O-linked glycans mediate apical sorting of human intestinal sucrase-isomaltase through association with lipid rafts.

The plasma membrane of polarised epithelial cells is characterised by two structurally and functionally different domains, the apical and basolateral domains. These domains contain distinct protein and lipid constituents that are sorted by specific signals to the correct surface domain [1]. The best characterised apical sorting signal is that of glycophosphatidylinositol (GPI) membrane anchors [2], although N-linked glycans on some secreted proteins [3] and O-linked glycans [4] also function as apical sorting signals. In the latter cases, however, the underlying sorting mechanisms remain obscure. Here, we have analysed the role of O-glycosylation in the apical sorting of sucrase-isomaltase (SI), a highly polarised N- and O-glycosylated intestinal enzyme, and the mechanisms underlying this process. Inhibition of O-glycosylation by benzyl-N-acetyl-alpha-D-galactosaminide (benzyl-GalNAc) was accompanied by a dramatic shift in the sorting of SI from the apical membrane to both membranes. The sorting mechanism of SI involves its association with sphingolipid- and cholesterol-rich membrane rafts because this association was eliminated when O-glycosylation was inhibited by benzyl-GaINAc. The results demonstrate for the first time that O-linked glycans mediate apical sorting through association with lipid rafts.     

Mitochondrial preprotein translocase of trypanosomatids has a bacterial origin

Mitochondria are found in all eukaryotic cells and derive from a bacterial endosymbiont [1, 2]. The evolution of a protein import system was a prerequisite for the conversion of the endosymbiont into a true organelle. Tom40, the essential component of the protein translocase of the outer membrane, is conserved in mitochondria of almost all eukaryotes but lacks bacterial orthologs [3-6]. It serves as the gateway through which all mitochondrial proteins are imported. The parasitic protozoa Trypanosoma brucei and its relatives do not have a Tom40-like protein, which raises the question of how proteins are imported by their mitochondria [7, 8]. Using a combination of bioinformatics and in vivo and in vitro studies, we have discovered that T. brucei likely employs a different import channel, termed ATOM (archaic translocase of the outer mitochondrial membrane). ATOM mediates the import of nuclear-encoded proteins into mitochondria and is essential for viability of trypanosomes. It is not related to Tom40 but is instead an ortholog of a subgroup of the Omp85 protein superfamily that is involved in membrane translocation and insertion of bacterial outer membrane proteins [9]. This suggests that the protein import channel in trypanosomes is a relic of an archaic protein transport system that was operational in the ancestor of all eukaryotes.     

Clathrin-mediated endocytosis is essential in Trypanosoma brucei

In Trypanosoma brucei, the plasma membrane is dominated by glycosylphosphatidylinositol (GPI)-anchored proteins. Endocytic activity correlates with expression levels of the clathrin heavy chain TbCLH, and additional evidence suggests that rapid endocytosis may play a role in evasion of the immune response. TbCLH is present on both endocytic vesicles and post-Golgi elements, suggesting a similar range of functions in trypanosomes to higher eukaryotes. We have assessed the role of TbCLH using RNA interference (RNAi). Suppression of TbCLH expression results in rapid lethality in the bloodstream stage, the form most active for endocytosis. The flagellar pocket, the site of both endocytosis and exocytosis, becomes massively enlarged, suggesting that membrane delivery is unaffected but removal is blocked. Endocytosis in TbCLHRNAi cells is essentially undetectable, suggesting that clathrin-mediated mechanisms are the major route for endocytosis in T.brucei and hence that GPI-anchored proteins are endocytosed by clathrin-dependent pathways in trypanosomes. In contrast, a massive internal accumulation of vesicles and significant alterations to trafficking of a lysosomal protein were observed in the procyclic stage, indicating developmental variation in clathrin function in trypanosomes.     

