Coated Vesicles from the Protozoan Parasite Trypanosoma brucei: Purification and Characterization

ABSTRACT. A procedure was developed to purify a coated vesicle fraction from the protozoan parasite Trypanosoma brucei. Electron microscopy revealed a difference between T. brucei coated vesicles and clathrin-coated vesicles from other eukaryotes: trypanosome vesicles were larger (100 to ISO nm in diameter) and contained an inner coat of electron-dense material in addition to the external coat. Evidence suggests that the internal coat is the parasite's variant surface glycoprotein (VSG) coat. The SDS-PAGE analysis shows the major protein of T. brucei coated vesicles has a molecular mass of 61 kD, similar to VSG; this protein was recognized in an immunoblot by anti-VSG serum. Trypanosome coated vesicles also contain a protein which comigrates with the major protein (clathrin) of coated vesicles purified from rat brains. However, this protein is a minor component and it is not serologically cross-reactive with mammalian clathrin. Immunoblot analysis demonstrated that the parasite vesicles contained host IgG, IgM, and serum albumin.     

Clathrin in Trypanosoma cruzi: in silico gene identification, isolation, and localization of protein expression sites

Clathrin is a scaffold protein found in different types of coated vesicles in most eukaryotic cells. Major forces that drive clathrin coat formation are the adaptor protein complexes. Trypanosoma cruzi is a flagellate protozoan that ingests macromolecules through receptor-mediated endocytosis, but the molecules involved in this process are still poorly known. Bioinformatics was used to identify proteins in the T. cruzi genome database, permitting discrimination of the genes involved in clathrin coat assembly. Clathrin expression was demonstrated in T. cruzi epimastigotes by using several experimental approaches. Western blot analysis showed a single 180-kDa protein band, which corresponds to the molecular mass of mammalian clathrin heavy chain. A flow cytometry assay demonstrated that the clathrin heavy chain was expressed in 97.74% of the cell population analyzed, with a high-fluorescence signal. Immunofluorescence observation showed labeling clustered at the flagellar pocket and Golgi complex region. Coated vesicles budding off from the flagellar pocket and the trans Golgi network membranes were identified by transmission electron microscopy. Our data demonstrate the expression of clathrin in T. cruzi epimastigotes and show the association of this polypeptide with the parasite endocytic and exocytic pathways.     

Phosphorylation of a clathrin light chain of coated vesicles in the presence of histones

Coated vesicles isolated from bovine brain contained a protein kinase(s) which phosphorylated phosvitin and an endogenous protein with a molecular weight (Mr) of 48,000. A clathrin light chain (Mr 33,000), a constituent of the coat structure of the coated vesicles, was also phosphorylated when histone was added to the incubation medium. The clathrin light chain was phosphorylated with GTP as well as ATP as the phosphoryl donor. The phosphorylation reaction was inhibited by heparin. An additional 1.35 mol of PO4/mol was incorporated into the clathrin light chain which had contained approximately 1.5 mol of PO4/mol when the coated vesicles were incubated with ATP, Mg2+, and histone. Phosphoamino acid determination revealed the presence of 32P-phosphorylated threonine and serine in phosvitin, threonine in the endogenous protein (Mr 48,000) and serine in the clathrin light chain (Mr 33,000).     

Structural characterization of labeled clathrin and coated vesicles

Clathrin (8 S) and coated vesicles have been covalently labeled by using the sulfhydryl-labeling fluorescent probe N-(1-anilinonaphthalene)maleimide. A large increase in energy transfer from Trp to anilinonaphthalene (AN) residues was observed in clathrin in the pH range approximately 6.5-6.0, where the rate of clathrin self-association increased rapidly. The change in energy transfer was indicative of a conformational rearrangement, which could be responsible for the initiation of the clathrin self-association reaction to form coat structure. The AN label was found in both the coat and membrane proteins after dissociation of coated vesicles at pH 8.5. The labeled coat and membrane proteins readily recombined to form coated vesicles after reducing the pH to 6.5, indicating that the labeling did not interfere with the ability of clathrin to self-associate and interact with uncoated vesicles to form coat structure. A comparison of the AN fluorescence with the Coomassie blue pattern after electrophoresis in sodium dodecyl sulfate-gels revealed that a 180,000-Da protein (clathrin) was mainly labeled in coated vesicles, while a 110,000-Da protein was also strongly labeled in uncoated vesicles. AN-labeled baskets and coated vesicles have been prepared. Trypsin digestion reduced the sedimentation rate of baskets from 150 S to 120 S and of coated vesicles from 200 S to 150 S. Gel electrophoresis of baskets and coated vesicles showed extensive conversion of clathrin (Mr 180,000) to a product of Mr approximately equal to 110,000, suggesting equivalent structural organization of the coat in coated vesicles as in baskets. In both cases, the peptide(s) released from the vesicles by digestion were essentially free of fluorescent label. In the case of the uncoated vesicles, tryptic digestion released most of the proteins remaining after coat removal.     

