Auxilin, a newly identified clathrin-associated protein in coated vesicles from bovine brain.

We have identified a new coat protein in clathrin-coated vesicles from bovine brain by urea-SDS gel electrophoresis. The protein was purified from Tris-solubilized coat proteins either by combination of hydroxyapatite chromatography and gel filtration or more rapidly in a single step by immunoaffinity chromatography. The purified protein binds to clathrin triskelia and thereby promotes clathrin assembly into regular 50-100-nm cages. We propose for the new protein the name auxilin (Latin auxilium, meaning support). Auxilin migrates as a 110-kD polypeptide in standard type SDS-PAGE, but in the presence of 6 M urea shifts to a position corresponding to 126 kD. Gel filtration in 6 M guanidinium hydrochloride gives a molecular weight of approximately 86,000. The native protein is monomeric in 0.5 M Tris. Antigenic reactivity and two-dimensional peptide maps gave no evidence of gross similarities between auxilin and any of the other known coated vesicle-associated proteins. Since the structural organization of auxilin does not resemble that of the ubiquitous heterotetrameric HA1 and HA2 adaptor complexes, that are believed to connect clathrin to receptors, it is unlikely that it functions as an adaptor. Immunoblotting did not reveal the presence of auxilin in tissues other than brain. If auxilin and AP 180 are indeed both confined to neuronal cells, as the immunochemical evidence suggests, it might be inferred that both serve to adapt clathrin-coated vesicles to an as yet undisclosed function unique to this cell type.

Subpopulations of endosomes generated at sequential stages in the endocytic pathway of asialoganglioside-containing ferrite ligands in rat liver.

Subpopulations of endosomes generated at different stages of the endocytic pathway were isolated by a high-gradient magnetic separation followed by a Percoll density gradient centrifugation. Rat livers were perfused for 5 min with asialoganglioside (ASG)-containing ferrite particles and chased at 37 degrees C. At various times after the internalization, the endocytic vesicles containing ferrite particles were isolated by the magnetic separation. Isolated fractions contained endosomes until 15-min perfusion, after which most of the particles were transported to lysosomes. The endosomal fractions isolated after the 5- or 15-min perfusions were further analyzed by 30% Percoll density gradient centrifugation. The endosomes after 5-min perfusion showed peaks around the density of 1.05 g/ml (peak I) and 1.07 g/ml (peak Is), both of which contained asialoglycoprotein receptors. In the 15-min perfusion, another peak of endosomes (peak II) was observed at the higher density of 1.09 g/ml without the receptors, in addition to peak I. These endosomes had their own characteristic proteins. Some proteins were common in the subgroups of endosomes. These results suggest that the endosome I containing the ligands and the receptors was first produced after endocytosis and, through the endosome is, was scissioned into the endosome II containing the ligands. The endosome II was then fused with primary lysosomes for proteolytic cleavage of ligands.

Analysis of 100-180-kDa phosphoproteins in clathrin-coated vesicles from bovine brain.

Protein kinases which co-purify with clathrin-coated vesicles are known to phosphorylate in vitro the 50-kDa subunit of the HA-II adaptor complex and upon inclusion of polylysine the beta-light chain of clathrin and polypeptides above 100 kDa. Here we relate the high molecular mass phosphoproteins to the known subunits of the adaptor protein complexes and to other clathrin-associated proteins by means of immunoprecipitation with monoclonal antibodies, two-dimensional electrophoresis, or electrophoresis in urea-sodium dodecyl sulfate-polyacrylamide gels. Our results show that some of the labeling of the 100-120-kDa region is accounted for by the beta'- and gamma-subunits of the HA-I adaptor complex, the alpha a-, and, to a lesser extent, by the beta-subunits of the HA-II adaptor complex. In addition, we found the assembly protein AP 180 and a hitherto undescribed 110-kDa coat polypeptide to be heavily phosphorylated upon release of these proteins from the coated vesicle membrane. In all cases, labeling was confined to serine residues.

Biochemical characterization of algal coated vesicles.