Clathrin-dependent targeting of receptors to the flagellar pocket of procyclic-form Trypanosoma brucei

In trypanosomatids, endocytosis and exocytosis occur exclusively at the flagellar pocket, which represents about 0.43% of the pellicle membrane and is a deep invagination of the plasma membrane where the flagellum extends from the cell. Receptor molecules are selectively retained at the flagellar pocket. We studied the function of clathrin heavy chain (TbCLH) in the trafficking of the flagellar pocket receptors in Trypanosoma brucei by using the double-stranded RNA interference approach. It appears that TbCLH is essential for the survival of both the procyclic form and the bloodstream form of T. brucei, even though structures resembling large coated endocytic vesicles are absent in procyclic-form trypanosomes. Down-regulation of TbCLH by RNA interference (RNAi) for 24 h rapidly and drastically reduced the uptake of macromolecules via receptor-mediated endocytosis in procyclic-form trypanosomes. This result suggested the importance of TbCLH in receptor-mediated endocytosis of the procyclic-form trypanosome, in which the formation of large coated endocytic vesicles may not be required. Surprisingly, induction of TbCLH RNAi in the procyclic T. brucei for a period of 48 h prohibited the export of the flagellar pocket-associated transmembrane receptor CRAM from the endoplasmic reticulum to the flagellar pocket, while trafficking of the glycosylphosphatidylinositol-anchored procyclin coat was not significantly affected. After 72 h of induction of TbCLH RNAi, procyclics exhibited morphological changes to an apolar round shape without a distinct structure of the flagellar pocket and flagellum. Although trypanosomes, like other eukaryotes, use similar organelles and machinery for protein sorting and transport, our studies reveal a novel role for clathrin in the secretory pathway of trypanosomes. We speculate that the clathrin-dependent trafficking of proteins to the flagellar pocket may be essential for the biogenesis and maintenance of the flagellar pocket in trypanosomes.     

Temporal association of the N- and O-linked glycosylation events and their implication in the polarized sorting of intestinal brush border sucrase-isomaltase, aminopeptidase N, and dipeptidyl peptidase IV.

The temporal association between O-glycosylation and processing of N-linked glycans in the Golgi apparatus as well as the implication of these events in the polarized sorting of three brush border proteins has been the subject of the current investigation. O-Glycosylation of pro-sucrase-isomaltase (pro-SI), aminopeptidase N (ApN), and dipeptidyl peptidase IV (DPPIV) is drastically reduced when processing of the mannose-rich N-linked glycans is blocked by deoxymannojirimycin, an inhibitor of the Golgi-located mannosidase I. By contrast, O-glycosylation is not affected in the presence of swainsonine, an inhibitor of Golgi mannosidase II. The results indicate that removal of the outermost mannose residues by mannosidase I from the mannose-rich N-linked glycans is required before O-glycosylation can ensue. On the other hand, subsequent mannose residues in the core chain impose no sterical constraints on the progression of O-glycosylation. Reduction or modification of N- and O-glycosylation do not affect the transport of pro-SI, ApN, or DPPIV to the cell surface per se. However, the polarized sorting of two of these proteins, pro-SI and DPPIV, to the apical membrane is substantially altered when O-glycans are not completely processed, while the sorting of ApN is not affected. The processing of N-linked glycans, on the other hand, has no influence on sorting of all three proteins. The results indicate that O-linked carbohydrates are at least a part of the sorting mechanism of pro-SI and DPPIV. The sorting of ApN implicates neither O-linked nor N-linked glycans and is driven most likely by carbohydrate-independent mechanisms.     

Sorting signals required for trafficking of the cysteine-rich acidic repetitive transmembrane protein in Trypanosoma brucei

