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.     

Electrostatic and hydrophobic interactions governing the interaction and binding of beta-lactoglobulin to membranes

The role of electrostatic and hydrophobic interactions in the binding and penetration of beta-lactoglobulin (betaLG) to preformed lipid membranes was studied using various phospholipid micelles and vesicles. Zwitterionic lysophospholipid micelles are able to induce the beta-sheet to alpha-helix transition, as judged by circular dichroism (CD), but the degree of transition is dramatically below and the amount of lipid required above that for anionic phospholipids with equivalent hydrocarbon chains. Anionic phospholipids with short hydrocarbon chains induce only low alpha-helical content in betaLG as compared to phospholipids with the same head group but longer hydrocarbon chains. These results suggest that both electrostatic and hydrophobic interactions are indispensable in betaLG-lipid interaction. Furthermore, air-water interface monolayer surface pressure and fluorescence anisotropy studies reveal that the membrane insertion of betaLG strongly depends on the nature of phospholipids, given the identical headgroup, particularly lipid packing. These results are supported by urea denaturation and acrylamide fluorescence quenching tests and by the FTIR-ATR polarization results for betaLG in multilayers on a surface. Under the same experimental conditions, the membrane binding and insertion of betaLG as well as the stability of the betaLG-lipid complexes can be enhanced by lowering the pH. Collectively, electrostatic interactions play a crucial role in all the processes involved in the betaLG-lipid interaction, while the presence of hydrophobic interaction remains necessary. Finally, betaLG biological function in the transport of fatty acids was tested by demonstrating the release of 2-AS from a 2-AS-betaLG complex on binding to lipids.     

Differential scanning calorimetric study of the effect of the antimicrobial peptide gramicidin S on the thermotropic phase behavior of phosphatidylcholine, phosphatidylethanolamine and phosphatidylglycerol lipid bilayer membranes

We have studied the effects of the antimicrobial peptide gramicidin S (GS) on the thermotropic phase behavior of large multilamellar vesicles of dimyristoylphosphatidylcholine (DMPC), dimyristoylphosphatidylethanolamine (DMPE) and dimyristoyl phosphatidylglycerol (DMPG) by high-sensitivity differential scanning calorimetry. We find that the effect of GS on the lamellar gel to liquid-crystalline phase transition of these phospholipids varies markedly with the structure and charge of their polar headgroups. Specifically, the presence of even large quantities of GS has essentially no effect on the main phase transition of zwitterionic DMPE vesicles, even after repeating cycling through the phase transition, unless these vesicles are exposed to high temperatures, after which a small reduction in the temperature, enthalpy and cooperativity of the gel to liquid-crystalline phase transitions is observed. Similarly, even large amounts of GS produce similar modest decreases in the temperature, enthalpy and cooperativity of the main phase transition of DMPC vesicles, although the pretransition is abolished at low peptide concentrations. However, exposure to high temperatures is not required for these effects of GS on DMPC bilayers to be manifested. In contrast, GS has a much greater effect on the thermotropic phase behavior of anionic DMPG vesicles, substantially reducing the temperature, enthalpy and cooperativity of the main phase transition at higher peptide concentrations, and abolishing the pretransition at lower peptide concentrations as compared to DMPC. Moreover, the relatively larger effects of GS on the thermotropic phase behavior of DMPG vesicles are also manifest without cycling through the phase transition or exposure to high temperatures. Furthermore, the addition of GS to DMPG vesicles protects the phospholipid molecules from the chemical hydrolysis induced by their repeated exposure to high temperatures. These results indicate that GS interacts more strongly with anionic than with zwitterionic phospholipid bilayers, probably because of the more favorable net attractive electrostatic interactions between the positively charged peptide and the negatively charged polar headgroup in such systems. Moreover, at comparable reduced temperatures, GS appears to interact more strongly with zwitterionic DMPC than with zwitterionic DMPE bilayers, probably because of the more fluid character of the former system. In addition, the general effects of GS on the thermotropic phase behavior of zwitterionic and anionic phospholipids suggest that it is located at the polar/apolar interface of liquid-crystalline bilayers, where it interacts primarily with the polar headgroup and glycerol-backbone regions of the phospholipid molecules and only secondarily with the lipid hydrocarbon chains. Finally, the considerable lipid specificity of GS interactions with phospholipid bilayers may prove useful in the design of peptide analogs with stronger interactions with microbial as opposed to eucaryotic membrane lipids.     