Calmodulin binding and protein phosphorylation in adrenal medulla coated vesicles

Coated vesicles from bovine adrenal medulla contained clathrin and major detergent-insoluble polypeptides of 120-100, 51 and 49 kDa. Intact coated vesicles and vesicles lacking clathrin light chains were bound by immobilized calmodulin in the presence of Ca2+. Clathrin in the form of 700 A cages was not bound. The calmodulin binding components in intact coated vesicles are therefore contributed by the enclosed vesicle or by the 120-100, 50 or 49 kDa polypeptides. The 51 kDa component incorporated 32Pi from labelled ATP by an endogenous kinase activity; no other coat or vesicle membrane protein was phosphorylated in vitro, either by intrinsic or exogenous kinases.     

Coat formation in coated vesicles

The proteins of Mr 100 000-110 000 present in the protein coat of coated vesicles have been shown to facilitate formation of a homogeneous small-size basket (coat) when added to clathrin [Zaremba, S., & Keen, J.H. (1983) J. Cell Biol. 97, 1339]. We have prepared this protein of coat proteins by two different methods and shown that they are very important for the binding of clathrin to uncoated vesicles to form coated vesicles. By labeling the three components (clathrin, 100 000-110 000 proteins, and uncoated vesicles) with different fluorescent markers and analyzing their distribution on sucrose gradients, we have been able to determine the composition of the products formed. In the presence of the 100 000-100 000 fraction of coat proteins, not only does the size distribution of the clathrin basket become uniform but also the rate of polymerization is strongly increased.     

Coated Vesicles From Locust Oocytes: Isolation and Characterization

Summary

This report demonstrates for the first time the isolation of coated vesicles from insect oocytes. Coated vesicles were purified from oocytes of Locusta migratoria by differential centrifugation and sucrose density centrifugation. The coated vesicles were characterized by electron microscopy, SDS-PAGE and scanning densitometry. Like coated vesicles isolated from pig brain and chicken oocytes, the coated vesicles from locust oocytes contained clathrin as the major protein component. Apart from clathrin, another major protein characteristic of coated vesicles had a molecular weight of about 115,000, and in addition, several minor unidentified bands were identified.     

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.     

Isolation and characterization of coated vesicles from filamentous fungi

Coated vesicles have been shown to exist in Neurospora crassa (Ascomycetes) and Uromyces phaseoli (Basidiomycetes) growing germlings. Separation of coated vesicles in both fungi was obtained when the high-speed (100,000g) pellet was fractioned on a Sephacryl S-1000 gel filtration column, according to the procedure of Mueller and Branton. Electron micrographs of negatively stained coated vesicles from fractions of gel filtration show the same striking lattice coated vesicles similar to vertebrate coated vesicles. We observe two major size classes of coated vesicles in both fungi: the larger class (100-180 nm) is similar in size to vertebrate coated vesicles; the smaller class (50-80 nm) is mostly found in both fungi. When examined by SDS-PAGE, the Sephacryl column fractions containing the maximum concentration of electron microscopically visible coated vesicles coincide with the bands of the protein coat reported as clathrin. The protein composition on SDS-PAGE of the coated vesicles indicates a major polypeptide species of 180 kDa and minor 30 to 36 kDa species. Polypeptides of 100 kDa and 64 kDa are also found in the fractions containing coated vesicles.     