In trypanosomatids, endocytosis and exocytosis are restricted to the flagellar pocket (FP). The cysteine-rich acidic repetitive transmembrane (CRAM) protein is located at the FP of Trypanosoma brucei and potentially functions as a receptor or an essential component for lipoprotein uptake. We characterized sorting determinants involved in efficient trafficking of CRAM to and from the FP of T. brucei. Previous studies indicated the presence of signals in the CRAM C terminus, specific for its localization to the FP and for efficient endocytosis (H. Yang, D. G. Russell, B. Zeng, M. Eiki, and M.G.-S. Lee, Mol. Cell. Biol. 20:5149-5163, 2000.) To delineate functional domains of putative sorting signals, we performed a mutagenesis series of the CRAM C terminus. Subcellular localization of CRAM mutants demonstrated that the amino acid sequence between -5 and -14 (referred to as a transport signal) is essential for exporting CRAM from the endoplasmic reticulum to the FP, and mutations of amino acids at -12 (V), -10 (V), or -5 (D) led to retention of CRAM in the endoplasmic reticulum. Comparison of the endocytosis efficiency of CRAM mutants demonstrated that the sequence from amino acid -5 to -23 (referred to as a putative endocytosis signal) is required for efficient endocytosis and overlaps with the transport signal. Apparently the CRAM-derived sorting signal can efficiently interact with the T. brucei micro1 adaptin, and mutations at amino acids essential for the function of the transport signal abolished the interaction of the signal with T. brucei micro1, strengthening the hypothesis of the involvement of the clathrin- and adaptor-dependent pathway in trafficking of CRAM via the FP.     

The endocytic apparatus of the kinetoplastida. Part II: machinery and components of the system

Endocytic systems within eukaryotic cells are a diverse set of intracellular transport pathways responsible for uptake, recycling, interaction with the exocytic system and degradation of molecules. Each of these pathways requires the interaction of distinct protein components that function in macromolecule sorting, control of transport rates and in membrane biogenesis. In the second of two articles on kinetoplastida endocytosis, the endocytic system in Trypanosoma brucei is considered as a model, and the molecules that control this system and the protein components of the endocytic pathway are discussed. We also consider novel mechanisms for sorting that have been proposed to operate in trypanosomes.     

Hydrodynamic flow-mediated protein sorting on the cell surface of trypanosomes

The unicellular parasite Trypanosoma brucei rapidly removes host-derived immunoglobulin (Ig) from its cell surface, which is dominated by a single type of glycosylphosphatidylinositol-anchored variant surface glycoprotein (VSG). We have determined the mechanism of antibody clearance and found that Ig-VSG immune complexes are passively sorted to the posterior cell pole, where they are endocytosed. The backward movement of immune complexes requires forward cellular motility but is independent of endocytosis and of actin function. We suggest that the hydrodynamic flow acting on swimming trypanosomes causes directional movement of Ig-VSG immune complexes in the plane of the plasma membrane, that is, immunoglobulins attached to VSG function as molecular sails. Protein sorting by hydrodynamic forces helps to protect trypanosomes against complement-mediated immune destruction in culture and possibly in infected mammals but likewise may be of functional significance at the surface of other cell types such as epithelial cells lining blood vessels.     

The majority of cellular fatty acid acylated proteins are localized to the cytoplasmic surface of the plasma membrane

The BC3Hl muscle cell line was previously reported to contain a broad array of fatty acid acylated proteins [Olson, E. N., Towler, D. A., & Glaser, L. (1985) J. Biol. Chem. 260, 3784-3790]. Palmitate was shown to be attached to membrane proteins posttranslationally through thiol ester linkages, whereas myristate was attached cotranslationally, or within seconds thereafter, to soluble and membrane-bound proteins through amide linkages [Olson, E. N., & Spizz, G. (1986) J. Biol. Chem. 261, 2458-2466]. The temporal and subcellular differences between palmitate and myristate acylation suggested that these two classes of acyl proteins might follow different intracellular pathways to distinct subcellular membrane systems or organelles. In this study, we examined the subcellular localization of the major fatty acylated proteins in BC3Hl cells. Palmitate-containing proteins were localized to the plasma membrane, but only a subset of myristate-containing proteins was localized to this membrane fraction. The majority of acyl proteins were nonglycosylated and resistant to digestion with extracellular proteases, suggesting that they were not exposed to the external surface of the plasma membrane. Many proteins were, however, digested during incubation of isolated membranes with proteases, which indicates that these proteins face the cytoplasm. Two-dimensional gel electrophoresis of proteins labeled with [3H]palmitate and [3H]myristate revealed that individual proteins were modified by only one of the two fatty acids and did not undergo both N-linked myristylation and ester-linked palmitylation. Together, these results suggest that the majority of cellular acyl proteins are routed to the cytoplasmic surface of the plasma membrane, and they raise the possibility that fatty acid acylation may play a role in intracellular sorting of nontransmembranous, nonglycosylated membrane proteins.     