Thin sections of tissue preparations from a green alga, Ulva lactuca (Ulvophyceae), and brown alga, Laminaria digitata (Pheophyceae) showed the presence of coated pits and coated vesicles in these 2 species. A discontinuous sucrose gradient after subcellular fractionation of the tissue homogenate resulted in an enriched coated vesicle fraction. Electron microscopy of negatively stained samples revealed the presence of coated vesicles of diameter ranging from 40-125 nm, together with large sheets of polygonal nets of clathrin. Electrophoresis of the CV purified fraction revealed various polypeptide components. Two of them, a 175 kDa and a 70 kDa, exhibited a positive response to bovine brain anticlathrin antibodies raised in goat or in rabbit. A third component of 30-40 kDa also gave a faint positive response. These 3 components corresponded to the clathrin heavy and light chains already described in higher plants. Clathrin was released from the CV algal preparations by treatment with 2M urea in Tris buffer, pH 8.5. Interestingly, in Ulva lactuca, the proportion of clathrin relative to the other proteins from the CV decreased with plant growth. Biochemical analysis of the purified CV revealed the presence of all the major phospholipids characterized in mammalian CV. The ratio of protein over lipid was also in the same range as that calculated for mammalian CV. Carbohydrate analysis demonstrated a high proportion of N-acetylgalactosamine and N-acetylglucosamine in both algal CV whereas these sugars were not detectable in the crude homogenate. These results demonstrate the presence of clathrin and coated vesicles in 2 species of algae.(ABSTRACT TRUNCATED AT 250 WORDS)

Fc receptor isoforms exhibit distinct abilities for coated pit localization as a result of cytoplasmic domain heterogeneity.

Mouse macrophages and lymphocytes express two distinct isoforms of a single class of Fc receptor for IgG. The macrophage isoform (FcRII-B2) is identical to the lymphocyte isoform (FcRII-B1) except for an inframe insertion in the cytoplasmic tail of FcRII-B1 that increases its length from 47 to 94 amino acids. To determine the functional significance of this cytoplasmic domain variation, presumably the result of alternative mRNA splicing, we expressed both isoforms in receptor-negative fibroblasts. While FcRII-B2 mediated the efficient ligand internalization and delivery to lysosomes, endocytosis via FcRII-B1--and via a tailminus mutant--was relatively inefficient. This difference reflected the inability of FcRII-B1 (and the tailminus mutant) to accumulate in clathrin-coated pits. Thus, the FcRII-B2 cytoplasmic tail contains a domain needed for accumulation in coated pits, and this domain is disrupted by the 47 amino acid insertion in FcRII-B1.

Cloning of cDNAs encoding two related 100-kD coated vesicle proteins (alpha-adaptins).

Coat proteins of approximately 100-kD (adaptins) are components of the adaptor complexes which link clathrin to receptors in coated vesicles. The alpha-adaptins, which are found exclusively in endocytic coated vesicles, separate into two bands on SDS gels, designated A and C (Robinson, M. S., 1987. J. Cell Biol. 104:887-895). Two distinct cDNAs (sequences 1 and 2) encoding the two alpha-adaptins were cloned from a mouse brain cDNA library. Southern blotting indicates that there is one copy of each of the two alpha-adaptin genes, and that there are no additional closely related genes. Based on the size of the predicted protein products of the two genes (108 and 104 kD), the relative abundance of the two messages in brain and liver, and the reactivity of a sequence 1 fusion protein with different antibodies, it was possible to conclude that sequence 1 codes for A and sequence 2 for C. The two protein sequences are strikingly homologous to each other (84% identical amino acids), the major difference being an additional stretch of 41 amino acids, rich in prolines and acidic residues, inserted into the COOH-terminal half of A. In situ hybridization carried out on mouse brain sections indicates that the same cell type may express both transcripts, but that their relative expressions vary. Antipeptide antibodies are now being raised to find out whether the proteins are localized in functionally distinct populations of endocytic coated vesicles.

A neuronal protein (NP185) associated with clathrin-coated vesicles. Characterization of NP185 with monoclonal antibodies.

