Immunohistochemical localization of 54,000 dalton sialoglycoprotein in the epididymal duct of the germ cell-free W/Wv mouse.

A monoclonal antibody T21 specifically recognizes the mouse epididymal sialoglycoprotein of 54,000 dalton (SGP54). The localization of SGP54 was studied in the epididymal duct of germ cell-free WBB6F1W/Wv mutant mice (W/Wv mice) by avidin biotin complex (ABC) immunohistochemistry using T21. None of the testis cells showed immunoreaction. No spermatozoa were present in the epididymal duct lumen. The duct luminal fluid was stained weakly in the proximal corpus epididymidis, and strongly in the cauda epididymidis. Degenerated cells appeared in the duct lumen. The degenerated cells located at the corpus epididymidis showed strong immunostaining in the cytoplasmic region, while the degenerated cells located at the cauda epididymidis showed weak immunostaining. Immunoreaction was also detected between and on microvilli along the epididymis, the intensity being very strong at the distal caput and proximal corpus epididymidis. Invaginations and coated vesicles at the luminal surface of the principal cells were frequently immunostained at the corpus epididymidis. Giant inclusions frequently occurred in the principal cells of the distal caput and corpus epididymidis, with these being very intensely immunostained. These inclusions are ultrastructurally confirmed to be giant multivesicular bodies reported by ABE et al. (1984) in the mouse with the efferent duct cutting. These results suggest that the majority of excess SGP54 are absorbed by the principal cells at the distal caput to corpus epididymidis and catalyzed in the giant multivesicular bodies.

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.

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)

Binding and transport of transthyretin-gold by the endothelium of the rat choriocapillaris.

The photoreceptors of the neural retina require retinol for synthesis of rhodopsin. In the plasma, retinol is bound to retinol binding protein which is carried by transthyretin (TTR; formerly called prealbumin). It is unknown whether, or how, retinol carrier proteins cross the endothelium of the choriocapillaris, the blood supply to the outer neural retina. This was examined in the present study with TTR-gold probes perfused into rats and localized by electron microscopic techniques. TTR-gold, often in clusters, was localized to diaphragmed fenestrae, parajunctional areas, coated pits, transendothelial channels, multivesicular bodies, and to vesicles close to the Golgi apparatus. The probe was also identified at the luminal and abluminal fronts and the interior of transendothelial channels in an apparent sequence of transit. TTR-gold was also found in a series of interconnected vesicles adjacent to the abluminal side of the endothelium. Localizations were not seen when rat albumin fraction V was substituted for TTR and when the rats were perfused with Pronase E before labeling with TTR-gold. These observations indicate that binding and receptor mediated-like transport of TTR by the endothelium of the choriocapillaris is present. This is similar to the processing of heparin-gold by this endothelium.

Coated vesicles from the protozoan parasite Trypanosoma brucei: purification and characterization.

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 150 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.

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.

Purification and characterization of clathrin-coated vesicles from Chlamydomonas.

Clathrin-coated vesicles, identified by negative staining with uranyl acetate, were purified from Chlamydomonas reinhardtii. Isolated coated vesicles had diameters ranging from 70 to 140 nm (mean diameter +/- SD of 95 +/- 17 nm, n = 300). These vesicles were markedly heterogeneous in both density and surface charge, as indicated by equilibrium density sedimentation and elution from anion-exchange columns. Highly-purified coated-vesicle fractions contained 2 major polypeptides, identified as the clathrin heavy chain (185 kDa) and the clathrin light chain (40 kDa). Chlamydomonas clathrin heavy chain cross-reacts weakly with an antibody against bovine brain clathrin heavy chain. Coat stability in several buffers was compared to that of bovine brain coated vesicles. Stability was similar, except for a greater stability of Chlamydomonas coated vesicles in 0.5 M Tris at pH 7.0.

Organization of clathrin coat structures.

