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.

An intermediate polymer in the assembly of clathrin baskets.

Clathrin (8 S) is known to polymerize into two varieties of basket structures (150 S or 300 S) under the normal buffer conditions [100 mM 2-(N-morpholino)ethanesulfonic acid (Mes), pH 5.9-6.7] used for the isolation of coated vesicles. However, it is now observed that under very low salt conditions (2 mM Mes, pH 5.9), it forms a homogeneous species with a sedimentation coefficient of 27 S. Increasing the salt concentration to 50 mM Mes completely converts all the 27S species into 150S baskets. Sedimentation equilibrium data show that this 27S species has a molecular weight that is 6 times that of the clathrin protomer and is the result of highly cooperative reversible self-association of the 8S protomer. Light-scattering studies show that the stabilities of 27S species and baskets (150 S or 300 S) are comparable. Fluorescent labeling of sulfhydryl groups with N-(1-anilinonaphthalenyl)maleimide indicates that the conformation of clathrin in 27S species and baskets (150 S or 300 S) is similar. Trypsin digestion reveals that in the 27S species clathrin has a conformation differing from that in both the 8S species and baskets.

In vivo biosynthesis of clathrin and other coated vesicle proteins from rat liver.

A biosynthetic study of rat liver coated vesicle (CV) proteins was undertaken by using in vivo labeling with L-[35S]methionine. CVs were isolated and purified by using standard procedures and characterized by electron microscopy, sedimentation, and sodium dodecyl sulfate polyacrylamide gel electrophoresis followed by fluorography, or by gel slicing and liquid scintillation counting. After 5 1/2 min of labeling (the earliest time examined), incorporation of radioactive clathrin heavy-chain (180-kD (kilodalton] subunits as well as a 90-kD CV-associated protein into purified CVs was demonstrated. The level of labeled 180-kD clathrin in coated vesicles increased rapidly during the first 2 hr of labeling and then continued to rise at a slower rate between 4 and 16 hr. This slow accumulation of labeled clathrin heavy chains in the CV pool may reflect early compartmental sequestration of a fraction of newly synthesized clathrin with delayed assembly into free CVs. By 16 hr of labeling, clathrin 180-kD chains and the 90-kD CV-associated protein accounted for approximately 48 and 26%, respectively, of the radioactivity in all CV proteins. Two proteins of MWa 68 kD and 53 kD showed marked declines in cpm/unit protein between 30 min and 4 hr, raising the possibility that these species may be transferred out of CVs during or after transport without loss of the other CV proteins. The possibility is also raised that clathrin heavy chains may be recycled during CV formation. Possible heterogeneity within individual CV preparations with respect to protein composition and derivation from both plasma membrane and Golgi regions are proposed.

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.

The stability and transitions of tryptic-digested clathrin.

The pH-dependence of dissociation of trypsin-digested baskets has been determined by light scattering and compared with that of undigested baskets. Essentially no difference was found between the two types of baskets. The molecular transitions of clathrin derived from digested baskets have been studied by fluorescence spectra and polarization measurements and compared with those of undigested baskets. The transitions in both forms of clathrin were very similar. It is clear, therefore, that removal of about 1/3 of the mass from the distal portions of the arms of the clathrin triskelion does not affect its structural transitions. The interactions between clathrin molecules in the basket structure and those within the molecule appear, therefore, to remain intact in the smaller clathrin chains remaining after tryptic digestion. The function of the distal portion of the clathrin chain still awaits elucidation.

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.

Interaction of carbohydrate and protein in thyroxine binding globulin.

The fluorescence properties of human thyroxine binding globulin were evaluated during enzymatic deglycosylation by using both neuraminidase and a mixture of glycosidases. Three fluorescent chromophores, one intrinsic and two extrinsic, were monitored, and all showed changes in fluorescent parameters that have been interpreted in terms of a loss of interactions between the carbohydrate and amino acid residues during deglycosylation. The loss of carbohydrates also results in a decrease in stability of the protein to both acid and guanidinium chloride inactivation. Since deglycosylation decreases the frictional ratio of thyroxine binding globulin, it is concluded that, although sialic acid and other sugar residues are in contact with the protein surface, the hydrated carbohydrate chains protrude partially into the solvent.

Interaction of basic compounds with coated vesicles.