Two monoclonal antibodies (S-8G8 and S-6G7) are characterized that react with an abundant neuronal protein associated with brain clathrin-coated vesicles (CCVs). This 185-kDa polypeptide (NP185) is not a transmembrane cargo molecule and is distinguishable from clathrin by several criteria including neuronal specificity, chymotryptic sensitivity, migration during two-dimensional gel electrophoresis, lack of cross-reactivity of S-8G8 or S-6G7 with purified clathrin, and lack of associated clathrin light chains. When 0.9 M NaCl extracts of CCVs were diluted and immunoprecipitated by either S-8G8 or S-6G7, NP185 precipitated as a complex with a fraction of the CCV assembly polypeptides. Immunofluorescence microscopy of PC12 cells cultured in nerve growth factor (NGF) revealed that NP185 was distributed in a punctate manner throughout the mature neurites. Immunoblot analysis of PC12 cell extracts, taken at various times during NGF-induced differentiation, revealed that steady-state accumulation of NP185 reaches significant levels 3 days after the addition of NGF and returns to undetectable levels when NGF is removed from the cultures. Significantly, the quantity of NP185 detected in differentiated PC12 cells exceeded the quantity of clathrin. These data indicate that while NP185 may be a specialized component of neuronal CCVs, its function in neuronal cells cannot be associated exclusively with these organelles.

In vivo phosphorylation of clathrin-coated vesicle proteins from rat reticulocytes.

We have studied the in vivo phosphorylation of clathrin-coated vesicle proteins from rat reticulocytes. The major 32P-labeled polypeptides of clathrin-coated vesicles isolated from metabolically labeled cells were the the 165-, 100-110-, and 50-kDa polypeptides of the assembly protein, the clathrin beta-light chain, and to a lesser extent the clathrin alpha-light chain. The phosphorylation of the assembled (particulate) and unassembled (soluble) pools of clathrin and assembly protein was compared by immunoprecipitating the respective protein complexes from particulate and soluble cell fractions. Although all the phosphorylated polypeptides were present in both fractions, the extent of labeling was protein and fraction specific: the apparent specific activities of the assembly protein 50-kDa polypeptide and clathrin light chain were higher in the unassembled pool, whereas those of the 100-110-kDa polypeptides were higher in the assembled pool. The amino acids and polypeptide fragments labeled in vivo appeared similar to those labeled in vitro.

Characterization of a 280-kD protein restricted to the coated pits of the renal brush border and the epithelial cells of the yolk sac. Teratogenic effect of the specific monoclonal antibodies.

Intermicrovillar areas and apical vesicles characterized by an extensive clathrin coat can be identified in some epithelial cell types. We describe a 280-kD protein, characteristic of these areas in the proximal tubule brush border and epithelial cells of the visceral yolk sac. When injected to 9-d pregnant rats, mAbs to the 280-kD protein regularly induced fetal resorption and/or malformations. Antibodies to a 330-kD protein that is also coated-pit-restricted had no effect. Our observations point to a key function for p280 and suggest that immunity to specific constituents of the receptor-mediated endocytotic system may be involved in the induction of fetal abnormalities.

Purification and characterization of two distinct complexes of assembly polypeptides from calf brain coated vesicles that differ in their polypeptide composition and kinase activities.

The binding and assembly of clathrin triskelions on vesicle membranes seem to be mediated by certain assembly polypeptides (Keen, J.H., Willingham, M.C., and Pastau, I.H. (1979) Cell 16, 303-312). These assembly polypeptides were further purified into two distinct complexes using hydroxylapatite chromatography. Peak 1 consists of two major bands of 98 and 112 kDa, two minor bands of 103 and 118 kDa, and a polypeptide of 46 kDa. Peak 2 consists of one major band of 100 kDa, two minor bands of 103 and 115 kDa, and a polypeptide of 50 kDa. Both complexes have a native molecular mass of 290 kDa as determined by gel filtration. Each 290-kDa complex contains two polypeptides of 98-118/100-115 kDa and two polypeptides of 46/50 kDa. The 46-kDa polypeptide is not phosphorylated, whereas the 50-kDa polypeptide is. Both peaks contain 50-kDa kinase-like activity. Time courses of the 50-kDa phosphorylation show that the activity in peak 1 saturates much faster than the activity in peak 2; there may be two 50-kDa kinase activities in coated vesicles. A kinase that phosphorylates the polypeptides in 98-118-kDa group is present in peak 1 but not in peak 2. Both peaks assemble clathrin triskelions into cages under conditions in which the clathrin alone would not assemble. Both rotary shadowed and negatively stained preparations of these reassembled cages as well as the purified complexes were examined by electron microscopy. Thus, two complexes have been identified that differ in their polypeptide composition and kinase activities, but are similar in their ability to assemble clathrin triskelions into cages.