Characterization of thermotropic structural transitions of the erythrocyte membrane: a biochemical and electron-paramagnetic resonance approach

The relationship between membrane structural properties and functions has been generally inferred from observed thermotropic phenomena. By the use of 16-dinyloxyl stearic acid spin probe we investigated the red blood cell membrane components involved in three characteristic thermotropic structural transitions occurring at 8, 20, and 40 degrees C. The transition at 8 degrees C is removed by chymotrypsin treatment at the cytoplasmic membrane layer. The 20 degrees C phase transition is unmodified after chymotrypsin treatment and occurs at 15 degrees C after complete proteolysis of intramembrane chymotrypsin-insensitive peptides. Liposomes from the total lipid extract of RBC show only one thermotropic transition at 15 degrees C. The 40 degrees C phase transition is absent in vesicles free of skeletal proteins, in vesicles obtained after RBC storage, and in low-ionic-strength resealed ghosts. Transitions at 8 degrees C and 40 degrees C appear to be due to the interactions of cytoplasmic exposed proteins with membrane, whereas the 20 degrees C transition is intrinsic to the lipid component.     

Clathrin-mediated endocytosis of the epithelial sodium channel. Role of epsin

Here we present evidence that the epithelial sodium channel (ENaC), a heteromeric membrane protein whose surface expression is regulated by ubiquitination, is present in clathrin-coated vesicles in epithelial cells that natively express ENaC. The channel subunits are ubiquitinated and co-immunoprecipitate with both epsin and clathrin adaptor proteins, and epsin, as expected, co-immunoprecipitates with clathrin adaptor proteins. The functional significance of these interactions was evaluated in a Xenopus oocyte expression system where co-expression of epsin and ENaC resulted in a down-regulation of ENaC activity; conversely, co-expression of epsin sub-domains acted as dominant-negative effectors and stimulated ENaC activity. These results identify epsin as an accessory protein linking ENaC to the clathrin-based endocytic machinery thereby regulating the activity of this ion channel at the cell surface.     

Studies on Freezing Injury of Plant Cells: I. Relation between Thermotropic Properties of Isolated Plasma Membrane Vesicles and Freezing Injury

The thermotropic transition of plasma membrane of Dactylis glomerata was studied by using fluorescence polarization of embedded fluorophore, 1,6-diphenyl-1,3,5-hexatriene. Under the presence of 35% ethylene glycol, reversible thermotropic transitions were observed in isolated plasma membrane vesicles in nearly the same temperature range as the temperature of freezing injury to cells. In liposomes prepared from isolated plasma membranes, however, the thermotropic transitions occurred at much lower temperatures in comparison with those of intact membrane vesicles. Following treatment with pronase, the thermotropic transition also shifted downward.

Thus, the thermotropic properties of plasma membranes appeared to be dependent on the membrane proteins. In vitro freezing of the isolated plasma membrane vesicles without addition of any cryoprotectant, such as sorbitol, resulted in an irreversible alteration both in the fluorescence anisotropy values and the temperatures for the thermotropic transition, suggesting an irreversible alteration in the membrane structure, presumably changes in lipid-protein interactions and protein conformation.

     

Incorporation of spin-labeled fatty acids into bovine brain clathrin coated vesicles

Stearic acids with a nitroxide radical at selected positions have been incorporated in the phospholipid bilayers of clathrin coated vesicles, uncoated vesicles and sonicated liposomes made from the lipids extracted from the uncoated vesicles. The extent of incorporation was found minimum for stearic acids labeled on C-12 and for bilayers of uncoated vesicles. The ESR spectra of the spin-labeled fatty acids incorporated in the bilayers showed a pronounced temperature dependence (without discontinuity) and a decrease in the hyperfine splitting as the nitroxide group was inserted deeper in the hydrophobic core of the membranes. An abrupt phospholipid phase transition or a phase separation could be excluded. The presence of the external proteins (the clathrin coat) on the membranes was not found to noticeably influence the gradient of flexibility of the fatty acid chains of the phospholipids. The influence of the internal proteins embedded in the bilayers was evidenced by a detailed analysis of the ESR spectra of (7,8)SA in terms of two components: one component arising from the labels surrounded exclusively by phospholipids, the other component arising from labels of reduced mobility perturbed by the vicinity of the proteins. These results support the persistence of lipidic domains in the endocytic vesicles despite the accumulation of receptors which follows their formation.     

Location of the 100 kd-50 kd accessory proteins in clathrin coats.