Calmodulin Affinity for Brain Coated Vesicle Proteins

A systematic characterization of the affinity of calmodulin for brain coated vesicles was undertaken. Binding of 125I-labeled calmodulin to coated vesicles was saturable and competed with unlabeled calmodulin, but not with troponin-C. Scatchard analysis revealed one high-affinity, low-capacity binding site, KD = 3.9 +/- 0.6 nM, Bmax = 16.3 +/- 2.4 pmol/mg, and one low-affinity, high-capacity binding site, KD = 102 +/- 15.0 nM, Bmax = 151 +/- 23.0 pmol/mg. Radioimmunoassay revealed that coated vesicles contain 1.05 microgram calmodulin/mg protein. Because this value remained constant even after removal of clathrin, the major coat protein, from the coated vesicle, it is apparent that calmodulin is associated with the vesicle per se rather than with its clathrin lattice. When a Triton X-100-treated extract of coated vesicles was passed through a Sepharose 4B-calmodulin affinity column, polypeptides with Mrs (molecular weights) of 100,000, 55,000, and 30,000 bound in a Ca2+-dependent manner. A 30,000 Mr protein doublet purified from coated vesicles was completely eluted by EGTA from the calmodulin affinity column, confirming that this protein doublet represents one of the coated vesicle calmodulin binding sites. Because calmodulin stimulated [Ca2+-Mg2+]-ATPase activity as well as Ca2+ uptake in coated vesicles, it is postulated that the 100,000 and 55,000 Mr calmodulin binding proteins represent the [Ca2+-Mg2+]-ATPase complex, the other coated vesicle calmodulin binding site.     

Light-chain-independent binding of adaptors, AP180, and auxilin to clathrin

Binding of coated vesicle assembly proteins to clathrin causes it to assemble into regular coat structures. The assembly protein fraction of bovine brain coated vesicles comprises AP180, auxilin, and HA1 and HA2 adaptors. Clathrin heavy chains, separated from their light chains, polymerize with unimpaired efficiency when assembly proteins are added. The reassembled coats were purified by sucrose gradient centrifugation and examined for composition by SDS-PAGE and immunoblotting. We found that all four major coat proteins are incorporated in the presence and absence of light chains. Moreover, each of the purified coat proteins is able to associate directly with clathrin heavy chains in preassembled cages as efficiently as with intact clathrin. We conclude that light chains are not essential for the interaction of AP180, auxilin, and HA1 and HA2 with clathrin.     

Clathrin functions in the absence of heterotetrameric adaptors and AP180-related proteins in yeast.

The major coat proteins of clathrin-coated vesicles are the clathrin triskelion and heterotetrameric associated protein (AP) complexes. The APs are thought to be involved in cargo capture and recruitment of clathrin to the membrane during endocytosis and sorting in the trans-Golgi network/endosomal system. AP180 is an abundant coat protein in brain clathrin-coated vesicles, and it has potent clathrin assembly activity. In Saccharomyces cerevisiae, there are 13 genes encoding homologs of heterotetrameric AP subunits and two genes encoding AP180-related proteins. To test the model that clathrin function is dependent on the heterotetrameric APs and/or AP180 homologs, yeast strains containing multiple disruptions in AP subunit genes, as well as in the two YAP180 genes, were constructed. Surprisingly, the AP deletion strains did not display the phenotypes associated with clathrin deficiency, including slowed growth and endocytosis, defective late Golgi protein retention and impaired cytosol to vacuole/autophagy function. Clathrin-coated vesicles isolated from multiple AP deletion mutants were morphologically indistinguishable from those from wild-type cells. These results indicate that clathrin function and recruitment onto membranes are not dependent upon heterotetrameric adaptors or AP180 homologs in yeast. Therefore, alternative mechanisms for clathrin assembly and coated vesicle formation, as well as the role of AP complexes and AP180-related proteins in these processes, must be considered.     

The glycoforms of a Trypanosoma brucei variant surface glycoprotein and molecular modeling of a glycosylated surface coat

The plasma membrane of the African sleeping sickness parasite Trypanosoma brucei is covered with a dense, protective surface coat. This surface coat is a monolayer of five million variant surface glycoprotein (VSG) dimers that form a macromolecular diffusion barrier. The surface coat protects the parasite from the innate immune system and, through antigenic variation, the specific host immune response. There are several hundred VSG genes per parasite, and they encode glycoproteins that vary in primary amino acid sequence, the number of N-glycosylation sites, and the types of N-linked oligosaccharides and glycosylphosphatidylinositol membrane anchors they contain. In this study, we show that VSG MITat.1.5 is glycosylated at all three potential N-glycosylation sites, and we assign the oligosaccharides present at each site. Using the most abundant oligosaccharides at each site, we construct a molecular model of the glycoprotein to assess the role of N-linked oligosaccharides in the architecture of the surface coat.     