Isolation of an endocytic compartment from A431 cells using a density modification procedure employing a receptor-specific monoclonal antibody complexed with colloidal gold.

Our objective was to isolate a prelysosomal compartment involved in receptor-mediated endocytosis in human epidermoid carcinoma (A431) cells. The isolation protocol involves density modification of endosome elements in A431 cells, caused by the receptor-dependent binding and internalization at 20 degrees C of colloidal gold-transferrin receptor antibody (B3/25) particles. The use of 125I-labelled gold-B3/25 provides a radioactive marker for the endosome compartment, the major peak being recovered at the bottom of a continuous sucrose gradient at a density of 1.23g ml-1. Enzyme markers characteristic of other cytoplasmic compartments are present only in negligible amounts in this fraction and L-[35S]methionine-labelling of the cells indicates approximately a 200-fold enrichment of 125I-labelled gold-B3/25 versus protein. Electron microscopy of the endosome-rich fraction reveals that we have isolated a highly purified population of small gold-containing vesicles and tubules from which the transferrin receptor can be immunoprecipitated using the B3/25 antibody. Gel electrophoresis and fluorography of L-[35S]-methionine-labelled cells suggests that these elements contain a characteristic profile of approximately 10 major proteins of which three appear to be specifically enriched. In cells incubated with [125I]transferrin, 12% of the ligand sediments with the gold-labelled elements. We conclude, therefore, that the components we have isolated play a role in the intracellular processing of the transferrin-transferrin receptor complexes.

Analysis of 100 kDa polypeptides from coated vesicles of bovine brain: two-dimensional tryptic and chymotryptic maps.

Two-dimensional peptide map analysis was used to determine the structural homology among the '100 kDa'-group of polypeptides. There are at least six distinct polypeptides whose apparent molecular weights are 116, 113, 111, 108, 105 and 100 kDa. The molar ratio of the '100 kDa'-group of polypeptides to three clathrin monomers (equivalent to one triskelion) is 1.2:1. There are three families of polypeptides in the '100 kDa'-group as determined by two-dimensional peptide map analysis. They are 116 and 113 kDa polypeptides, 111, 108, and 105 kDa polypeptides and 100 kDa polypeptide. However, all six polypeptides apparently show a series of homologous peptides. It is suggested that the 100-116 kDa polypeptides may bind to triskelions at the area of homology that is found in the 100-116 kDa polypeptides.

Purification and characterization of a molecular weight 100,000 coat protein from coated vesicles obtained from bovine brain.

A protein designated as a 100-kDa protein on the basis of sodium dodecyl sulfate gel electrophoresis was purified from coated vesicles obtained from bovine brain, with uncoated vesicles as starting material. Two gel filtration steps, one involving 0.5 M tris(hydroxymethyl)aminomethane, pH 8.0, buffer, and the other 0.01 M tris(hydroxymethyl)aminomethane, pH 8.0, and 3 M urea buffer, were employed. The purified protein has a native molecular weight of 114,000 as determined by sedimentation equilibrium analysis. Circular dichroism data showed that the protein has 28% helical structure, 29% beta-structure, and 15% beta-turns, and the rest is random coil. Addition of the purified protein to clathrin results in the polymerization of clathrin to homogeneous size baskets of sedimentation velocity 150 S. A scan of the Coomassie Blue stained electrophoresis gels of the polymerized baskets shows that, for every clathrin trimer, there is approximately one 100-kDa protein molecule.

[Isolation and characteristics of endocytic vacuoles containing the influenza virus]. / Vydelenie i kharakteristika éndotsitarnykh vakuolei, soderzhashchikh virus grippa.