Late endosomal Rab7 regulates lysosomal trafficking of endocytic but not biosynthetic cargo in Trypanosoma brucei

We present the first functional analysis of the small GTPase, TbRab7, in Trypanosoma brucei. TbRab7 defines discrete late endosomes closely juxtaposed to the terminal p67(+) lysosome. RNAi indicates that TbRab7 is essential in bloodstream trypanosomes. Initial rates of endocytosis were unaffected, but lysosomal delivery of cargo, including tomato lectin (TL) and trypanolytic factor (TLF) were blocked. These accumulate in a dispersed internal compartment of elevated pH, likely derived from the late endosome. Surface binding of TL but not TLF was reduced, suggesting that cellular distribution of flagellar pocket receptors is differentially regulated by TbRab7. TLF activity was reduced approximately threefold confirming that lysosomal delivery is critical for trypanotoxicity. Unexpectedly, delivery of endogenous proteins, p67 and TbCatL, were unaffected indicating that TbRab7 does not regulate biosynthetic lysosomal trafficking. Thus, unlike mammalian cells and yeast, lysosomal trafficking of endocytosed and endogenous proteins occur via different routes and/or are regulated differentially. TbRab7 silencing had no effect on a cryptic default pathway to the lysosome, suggesting that the default lysosomal reporters p67ΔTM, p67ΔCD and VSGΔGPI do not utilize the endocytic pathway as previously proposed. Surprisingly, conditional knockout indicates that TbRab7 may be non-essential in procyclic insect form trypanosomes.     

Clathrin-mediated constitutive endocytosis of PIN auxin efflux carriers in Arabidopsis

Endocytosis is an essential process by which eukaryotic cells internalize exogenous material or regulate signaling at the cell surface [1]. Different endocytic pathways are well established in yeast and animals; prominent among them is clathrin-dependent endocytosis [2, 3]. In plants, endocytosis is poorly defined, and no molecular mechanism for cargo internalization has been demonstrated so far [4, 5], although the internalization of receptor-ligand complexes at the plant plasma membrane has recently been shown [6]. Here we demonstrate by means of a green-to-red photoconvertible fluorescent reporter, EosFP [7], the constitutive endocytosis of PIN auxin efflux carriers [8] and their recycling to the plasma membrane. Using a plant clathrin-specific antibody, we show the presence of clathrin at different stages of coated-vesicle formation at the plasma membrane in Arabidopsis. Genetic interference with clathrin function inhibits PIN internalization and endocytosis in general. Furthermore, pharmacological interference with cargo recruitment into the clathrin pathway blocks internalization of PINs and other plasma-membrane proteins. Our data demonstrate that clathrin-dependent endocytosis is operational in plants and constitutes the predominant pathway for the internalization of numerous plasma-membrane-resident proteins including PIN auxin efflux carriers.     