Clathrin (8S), when purified, polymerizes under low-pH conditions (0.1 M MES, pH 6.0-6.2) into a heterogeneous population of baskets with sedimentation coefficients ranging from 150 to 400 S. Several groups of proteins of molecular masses 180, 110, 100, 50, and 47 kDa (based on sodium dodecyl sulfate gel electrophoresis) present in the isolated coated vesicles are involved in polymerizing clathrin under physiological conditions to a homogeneous population of baskets [Zaremba, S., & Keen, J. H. (1983) J. Cell Biol. 97, 1339; Ahle, S., & Ungewickell, E. (1986) EMBO J. 5, 3143]. We now report that in 0.1 M MES, pH 6.0, where pure clathrin polymerizes by itself, the above proteins (together known as associated proteins or APs) induce polymerization of clathrin into three distinct sizes of baskets with sedimentation coefficients of 150, 220, and 300 S. Low ratios of clathrin to APs give rise to smaller sizes, whereas higher ratios give rise to predominantly the larger sizes. The smaller size baskets (150S) are intermediates in the polymerization of clathrin to larger size baskets (300S) as inferred from the dissociation of larger size baskets into smaller size baskets and the formation of larger size baskets from smaller size baskets upon the addition of pure clathrin.

Structural and functional division into two domains of the large (100- to 115-kDa) chains of the clathrin-associated protein complex AP-2.

The clathrin-associated protein complex 2 (AP-2 complex) is a group of proteins associated with clathrin-coated vesicles and believed to interact with cytoplasmic domains of receptors found in the plasma membrane. AP-2 was purified as an assembly of several polypeptide chains (alpha, beta, AP50, and AP17), of which only the alpha and beta chains (100-115 kDa) show significant heterogeneity. We have obtained cDNA clones for two distinct rat brain beta chains. We have also studied the domain organization of bovine brain AP-2 complexes by selective proteolysis. Results of these studies show that the alpha and beta chains have a similar two-domain organization. Their amino-terminal domains are relatively invariant whereas their carboxyl-terminal domains are variable in both sequence and length. We propose that the variable domains select receptors for inclusion in coated vesicles.

Interaction of clathrin coat proteins with unilamellar and multilamellar vesicles of phosphatidylcholine.

The binding of clathrin and accessory coat proteins to small unilamellar vesicles and to liposomes of uncharged phospholipids has been followed by chromatography, 31P-NMR, ESR and fluorescence anisotropy. At pH 6.5 and at an ionic strength value (0.1 M Mes) close to that used during the purification of clathrin-coated vesicles, the proteins do not restore the characteristic network found around the natural vesicles. Instead, a limited fusion leads to enlarged structures in which the perturbation of the dynamics of the phospholipids decreases gradually with the depth in the membrane. While the rate of motion of the outer polar heads is lowered, the order parameter of doxyl groups located either under or in the vicinity of the glycerol backbone is not affected by the proteins. In the inner core of the membrane, the main thermotropic transition of the hydrocarbon chains is unchanged. All the effects are the results of interactions limited to the membrane surface. The electrostatic nature of these interactions is evidenced when the embedded spin labels have a charge protruding at the membrane surface. An 'anchoring' effect appears which is due to the charged groups of the proteins. The lateral diffusion of the probes is reduced and, at low ionic strength, a cationic derivative no longer detects the thermotropic transition of the hydrocarbon chains. These results indicate that, although it is known that clathrin and accessory proteins bind to membranes by a series of protein-protein interactions, this system is not devoid of lipid-protein interactions, at least when it is not organized as in the natural system.

Use of dinitrophenol-IgG conjugates to detect sparse antigens by immunogold labeling.

We describe a novel method for localizing sparse antigens in thin sections by protein A-gold labeling. The primary antibody is applied to fixed and detergent-permeabilized cells. The cells are then incubated with an anti-antibody that has been labeled with multiple dinitrophenol residues. The cells are next fixed again with glutaraldehyde and osmium tetroxide fixatives before embedding in Eponate. When thin sections are prepared, the dinitrophenol residues are readily detected with a tertiary anti-DNP antibody followed by protein A-gold labeling. This method offers good sensitivity along with superior morphology. Our test antigen for this method was the receptor for low-density lipoprotein, an antigen which had evaded detection by protein A-gold using ultra-thin cryosections.

