanced gold particles and can lead to disintegrated silver/gold particles. Therefore, this technique is useful for pre-embedding IEM, on-(resin)section, and ultrathin cryosection labeling experiments. However, it appears to be unsuitable for double-labeling studies using different gold sizes, for quantitation experiments, and in SEM.