Rab4 is an essential regulator of lysosomal trafficking in trypanosomes

Rapid endocytosis and recycling of surface proteins are important processes common to most nucleated eukaryotic cells. The best characterized membrane recycling routes are mediated by the small GTPases Rab4 and Rab11, but the precise roles that these pathways play have not been fully elucidated. The protozoan Trypanosoma brucei has a highly developed endocytic system that is similar to that found in metazoans, albeit with an accelerated rate of membrane turnover. We have used this organism to investigate the function of the trypanosome orthologue of Rab4 (TbRAB4) by a combination of RNA interference, microscopy, and quantitative trafficking assays. RNA interference-mediated suppression of TbRAB4 expression inhibited the growth of trypanosomes without affecting receptor-mediated endocytosis or ligand recycling. Ultrastructural analysis indicated a major defect in membrane transport events. The accumulation of fluorescent dextran, a fluid-phase marker, was blocked in cells lacking TbRAB4 protein. Since most fluid-phase markers are transported to the lysosome in T. brucei, the effects of TbRAB4 RNA interference on lysosomal function were investigated. By immunofluorescence, the major lysosomal protein p67 became progressively dispersed in cells lacking the TbRAB4 protein. Pulse-chase analysis demonstrated that initial proteolytic cleavage and glycan processing of p67 were unaffected but that cells failed to accumulate the later p67 proteolyzed products associated with the lysosome. To confirm the role of TbRAB4 in lysosomal trafficking, a constitutively active mutant, TbRAB4QL, was expressed. TbRAB4QL was closely associated with an enlarged multivesicular body that contained p67. In addition, cells expressing TbRAB4QL showed increased fluid-phase uptake when compared with the parental line. Taken together, these data suggest that TbRAB4 is involved in regulation of fluid-phase traffic to the lysosome in T. brucei but not in receptor-mediated endocytosis or recycling. These data have implications for the role of Rab4 in other cell systems.     

A conserved flagellar pocket exposed high mannose moiety is used by African trypanosomes as a host cytokine binding molecule

Trypanosomes use antigenic variation of their variant-specific surface glycoprotein (VSG) coat as defense against the host immune system. However, in order to sustain their growth, they need to expose conserved epitopes, allowing host macromolecule binding and receptor-mediated endocytosis. Here we show that Trypanosoma brucei uses the conserved chitobiose-oligomannose (GlcNAc(2)-Man(5-9)) moieties of its VSG as a binding ligand for tumor necrosis factor (TNF), a host cytokine with lectin-like properties. As endocytosis in trypanosomes is restricted to the flagellar pocket, we show that soluble flagellar pocket extracts, and in particular soluble VSG, inhibit the binding of (125)I-TNF to trypanosomes. The interaction between TNF and VSG is confirmed by affinity chromatography, biosensor, and dot-blot affinity measurements, and soluble VSG inhibition of TNF-mediated trypanolysis. In all approaches, removal of N-linked carbohydrates abrogates the TNF-VSG interaction. In addition, synthetic high mannose oligosaccharides can block TNF-VSG interactions, and a VSG glycopeptide carrying the GlcNAc(2)-Man(5-9) moiety is shown to inhibit TNF-mediated trypanosome killing in mixed parasite/macrophage cell cultures. Together, these results support the observation that TNF plays a role in growth control of trypanosomes and, moreover, suggest that, by the use of conserved VSG carbohydrates as lectin-binding epitopes, trypanosomes can limit the necessity to express large numbers of invariant surface exposed receptors.     

Endocytosis of thyroglobulin is not mediated by mannose-6-phosphate receptors in thyrocytes. Evidence for low-affinity-binding sites operating in the uptake of thyroglobulin.

Thyroglobulin, the major secretory product of thyrocytes, is the macromolecular precursor of thyroid hormones. After its synthesis, thyroglobulin follows a complex secretion, storage and recapture pathway to lysosomes. Porcine thyroglobulin was shown to carry the mannose 6-phosphate-(Man6P)-recognition marker on its N-linked glycans. Since the cation-independent Man6P receptor could also be found on the apical plasma membrane of porcine thyrocytes, we examined the significance of the Man6P signal for the transport of thyroglobulin. Here, we present data implying that Man6P receptors are not relevant for endocytosis of thyroglobulin in thyrocytes. Instead, we provide evidence for the existence of specific, low-affinity-binding sites for thyroglobulin on the apical plasma membrane of thyrocytes responsible for endocytosis of thyroglobulin. Binding studies with intact, polar-organized porcine thyrocytes grown on collagen-coated filters revealed cooperative and saturable binding of thyroglobulin to the apical-plasma-membrane domain at relatively high concentrations of thyroglobulin (20 microM). These observations show that low-affinity interactions between thyroglobulin and the apical plasma membrane play a key role in endocytosis of thyroglobulin and hormone formation in the thyroid. The data in this publication have been published as an abstract [Lemansky, P. and Herzog, V. (1991) J. Cell Biol. 115, 261a].     