The effect of poly- and dibasic amines, including chloroquine and quinacrine, on the dissociation of coated vesicles at pH 7.4 in 0.01 M 2-(N-morpholino)ethanesulfonic acid has been evaluated by light scattering and sucrose gradient centrifugation. The degree of inhibition of dissociation by the polybases is proportional to the number of amine groups in each compound. However, very little difference in effectiveness was found in a series of dibasic compounds, NH2(CH2)2-5NH2. Chloroquine and quinacrine contain dibasic aliphatic chains as well as aromatic ring systems. These two antimalarials are more effective in inhibiting dissociation of coated vesicles than the dibasic aliphatic amines. The ring systems therefore appear to be contributing, independently, to the free energy of stabilization of the coat structure of coated vesicles. It is suggested that the interaction of poly- or dibasic compounds with clathrin or coated vesicles could influence the turnover of ligands in receptor-mediated endocytosis.

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.

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.

Stability and structure of clathrin.

The effects of urea on the dissociation and structural transitions of clathrin (8 S) have been evaluated by various techniques. The dissociation of the light chains in 3 M urea has been shown by light scattering, ultracentrifugation, and column chromatography. The dissociated components still retain the capacity to form the characteristic polygonal structure of the coat after removal of the urea. At higher concentrations of urea, the secondary and tertiary structures are eliminated, as documented by various spectroscopic techniques, i.e., tryptophan polarization and emission maxima, circular dichroism, and difference spectra. Two distinct transitions are observed by all techniques, one between 3 and 6 M urea and a second one which starts at 7 M but is still incomplete by 9.6 M urea. A concentration-dependent aggregation of clathrin chains occurs in 4 and 5 M urea solutions, as observed by light scattering and sedimentation. The results indicate that there are two large, independent domains in clathrin heavy chains and that each domain may have a single, highly cooperative transition.

Organization and dynamics of lipids in bovine brain coated and uncoated vesicles.

Three characteristics have been demonstrated by the chemical analysis of bovine brain coated vesicles following removal of the coat proteins: a high protein content, a high cholesterol/lipid ratio and a high percentage of phosphatidylethanolamine amongst the phospholipids. The study of lipid bilayer organization and dynamics has been performed using the fluorescent probes pyrene and parinaric acid (cis and trans). This has allowed the study of both lateral mobility and rotational motion in the lipid bilayer of the coated and uncoated vesicles. Lateral mobility in the fluid phase of the lipid is slightly reduced by the presence of the clathrin coat, as indicated by the lower diffusion coefficient of pyrene in coated compared with uncoated vesicles. At all temperatures from 6 degrees to 30 degrees C, solid-phase domains, probed by trans parinaric acid, coexist with fluid-phase domains in the lipid bilayer. The temperature dependence of the parinaric acid lifetimes and of their amplitudes strongly suggests that the solid phase domains decrease in size with temperature, both in coated and uncoated vesicles. However, the difference in the value of the anisotropy at long times (r infinity), between coated and uncoated vesicles (a difference which is more pronounced for cis than for trans parinaric acid), indicates that the presence of the clathrin coat introduces disorder in the surrounding lipids, thus suggesting a possible role of the clathrin in the formation of the pits on the plasma membrane.

Sizes and mass distributions of clathrin-coated vesicles from bovine brain.

Clathrin-coated vesicles obtained from bovine brain have been studied by ultracentrifugation and dynamic light scattering techniques to provide information on their sedimentation and mass distributions and their average diffusion coefficients. "Uncoated" vesicles, obtained by removing the protein coat from coated vesicles, have been similarly characterized. For typical preparations, maximal values of approximately 210 and 95 S are observed for the sedimentation coefficients of coated and uncoated vesicles, respectively. Corresponding values for the average molecular weights, determined from values of average sedimentation and diffusion coefficients, are 49 X 10(6) and 13 X 10(6); values obtained by equilibrium sedimentation are 37.2 X 10(6) and 10.6 X 10(6). In order to obtain these results, some minor modifications of sedimentation and light-scattering techniques have been devised which may have application to other studies of size distributions of large particles.

Characterization of the major polypeptide chains of reduced bovine thyroglobulin.