Diffusion properties of clathrin on the surface of isolated mouse liver nuclei by the fluorescence recovery after photobleaching technique.

Clathrin labeled with eosin maleimide showed physicochemical properties similar to the native clathrin. The diffusion coefficients of clathrin protomers and cages measured at 20 degrees C by the fluorescence recovery after photobleaching technique (FRAP) were found equal to (9 +/- 1).10(-8) cm2.s-1 and (3 +/- 0.4.10(-8) cm2.s-1, respectively. After incubation with isolated mouse liver nuclei suspended in an aqueous buffer, FRAP measurements showed that 78% of clathrin was immobilized on the nuclear surface. This immobile fraction might correspond to aggregates of molecules resembling coated pits. The mobile fraction had a diffusion coefficient of 2.5.10(-9) cm2.s-1 which was reduced seven times when the suspension medium of the nuclei contained 50% sucrose, showing that the aqueous phase exerted an important drag on the clathrin molecules motion diffusing on the nuclear surface.

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.

Coated pits and vesicles in the osteoclast.

The osteoclast is a cell with a phagocytic ability not dissimilar to the macrophage. Nevertheless, the mechanisms by which it resorbs bone are poorly understood. The aim of this study was to examine the distribution of coated membrane structures in the osteoclast in order to gain further information about endocytosis in this cell. Osteoclasts around the developing tooth germs of young rats were examined using transmission electron microscopy, and immunocytochemistry. Results showed that previously described coated membrane structures within the ruffled border do not appear to be associated with coated pits or vesicles. Coated pits were, however, evident on the dorsal and lateral surfaces of the cell, particularly opposite the clear zone areas. Immunogold staining for clathrin confirmed that coated pits and vesicles are absent within both the clear zone and ruffled border areas, but present on the lateral and dorsal surfaces of the actively resorbing cell. It is suggested that clathrin-associated receptor-mediated endocytosis occurs along the lateral and dorsal surfaces of the osteoclast for the uptake of nutrients and macromolecules, while endocytosis of bone mineral by the ruffled border is mediated by a non-clathrin associated coated membrane structure.

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.

Clathrin coated vesicles in Neurospora crassa.

Electropherograms of Neurospora crassa homogenates showed a polypeptide with a mobility slightly lower than that of a standard sample of clathrin (from bovine brain). Subcellular fractionation of the homogenate resulted in a 20-fold enrichment of the putative N. crassa clathrin in the microsomal fraction. Further fractionation of the microsomal fraction by glass bead permeation chromatography yielded a fraction enriched about 150-fold relative to the homogenate. Coated vesicles (42.5 +/- 2.5 nm diameter) were found in this preparation by electron microscopy of negatively stained specimens. Ribosomes were virtually absent from this sample. N. crassa clathrin remained associated with the coated vesicles after repeated centrifugation and homogenization steps, even in the presence of 0.4 M-NaCl, but was released by treatment with Tris buffer pH 8.5. However the polypeptide was again sedimentable after dialysis against Mes buffer pH 6.5. Under the electron microscope this sediment resembled the empty coats of higher eukaryotes. The results taken together indicate that a clathrin-like protein occurs in wild type cells of N. crassa.

Clustering in coated vesicles of polyunsaturated phospholipids segregated from plasma and Golgi membranes of adrenocortical cells.

In bovine adrenocortical cells, the fatty acyl chains of the phospholipids have been identified in the membranes of the different cell compartments: plasma membranes, Golgi complex and coated vesicle membranes. An increase in the total number of unsaturation in the fatty acid is demonstrated in the coated vesicle membranes as compared with the plasma and Golgi membranes. Furthermore, it appears that phosphatidylcholine and phosphatidylethanolamine are both enriched in polyunsaturated fatty acyl chains, namely arachidonic and adrenic acids in both types of coated vesicles. Only two of the fatty acids are characteristic of Golgi complex and small coated vesicles, 22:5 (n-6) in PC and 22:6 (n-3) in PE, suggesting that the SCV could originate from the Golgi stacks. A high value of the ratio 22:5 (n-3)/22:6 (n-3) is observed which is, as far as we know, characteristic of adrenal cells.