Lysosomal metabolism of glycoproteins

The lysosomal catabolism of glycoproteins is part of the normal turnover of cellular constituents and the cellular homeostasis of glycosylation. Glycoproteins are delivered to lysosomes for catabolism either by endocytosis from outside the cell or by autophagy within the cell. Once inside the lysosome, glycoproteins are broken down by a combination of proteases and glycosidases, with the characteristic properties of soluble lysosomal hydrolases. The proteases consist of a mixture of endopeptidases and exopeptidases, which act in concert to produce a mixture of amino acids and dipeptides, which are transported across the lysosomal membrane into the cytosol by a combination of diffusion and carrier-mediated transport. Although the glycans of all mature glycoproteins are probably degraded in lysosomes, the breakdown of N-linked glycans has been studied most intensively. The catabolic pathways for high-mannose, hybrid, and complex glycans have been established. They are bidirectional with concurrent sequential removal of monosaccharides from the nonreducing end by exoglycosidases and proteolysis and digestion of the carbohydrate-polypeptide linkage at the reducing end. The process is initiated by the removal of any core and peripheral fucose, which is a prerequisite for the action of the peptide N-glycanase aspartylglucosaminidase, which hydrolyzes the glycan-peptide bond. This enzyme also requires free alpha carboxyl and amino groups on the asparagine residue, implying extensive prior proteolysis. The catabolism of O-linked glycans has not been studied so intensively, but many lysosomal glycosidases appear to act on the same linkages whether they are in N- or O-linked glycans, glycosaminoglycans, or glycolipids. The monosaccharides liberated during the breakdown of N- and O-linked glycans are transported across the lysosomal membrane into the cytosol by a combination of diffusion and carrier-mediated transport. Defects in these pathways lead to lysosomal storage diseases. The structures of some of the oligosaccharides that accumulate in these diseases are not digestion intermediates in the lysosomal catabolic pathways but correspond to intermediates in the biosynthetic pathway for N-linked glycans, suggesting another route of delivery of glycans to the lysosome. Incorrectly folded or glycosylated proteins that are rejected by the quality control mechanism are broken down in the ER and cytoplasm and the end product of the cytosolic degradation of N-glycans is delivered to the lysosomes. This route is enhanced in cells actively secreting glycoproteins or producing increased amounts of aberrant glycoproteins. Thus interaction between the lysosome and proteasome is important for the regulation of the biosynthesis and distribution of N-linked glycoproteins. Another example of the extralysosomal function of lysosomal enzymes is the release of lysosomal proteases into the cytosol to initiate the lysosomal pathway of apoptosis.     

Multiple motifs regulate trafficking of the LAMP-like protein p67 in the ancient eukaryote Trypanosoma brucei

p67 is a lysosome-associated membrane protein-like lysosomal type I transmembrane glycoprotein in African trypanosomes. The p67 cytoplasmic domain (CD) is both necessary and sufficient for lysosomal targeting in procyclic insect-stage parasites. The p67CD contains two [DE]XXXL[LI]-type dileucine motifs, which function as lysosomal targeting signals in mammalian cells. Using a green fluorescent protein fusion to the p67 transmembrane and cytoplasmic domains as a reporter system, we investigated the role of these motifs in lysosomal targeting in procyclic trypanosomes. Pulse-chase turnover studies, steady-state immunolocalization and quantitative flow cytometry all gave consistent results. Mutagenesis of the membrane-distal dileucine motif impairs lysosomal trafficking leading to partial appearance of the reporter on the cell surface. Mutagenesis of the membrane-proximal motif has little effect on proper targeting. Simultaneous mutagenesis of both motifs results in quantitative delivery to the cell surface. Thus, the distal motif plays a dominant role, but both dileucine motifs are necessary for maximal lysosomal targeting. Additional studies suggest that the upstream acidic residues in each motif influence lysosomal targeting and may also affect forward trafficking in the early secretory pathway. These results strongly suggest an evolutionary conservation in lysosomal trafficking mechanisms in the ancient eukaryote Trypanosoma brucei.     