We present a three-dimensional map of the clathrin coat of coated vesicles, generated from tilt series of electron micrographs of unstained specimens embedded in vitreous ice. We have examined native placental coated vesicles and coats reassembled from their purified constituents, namely clathrin triskelions and accessory proteins of approximate mol. wts 100 kd and 50 kd. Our results show that the accessory proteins contribute a further shell of density within the double shell of the clathrin cage, extending from the terminal domains of the clathrin to the membrane of the vesicle. The thickness of the complete coat is approximately 22 nm.     

Clathrin- and AP-2-binding sites in HIP1 uncover a general assembly role for endocytic accessory proteins

Clathrin-mediated endocytosis is a major pathway for the internalization of macromolecules into the cytoplasm of eukaryotic cells. The principle coat components, clathrin and the AP-2 adaptor complex, assemble a polyhedral lattice at plasma membrane bud sites with the aid of several endocytic accessory proteins. Here, we show that huntingtin-interacting protein 1 (HIP1), a binding partner of huntingtin, copurifies with brain clathrin-coated vesicles and associates directly with both AP-2 and clathrin. The discrete interaction sequences within HIP1 that facilitate binding are analogous to motifs present in other accessory proteins, including AP180, amphiphysin, and epsin. Bound to a phosphoinositide-containing membrane surface via an epsin N-terminal homology (ENTH) domain, HIP1 associates with AP-2 to provide coincident clathrin-binding sites that together efficiently recruit clathrin to the bilayer. Our data implicate HIP1 in endocytosis, and the similar modular architecture and function of HIP1, epsin, and AP180 suggest a common role in lipid-regulated clathrin lattice biogenesis.     

Dynamic behavior of clathrin in Arabidopsis thaliana unveiled by live imaging

Clathrin-coated vesicles (CCV) are necessary for selective transport events, including receptor-mediated endocytosis on the plasma membrane and cargo molecule sorting in the trans-Golgi network (TGN). Components involved in CCV formation include clathrin heavy and light chains and several adaptor proteins that are conserved among plants. Clathrin-dependent endocytosis has been shown to play an integral part in plant endocytosis. However, little information is known about clathrin dynamics in living plant cells. In this study, we have visualized clathrin in Arabidopsis thaliana by tagging clathrin light chain with green fluorescent protein (CLC-GFP). Quantitative evaluations of colocalization demonstrate that the majority of CLC-GFP is localized to the TGN, and a minor population is associated with multivesicular endosomes and the Golgi trans-cisternae. Live imaging further demonstrated the presence of highly dynamic clathrin-positive tubules and vesicles, which appeared to mediate interactions between the TGNs. CLC-GFP is also targeted to cell plates and the plasma membrane. Although CLC-GFP colocalizes with a dynamin isoform at the plasma membrane, these proteins exhibit distinct distributions at newly forming cell plates. This finding indicates independent functions of CLC (clathrin light chains) and dynamin during the formation of cell plates. We have also found that brefeldin A and wortmannin treatment causes distinctly different alterations in the dynamics and distribution of clathrin-coated domains at the plasma membrane. This could account for the different effects of these drugs on plant endocytosis.     

Studies of protein-phospholipid interaction in isolated mitochondrial ubiquinone-cytochrome c reductase

The interaction between phospholipids, ubiquinone and highly purified ubiquinol-cytochrome c reductase was studied using differential scanning calorimetry. The enzyme complex and its delipidated forms undergo thermodenaturation at 337.3 and 322.7 K, respectively. The reduced reductase is more stable toward thermodenaturation than is the oxidized enzyme. While phospholipids restored enzymatic activity to the delipidated enzyme complex and stabilized the enzyme toward thermodenaturation, ubiquinone showed little effect on the thermostability of ubiquinol-cytochrome c reductase. The effect of phospholipids on the thermotropic properties of ubiquinol-cytochrome c reductase is dependent upon the molecular properties of the phospholipid. When ubiquinol-cytochrome c reductase was embedded in closed asolectin vesicles, an exothermic transition peak was observed upon thermodenaturation. When the asolectin concentration in the reconstituted preparation was less than 0.3 mg/mg protein, an amorphous structure was observed in the electron micrograph and the preparation showed an endothermic transition upon thermodenaturation. The thermotropic properties of the enzyme-phospholipid vesicles were affected by the phospholipid head groups as well as the fatty-acyl chains, with those phospholipids having the most highly unsaturated fatty-acyl chains having the greatest effect. The energy for the exothermic transition may be derived from the collapse, upon thermodenaturation, of a strained interaction between the unsaturated fatty-acyl groups of phospholipids and protein molecules resulting from vesicle formation. The exothermic transition of the enzyme-phospholipid vesicle was abolished when cholesterol was included in the vesicles and when reductase was treated with a proteolytic enzyme prior to incorporation into the phospholipid vesicles.     