The finding that reduced 19S bovine thyroglobulin showed two major, closely migrating polypeptide bands during electrophoresis in SDS-polyacrylamide gels was presented as evidence that thyroglobulin consists of two nonidentical subunits, referred to as S and F, of approximately the same size (J. Biol. Chem. 253, 1853-1858 (1978)). It was, however, not clear whether the difference in migration rates of the two subunits was due to differences in amino acid or carbohydrate composition or a small difference in size. In this study, several physical and chemical properties of purified S and F polypeptides were investigated. Three methods revealed important differences between them. It was found by cyanogen bromide degradation that S contained one fragment (Mr approximately equal to 20 000) more than F. S eluted ahead of F by column chromatography in SDS. The sedimentation coefficient of S was found to be greater than that of F. However, the two subunits were shown to have similar amino acid and carbohydrate compositions and to be indistinguishable in their interaction with SDS. The different migration rates of S and F can therefore be explained by a small difference in size which agrees with the finding (Endocrinology 108, 1285-1292 (1981)) that a proteolytic enzyme present in commercial horseradish peroxidase can convert S into F.

Reversibility of coated vesicle dissociation.

The dissociation of the coated vesicles to clathrin and uncoated vesicles and their reassociation have been studied under various conditions. The extent of reassociation is pH dependent and increases slightly with increasing concentrations of the components. Unlike the self-association of clathrin which is strongly salt dependent, the reassociation of clathrin and uncoated vesicles is practically independent of salt concentration. The coated vesicle gradually loses its coat with increasing pH, and the dissociation process is not an all or none reaction. Ca2+ inhibits dissociation of the coated vesicles and enhances the reassociation of clathrin and uncoated vesicles. Our results show that, although many conditions result in reassociation of protein and lipid vesicle, few conditions result in vesicles of both the same size and composition as native coated vesicles.

Properties of clathrin coat structures.

Clathrin polymerizes to form characteristic coat structures (baskets) closely resembling those found on coated vesicles. Two sizes of baskets are formed from clathrin, depending on the purity of the preparation and on other factors. A protein of Mr 110 000 has been separated from clathrin by lysine-Sepharose chromatography which is needed for the formation of 150S baskets. In its absence, polymerization results in the larger size baskets, i.e., 300S. Addition of Ca2+ or Mg2+ stimulates 300S formation in the presence of the 110 000 protein.

Instability of coated vesicles in concentrated sucrose solutions.

A method of preparing homogeneous coated vesicles that eliminates the high sucrose concentrations heretofore used is presented. It is shown that sucrose at high concentrations dissociates the coat from coated vesicles. This reaction can explain the presence of empty coats observed with preparations obtained with high concentrations of sucrose. The protein and membrane lipid components have been analyzed by the intrinsic tryptophan and extrinsic diphenylhexatriene fluorescence, respectively. Analysis of mixtures of coated vesicles and baskets resolved the contributions of the two species to the fluorescence curves.

The isoelectric focusing of human thyroglobulin.

The isoelectric focusing of human thyroglobulin has been studied on slab gels. Three bands, focusing between pH 4.4 and 4.7, are observed. Deglycosylation of thyroglobulin does not affect the distribution of focused bands, but increases the pH range of focusing slightly. Native thyroglobulin and its half-sized subunit show the same distribution of isoelectric bands. Refocusing of one band results in the appearance of the three original bands. It appears that soluble complexes of thyroglobulin with ampholyte account for the apparent heterogeneity observed on isoelectric focusing.

Effects of several antimalarials and phenothiazine compounds on the formation of coat structure from clathrin.

We have evaluated the effects of two phenothiazine and several antimalarial drugs on the rates of polymerization of 8S clathrin molecules to 300S coat structures. Most of the drugs investigated have been shown in other studies to inhibit receptor-mediated endocytosis through the coated pit regions of plasma membranes. The two types of drugs were found to accelerate the polymerization rate without having much effect on the size distribution of the polymer species. The activities of the drugs appear to depend on the dibasic moiety and a large, hydrophobic aromatic ring in their structures.

Effects of thyroxine binding on the stability, conformation, and fluorescence properties of thyroxine-binding globulin.

The effects of thyroxine (T4) on several molecular properties of human thyroxine-binding globulin (TBG) have been evaluated. Changes in the sedimentation constant and relaxation time indicate that TBG becomes more symmetric and compact when T4 is bound. This modification in structure is associated with an increase in the stability of TBG to denaturation by either acid or guanidinium chloride. T4 binding also produces changes in the emission and excitation spectra of TBG, reflecting different environments of the four tryptophanyl residues. T4 preferentially quenches residues in a less polar environment. In addition, it alters the effect of the collisional quencher, acrylamide, so as to indicate a shift in the environment of some of the exposed tryptophanyl residues.