Identification and characterization of three peroxins--PEX6, PEX10 and PEX12--involved in glycosome biogenesis in Trypanosoma brucei

Protozoan Kinetoplastida such as the pathogenic trypanosomes compartmentalize several important metabolic systems, including the glycolytic pathway, in peroxisome-like organelles designated glycosomes. Genes for three proteins involved in glycosome biogenesis of Trypanosoma brucei were identified. A preliminary analysis of these proteins, the peroxins PEX6, PEX10 and PEX12, was performed. Cellular depletion of these peroxins by RNA interference affected growth of both mammalian bloodstream-form and insect-form (procyclic) trypanosomes. The bloodstream forms, which rely entirely on glycolysis for their ATP supply, were more rapidly killed. Both by immunofluorescence studies of intact procyclic T. brucei cells and subcellular fractionation experiments involving differential permeabilization of plasma and organellar membranes it was shown that RNAi-dependent knockdown of the expression of each of these peroxins resulted in the partial mis-localization of different types of glycosomal matrix enzymes to the cytoplasm: proteins with consensus motifs such as the C-terminal type 1 peroxisomal targeting signal PTS1 or the N-terminal signal PTS2 and a protein for which the sorting information is present in a polypeptide-internal fragment not containing an identifiable consensus sequence.     

N-linked glycans with similar location in the fusion protein head modulate paramyxovirus fusion

N-linked glycans not only orchestrate the folding and intracellular transport of viral glycoproteins but also modulate their function. We have characterized the three glycans attached to fusion (F) proteins of the morbilliviruses canine distemper virus and measles virus. The individual Morbillivirus glycans have similar functional properties: the glycan at position 68 is essential for protein transport, and those at positions 36 and 75 modulate fusion (numbering according to the Newcastle disease virus [NDV] F protein sequence). Based on the crystal structure of the NDV F protein, we then predicted the locations of the Morbillivirus glycans: the glycan at position 36 is located in the F protein head, and those at positions 68 and 75 are located near the neck-stalk interface. NDV position 36 is not occupied by a glycan; the only glycan in that F protein head also has a fusion control function and grows from residue 366, located only 6 A from residue 36. We then exchanged the glycan at position 36 with the glycan at position 366 and showed functional complementation. Thus, structural information about the F proteins of Paramyxoviridae coupled with functional analysis disclosed a location in the protein head into which fusion-modulating glycans independently evolved.     

Kinetics of S-adenosylmethionine cellular transport and protein methylation in Trypanosoma brucei brucei and Trypanosoma brucei rhodesiense

African trypanosomes of the Trypanosoma brucei group are agents of disease in man and animals. They present unique biochemical characteristics such as the need for preformed purines and have extensive salvage mechanisms for nucleoside recovery. In this regard we have shown that trypanosomes have a dedicated transporter for S-adenosylmethionine (AdoMet), a key metabolite in transmethylation reactions and polyamine synthesis. In this study we compared the apparent kinetics of AdoMet transport, cytosolic AdoMet pool formation, and utilization of AdoMet in protein methylation reactions using two isolates: Trypanosoma brucei brucei, a veterinary parasite, and Trypanosoma brucei rhodesiense, a human pathogen that is highly refractory and has greatly reduced susceptibility to standard trypanocidal agents active against T. b. brucei. The apparent Km values for [methyl-3H]AdoMet transport, derived by Hanes-Woolf analysis, for T. b. brucei was 4.2 and 10 mM for T. b. rhodesiense, and the Vmax values were 124 and 400 micromol/liter/min, respectively. Both strains formed substantial cytosolic pools of AdoMet, 1600 nmol/10(9) T. b. brucei and 3500 nmol/10(9) T. b. rhodesiense after 10 min incubation with 25 mM exogenous AdoMet. Data obtained from washed trichloroacetic acid precipitates of cells incubated with [methyl-3H]AdoMet indicated that the rate of protein methylation in T. b. brucei was fourfold greater than in T. b. rhodesiense. These results demonstrate that the unique rapid uptake and utilization of AdoMet by African trypanosomes is an important consideration in the design and development of new agents of potential use in chemotherapy.