Depolarized light-scattering studies of bilayer structures in phospholipid vesicles

Depolarized light scattering has been used to investigate the hydrocarbon chain packing of phospholipids in vesicles below the phase transition and ordering of their chains above the phase transition. The chain packing and ordering have been demonstrated for vesicles of l-α-dipalmitoylphosphatidylethanolamine and some phosphatidylcholines of different hydrocarbon chain lenghts. Anisotropy ratios for phospholipid vesicles could be determined by measuring depolarization ratios for several vesicle sizes at low concentrations of the lipids. The following results were obtained. Hydrocarbon chains of l-α-dimyristoyl and distearoylphosphatidylcholines below their phase transitions pack at tilting angles in good agreement with X-ray diffraction data. On the other hand, hydrocarbon chains of dipalmitoylphosphatidylethanolamine pack perpendicular to the bilayer surface. Values of the averaged order parameter for dimyristoyl, dipalmitoyl and distearoylphosphatidylcholines at 2.5°C above their phase transition are all the same and the value for dipalmitoylphosphatidylcholine is in agreement with results from ²H-NMR experiments. The value of the order parameter for dipalmitoylphosphatidylethanolamine is slightly larger than that for dipalmitoylphosphatidylcholine.     

Membrane binding properties of blood coagulation Factor V and derived peptides

The interactions of factor V and factor Va light chain with phospholipid vesicles were compared. The results showed that the factor Va light chain bound with the same parameters as factor V when the proteins were present at similar densities on the membrane. The protein-vesicle collisional efficiency was 30-50% for both factor V and factor Va light chain. The factor Va light chain bound at a higher density, and the additional binding interactions had lower affinity. The dissociation process showed negative cooperativity, possibly due to competition for acidic phospholipids in the membrane. The higher molar packing density produced more rapid protein-membrane dissociation rate constants. However, when factor V and Va light chains were present at similar molar densities on the vesicle, the dissociation rates, estimated by two methods, were similar. Analysis of dissociation rates also showed that factor Va interacted with factor Xa on the membrane surface while factor Va light chain did not. Factor Va generated by thrombin digestion of factor V did not result in a major loss of membrane-bound protein mass unless ethylenenediaminetetraacetic acid was present; in the latter case the mass changes indicated that all peptides were removed from the membrane except factor Va light chain. Equilibrium and dynamic measurements showed that ionic strength had a major effect on the dissociation rate but not on the association process. The salt effect indicated interaction between oppositely charged species with the product of the number of charges equal to at least -5.5. Factor Va light chain appeared to interact with phospholipids via a general charge interaction rather than via a specific charge stoichiometry.(ABSTRACT TRUNCATED AT 250 WORDS)     

Close association between clathrin and the hydrophobic domain of boundary membranes in brain endocytic vesicles

Purified bovine brain clathrin binds readily, in a pH-dependent fashion, to protein-free phospholipid bilayers. The association is tight and leads to inter-bilayer fusion, however, photolabeling studies using the amphiphilic photoreactive glycolipid 12-(4-azido-2-nitrophenoxy)stearoyl[1-14C]glucosamine provide no evidence for direct insertion of clathrin into the central, hydrophobic domain of of these target membranes. In contrast, similar photolabeling studies of isolated, intact clathrin-coated vesicles show that, in these structures, clathrin is readily accessible to a probe which is known to reside preferentially within the hydrophobic domain of the membrane. The results are consistent with a natural requirement, by clathrin, for accessory proteins in order to effect membrane penetration.     

Visualization of the binding of Hsc70 ATPase to clathrin baskets: implications for an uncoating mechanism

Clathrin assembly into coated pits and vesicles is promoted by accessory proteins such as auxilin and AP180, and disassembly is effected by the Hsc70 ATPase. These interactions may be mimicked in vitro by the assembly and disassembly of clathrin "baskets." The chimera C58J is a minimal construct capable of supporting both reactions; it consists of the C58 moiety of AP180, which facilitates clathrin assembly, fused with the J domain of auxilin, which recruits Hsc70 to baskets. We studied the process of disassembly by using cryo-electron microscopy to identify the initial binding site of Hsc70 on clathrin-C58J baskets at pH 6, under which conditions disassembly does not proceed further. Hsc70 interactions involve two sites: (i) its major interaction is with the sides of spars of the clathrin lattice, close to the triskelion hubs and (ii) there is another interaction at a site at the N-terminal hooks of the clathrin heavy chains, presumably via the J domain of C58J. We propose that individual triskelions may be extricated from the clathrin lattice by the concerted action of up to six Hsc70 molecules, which intercalate between clathrin leg segments, prying them apart. Three Hsc70s remain bound to the dissociated triskelion, close to its trimerization hub.     