Endocytic vacuoles (receptosomes) containing influenza virus were isolated from the cytoplasm of Ehrlich ascitic carcinoma cells and characterized. In the sucrose density gradient, the virus-containing material was detected in two peaks with a buoyant density of 1.175-1.16 and 1.155-1.135 g/cm3 with which the activity of marker enzymes of cell plasma membranes was associated. The virus was present in receptosomes in morphologically and electrophoretically intact condition. Examinations for the lipid composition of endocytic vacuoles showed the presence in their membranes of large amounts of cholesterol and glycolipids, particularly asialo-GM1 which, according to some authors may enhance the fusion of viral and cell membranes.

Separation of endocytic from exocytic coated vesicles using a novel cholinesterase mediated density shift technique.

Coated vesicles isolated from rat liver perfused with diisopropylfluorophosphate (DFP) to inactivate endogenous cholinesterase contained newly synthesized secretory cholinesterase after a 30 min recovery. The cholinesterase is found in coated vesicles of presumed endocytic origin following DFP treatment and perfusion for 3 min with galactosylated cholinesterase, a ligand for the asialoglycoprotein receptor. Highly enriched populations of endocytic and exocytic coated vesicles can be separated by use of a novel cholinesterase mediated density shift technique. The two coated vesicle classes have very similar polypeptide compositions but differ significantly in the ratio of cholesterol to phospholipid.

Evidence for the interaction of alpha-actinin and calmodulin with the clathrin heavy chain.

In the present study protein overlays were used to study the molecular interactions of clathrin with clathrin coat-associated proteins. Coated vesicles (CV) were isolated, subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and transferred to nitrocellulose. The transfers were quenched and equilibrated in buffer, containing 1% Triton X-100. Alpha-Actinin and calmodulin, proteins known to interact with coated vesicles, were iodinated, placed in buffer, and incubated over the transfer for 1 h. After being rinsed extensively, the total amount of 125I associated with the filters was measured, and the filters were then processed for autoradiography. For alpha-actinin, the clathrin heavy chain and a series of lower molecular weight proteins were labeled. The binding of 125I alpha-actinin was inhibited with cold ligand and selectively released from the transfer with a buffer know to strip alpha-actinin from plasma membrane preparations. For 125I calmodulin the predominant binding site was also the clathrin heavy chain. Cold ligand inhibited binding and 60% of the detectable binding were calcium dependent. In addition, when these ligands were used in competition with each other, no significant inhibition was detected in the amount of binding associated with the clathrin heavy chain. These studies show that the clathrin heavy chain is a primary site of the clathrin cage receptive to intracellular interactions and furthermore suggest that the clathrin heavy chain consists of domains of biochemical specificity which may selectively affect the activities of coated vesicles.

Transmembrane orientation of the mannose 6-phosphate receptor in isolated clathrin-coated vesicles.

The mannose 6-phosphate (Man-6-P) receptor is an integral membrane glycoprotein which mediates intracellular transport and receptor-mediated endocytosis of lysosomal proteins. Clathrin-coated vesicles, which have been shown to be significantly involved in these processes, have also been shown to be a major subcellular site of the receptor. In order to define the orientation of the Man-6-P receptor within the coated vesicle membrane, highly purified preparations of coated vesicles were prepared from bovine brain employing D2O/sucrose gradient centrifugation and Sephacryl S-1000 column chromatography. Using [35S]methionine-labeled lysosomal enzymes secreted by Chinese hamster ovary cells as receptor ligand, significant binding activity was detected only upon permeabilization of the coated vesicle membranes with detergent. Prior treatment of intact vesicles with proteinase K resulted in similar binding activity upon permeabilization. However, examination of the receptor by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting with rabbit anti-receptor serum revealed that proteinase K treatment of intact vesicles reduced the size of the receptor by 12,000 daltons. A similar decrease in size was obtained when the vesicles were treated with carboxypeptidase Y. These results suggest that the Man-6-P receptor is a transmembrane protein with its lysosomal enzyme binding site oriented toward the lumen of the coated vesicle and its C-terminal end exposed to the exterior or cytoplasmic portion of the vesicle membrane.

Coated vesicles from the thyroid gland: isolation, characterization, and a search for a possible role in thyroglobulin transport.