Immunocytochemical visualization of coated pits and vesicles in human fibroblasts: relation to low density lipoprotein receptor distribution.

The coated pit-coated vesicle system has a key role in the uptake of plasma low density lipoprotein (LDL) and other receptor-bound proteins in human fibroblasts. To study the distribution of coated pits and coated vesicles in fibroblasts by immunochemical techniques at both the light and electron microscopic levels, we immunized rabbits with coat protein extracted from bovine brain-coated vesicles. The resulting anti-coat protein antibody was directed predominantly against clathrin, the 180,ooo dalton protein that constitutes the major component of coat protein. By indirect immunoperoxidase electron microscopy, the anti-coat protein antibody was observed to bind specifically to coated pits on the surface of human fibroblasts and to coated vesicles within the cell. Indirect immunofluorescence and immunoperoxidase staining techniques at the light microscopic level revealed that the coat protein was distributed in fibroblasts in two distinctive patterns: as discrete foci on or near the cell surface that were linearly aligned in association with phase-dense cellular fibers (first pattern), and as intracellular foci that were randomly arranged around the cell nucleus (second pattern). The distribution of coat protein in fibroblasts was compared with the distribution of ferritin-labeled LDL, which was studied with the use of similar electron microscopic and immunofluorescence techniques. As previously reported, electron microscopic studies revealed that the LDL-ferritin binding sites at 4 degrees C were clustered in coated pits. By immunofluorescence microscopy, the LDL-ferritin that was bound to receptors within coated pits was shown to be arranged linearly over the cell surface in a pattern that was similar to the linear arrangement of coat protein (first pattern). Considered together, the current data indicate that coated pits in human fibroblasts contain a protein analogous to clathrin, and that those coated pits which contain receptors for LDL are located over intracellular fibers most likely corresponding to stress fibers. These observationa may have relevance to the mechanisms by which the coated pit-coated vesicle system efficiently delivers recptor-bound ligands to lysosomes.     

Coat proteins isolated from clathrin coated vesicles can assemble into coated pits

Isolated human fibroblast plasma membranes that were attached by their extracellular surface to a solid substratum contained numerous clathrin coated pits that could be removed with a high pH buffer (Moore, M.S., D.T. Mahaffey, F.M. Brodsky, and R.G.W. Anderson. 1987. Science [Wash. DC]. 236:558-563). When these membranes were incubated with coat proteins extracted from purified bovine coated vesicles, new coated pits formed that were indistinguishable from native coated pits. Assembly was dependent on the concentration of coat protein with half maximal assembly occurring at 7 micrograms/ml. Assembly was only slightly affected by the presence of divalent cations. Whereas normal appearing lattices formed in a low ionic strength buffer, when assembly was carried out in a low pH buffer, few coated pits were evident but numerous small clathrin cages decorated the membrane. Coated pits did not form randomly on the surface; instead, they assembled at differentiated regions of membrane that could be distinguished in carbon/platinum replicas of frozen and etched membranes by the presence of numerous particles clustered into patches the size and shape of a coated pit.     

Purification and properties of a new clathrin assembly protein.

A clathrin assembly protein (AP180) has been purified and characterized from coated vesicles of bovine brain. This protein has hitherto escaped detection because in SDS-gel electrophoresis it is obscured by the 180 kd heavy chain of clathrin. Despite the similarity in electrophoretic mobility, AP180 differs from clathrin in both its subunit and native mol. wt, as well as hydrodynamic properties, surface charge and tryptic peptide composition. It also appears immunologically distinct from clathrin, since neither a polyclonal antiserum nor a monoclonal antibody, that have been shown to be specific for AP180, cross-react with the heavy chain of clathrin. AP180 binds to clathrin triskelia and thereby promotes clathrin assembly into regular polyhedral structures of narrow size-distribution (60-90 nm), reminiscent of the surface coat of coated vesicles. In this respect AP180 bears a functional resemblance to the 100-110 kd clathrin assembly polypeptides that have been previously described.