Calorimetric, x-ray diffraction, and spectroscopic studies of the thermotropic phase behavior and organization of tetramyristoyl cardiolipin membranes

The thermotropic phase behavior and organization of aqueous dispersions of the quadruple-chained, anionic phospholipid tetramyristoyl diphosphatidylglycerol or tetramyristoyl cardiolipin (TMCL) was studied by differential scanning calorimetry, x-ray diffraction, (31)P NMR, and Fourier-transform infrared (FTIR) spectroscopy. At physiological pH and ionic strength, our calorimetric studies indicate that fully equilibrated aqueous dispersions of TMCL exhibit two thermotropic phase transitions upon heating. The lower temperature transition is much less cooperative but of relatively high enthalpy and exhibits marked cooling hysteresis, whereas the higher temperature transition is much more cooperative and also exhibits a relatively high enthalpy but with no appreciable cooling hysteresis. Also, the properties of these two-phase transitions are sensitive to the ionic strength of the dispersing buffer. Our spectroscopic and x-ray diffraction data indicate that the lower temperature transition corresponds to a lamellar subgel (L(c)') to gel (L(beta)) phase transition and the higher temperature endotherm to a L(beta) to lamellar liquid-crystalline (L(alpha)) phase transition. At the L(c)'/L(beta) phase transition, there is a fivefold increase of the thickness of the interlamellar aqueous space from approximately 11 A to approximately 50 A, and this value decreases slightly at the L(beta)/L(alpha) phase transition. The bilayer thickness (i.e., the mean phosphate-phosphate distance across the bilayer) increases from 42.8 A to 43.5 A at the L(c)'/L(beta) phase transition, consistent with the loss of the hydrocarbon chain tilt of approximately 12 degrees , and decreases to 37.8 A at the L(beta)/L(alpha) phase transition. The calculated cross-sectional areas of the TMCL molecules are approximately 79 A(2) and approximately 83 A(2) in the L(c)' and L(beta) phases, respectively, and we estimate a value of approximately 100 A(2) in the L(alpha) phase. The combination of x-ray and FTIR spectroscopic data indicate that in the L(c)' phase, TMCL molecules possess tilted all-trans hydrocarbon chains packed into an orthorhombic subcell in which the zig-zag planes of the chains are parallel, while in the L(beta) phase the untilted, all-trans hydrocarbon chains possess rotational mobility and are packed into a hexagonal subcell, as are the conformationally disordered hydrocarbon chains in the L(alpha) phase. Our FTIR spectroscopic results demonstrate that the four carbonyl groups of the TMCL molecule become progressively more hydrated as one proceeds from the L(c)' to the L(beta) and then to the L(alpha) phase, while the two phosphate moieties of the polar headgroup are comparably well hydrated in all three phases. Our (31)P-NMR results indicate that although the polar headgroup retains some mobility in the L(c)' phase, its motion is much more restricted in the L(beta) and especially in the L(alpha) phase than that of other phospholipids. We can explain most of our experimental results on the basis of the relatively small size of the polar headgroup of TMCL relative to other phospholipids and the covalent attachment of the two phosphate moieties to a single glycerol moiety, which results in a partially immobilized polar headgroup that is more exposed to the solvent than in other glycerophospholipids. Finally, we discuss the biological relevance of the unique properties of TMCL to the structure and function of cardiolipin-containing biological membranes.     

Novel binding sites on clathrin and adaptors regulate distinct aspects of coat assembly

Clathrin-coated vesicles (CCVs) sort proteins at the plasma membrane, endosomes and trans Golgi network for multiple membrane traffic pathways. Clathrin recruitment to membranes and its self-assembly into a polyhedral coat depends on adaptor molecules, which interact with membrane-associated vesicle cargo. To determine how adaptors induce clathrin recruitment and assembly, we mapped novel interaction sites between these coat components. A site in the ankle domain of the clathrin triskelion leg was identified that binds a common site on the appendages of tetrameric [AP1 and AP2] and monomeric (GGA1) adaptors. Mutagenesis and modeling studies suggested that the clathrin-GGA1 appendage interface is nonlinear, unlike other peptide-appendage interactions, but overlaps with a sandwich domain binding site for accessory protein peptides, allowing for competitive regulation of coated vesicle formation. A novel clathrin box in the GGA1 hinge region was also identified and shown to mediate membrane recruitment of clathrin, while disruption of the clathrin-GGA1 appendage interaction did not affect recruitment. Thus, the distinct sites for clathrin-adaptor interactions perform distinct functions, revealing new aspects to regulation of CCV formation.     