We report the first isolation of purified coated vesicles (CVs) from thyroid gland. Bovine thyroid CVs were isolated by differential centrifugation, including a step through sucrose-D2O, using a modification of the method described by Nandi et al. (1) for bovine brain CVs. The CVs were characterized by electron microscopy, sedimentation properties, and SDS-PAGE of the protein components. Thyroglobulin (Tg) was found to be associated with the purified CVs. When the thyroid CVs were exposed to conditions known to remove the protein coat from brain CVs, such as low ionic strength at pH 8.5, most of the Tg dissociated from the vesicles along with the coat proteins. Moreover, the Tg remaining with the uncoated vesicles (UVs) was trypsin sensitive, and therefore judged to be associated with the external surface of the vesicle. Since ligand-receptor complexes are normally located within CVs and not on their outer surface, no evidence was found for Tg-receptor complexes within thyroid CVs. Thyroid slices were incubated in the presence of [35S] methionine with subsequent isolation of labeled CVs in order to study the incorporation of newly-synthesized proteins into these structures. At 0.5 and 2 hours of incubation, the 180K MW subunit of clathrin, as well as other proteins, but not Tg, had become labeled in the purified CVs. Extracellular 19S-[35S] thyroglobulin was isolated from the incubation medium, however, demonstrating release of newly-synthesized Tg (presumably into cut follicles). It is concluded that thyroid CVs do not seem to be involved in the secretion of newly-synthesized Tg from the rough endoplasmic reticulum into the follicular lumen. While a possible role of thyroid CVs in the reabsorption of small quantities Tg by micropinocytosis cannot be completely excluded, the present data do not support a primary role for thyroid CVs in either endocytosis or exocytosis of Tg.

Solubilization of proteins from bovine brain coated vesicles by protein perturbants and Triton X-100.

To identify integral and peripheral membrane proteins, highly purified coated vesicles from bovine brain were exposed to solutions of various pH, ionic strength, and concentrations of the nonionic detergent Triton X-100. At pH 10.0 or above most major proteins were liberated, but four minor polypeptides sedimented with the vesicles. From quantitative analysis of phospholipids in the pellet and extract, we determined that at a pH of up to 12 all phospholipids could be recovered in the pellet. Electron microscopic examination of coated vesicles at pH 12.0 showed all vesicles devoid of coat structures. Treatment with high ionic strength solutions (0-1.0 M KCl) at pH 6.5-8.5 also liberated all major proteins, except tubulin, which remained sedimentable. The addition of Triton X-100 to coated vesicles or to stripped vesicles from which 90% of the clathrin had been removed resulted in the release of four distinct polypeptides of approximate Mr 38,000, 29,000, 24,000 and 10,000. The 38,000-D polypeptide (pK approximately 5.0), which represents approximately 50% of the protein liberated by Triton X-100, appears to be a glycoprotein on the basis of its reaction with periodic acid-Schiff reagent. Extraction of 90% of the clathrin followed by extraction of 90% of the phospholipids with Triton X-100 produced a protein residue that remained sedimentable and consisted of structures that appeared to be shrunken stripped vesicles. Together our data indicate that most of the major polypeptides of brain coated vesicles behave as peripheral membrane proteins and at least four polypeptides behave as integral membrane proteins. By use of a monoclonal antibody, we have identified one of these polypeptides (38,000 mol wt) as a marker for a subpopulation of calf brain coated vesicles.

The effects of lyotropic (Hofmeister) salts on the stability of clathrin coat structure in coated vesicles and baskets.

The pH dependence of the stability of the clathrin coat structure of coated vesicles and baskets has been evaluated by light scatter and sucrose gradient centrifugation. The influence of several lyotropic (Hofmeister) salts has also been studied by the same methods in order to distinguish between electrostatic and hydrophobic contributions to the free energy of clathrin association. In accord with the Hofmeister ranking, sulfate stabilizes whereas perchlorate destabilizes coat structure. Both types of interactions contribute to the stability of the coat structure in coated vesicles and baskets since both ionic strength and Hofmeister effects have an important influence. The properties of clathrin in both types of particles are similar, with the coat being slightly more stable in coated vesicles than in baskets.