Structural insights into the clathrin coat

Clathrin is a cytoplasmic protein best known for its role in endocytosis and intracellular trafficking. The diverse nature of clathrin has recently become apparent, with strong evidence available suggesting roles in both chromosome segregation and reassembly of the Golgi apparatus during mitosis. Clathrin functions as a heterohexamer, adopting a three-legged triskelion structure of three clathrin light chains and three heavy chains. During endocytosis clathrin forms a supportive network about the invaginating membrane, interacting with itself and numerous adapter proteins. Advances in the field of structural biology have led us to a greater understanding of clathrin in its assembled state, the clathrin lattice. Combining techniques such as X-ray crystallography, NMR, and cryo-electron microscopy has allowed us to piece together the intricate nature of clathrin-coated vesicles and the interactions of clathrin with its many binding partners. In this review I outline the roles of clathrin within the cell and the recent structural advances that have improved our understanding of clathrin-clathrin and clathrin-protein interactions.     

Adaptor proteins in protein trafficking between endomembrane compartments in plants

Clathrin is a highly conserved coat protein that plays a critical role in lipid vesicle-mediated trafficking at multiple routes in various post-Golgi compartments. It consists of large and small subunits, and exists in the cytosol as triskelions composed of three pairs of small and large subunits. For vesicle formation, the triskelions are recruited to the membrane of specific compartments where they undergo self-polymerization to produce coats for lipid vesicles. However, clathrin has no ability to bind directly to lipid membranes. Therefore, accessory proteins are necessary for its recruitment to the donor compartment where vesicles are formed. A large number of accessory proteins, called adaptor proteins, have been identified and characterized extensively at the molecular and cellular levels in animal cells and yeast. Recently, the roles of many adaptor proteins have been elucidated in plant cells. As expected from the conserved nature of lipidmediated trafficking in eukaryotic cells, these plant adaptor proteins for clathrin show a high degree of functional conservation with those found in animal cells and yeast. At the same time, they are also involved in plant-specific processes such as the transition from the PSV to the lytic vacuole and cell-plate formation. Here, we summarize recent advances in the physiological roles of adaptor proteins in plant cells.     

Human erythrocyte clathrin and clathrin-uncoating protein

Clathrin, a Mr = 72,000 clathrin-associated protein, and myosin were purified in milligram quantities from the same erythrocyte hemolysate fraction. Erythrocyte clathrin closely resembled brain clathrin in several respects: (a) both are triskelions as visualized by electron microscopy with arms 40 nm in length with globular ends and a flexible hinge region in the middle of each arm, and these triskelions assemble into polyhedral "cages" at appropriate pH and ionic strength; (b) both molecules contain heavy chains of Mr = 170,000 that are indistinguishable by two-dimensional maps of 125I-labeled peptides; and (c) both molecules contain light chains of Mr approximately 40,000 in a 1:1 molar ratio with the heavy chain. Erythrocyte clathrin is not identical to brain clathrin since antibody raised against the erythrocyte protein reacts better with erythrocyte clathrin than with brain clathrin and since brain clathrin contains two light chains resolved on sodium dodecyl sulfate gels while the light chain of erythrocyte clathrin migrates as a single band. The erythrocyte Mr = 72,000 clathrin-associated protein is closely related to a protein in brain that mediates ATP-dependent disassembly of clathrin from coated vesicles and binds tightly to clathrin triskelions (Schlossman, D. M., Schmid, S. L., Braell, W. A., and Rothman, J. E. (1984) J. Cell Biol. 99, 723-733). The erythrocyte and brain proteins have identical Mr on sodium dodecyl sulfate gels and identical maps of 125I-labeled peptides, share antigenic sites, and bind tightly to ATP immobilized on agarose. Clathrin and the uncoating protein are not restricted to reticulocytes since equivalent amounts of these proteins are present in whole erythrocyte populations and reticulocyte-depleted erythrocytes. Clathrin is present at 6,000 triskelions/cells, while the uncoating protein is in substantial excess at 250,000 copies/cell.