Dissociation of clathrin coats coupled to the hydrolysis of ATP: role of an uncoating ATPase

ATP hydrolysis was used to power the enzymatic release of clathrin from coated vesicles. The 70,000-mol-wt protein, purified on the basis of its ATP-dependent ability to disassemble clathrin cages, was found to possess a clathrin-dependent ATPase activity. Hydrolysis was specific for ATP; neither dATP nor other ribonucleotide triphosphates would either substitute for ATP or inhibit the hydrolysis of ATP in the presence of clathrin cages. The ATPase activity is elicited by clathrin in the form of assembled cages, but not by clathrin trimers, the product of cage disassembly. The 70,000-mol-wt polypeptide, but not clathrin, was labeled by ATP in photochemical cross-linking, indicating that the hydrolytic site for ATP resides on the uncoating protein. Conditions of low pH or high magnesium concentration uncouple ATP hydrolysis from clathrin release, as ATP is hydrolyzed although essentially no clathrin is released. This suggests that the recognition event triggering clathrin-dependent ATP hydrolysis occurs in the absence of clathrin release, and presumably precedes such release.     

Crystals of an ATPase fragment of bovine clathrin uncoating ATPase

A 44,000 Mr amino-terminal, clathrin-independent ATPase fragment of the bovine clathrin uncoating ATPase has been crystallized in a form suitable for X-ray diffraction studies. The crystals are orthorhombic, space group P2(1)2(1)2(1), a = 145.3 A, b = 65.0 A, c = 46.9 A, with one protein molecule per asymmetric unit (1 A = 0.1 nm).     

The ATPase core of a clathrin uncoating protein

Chymotryptic digestion of bovine brain uncoating ATPase produced a 60-kDa fragment that was subsequently proteolyzed to 44 kDa. Loss of clathrin cage uncoating activity paralleled the conversion of the intact 70-kDa enzyme to the 60-kDa fragment, while clathrin binding activity was lost as the 60-kDa fragment was degraded to 44 kDa. This 44-kDa fragment has been purified to homogeneity and characterized as a clathrin-independent ATPase. The 44-kDa ATPase domain has been localized within the intact enzyme by the use of amino-terminal specific antibodies. This localization relates to the conserved nature of the 70-kDa heat shock protein family, of which bovine brain uncoating ATPase is a constitutively expressed member.     

Dissociation of clathrin from coated vesicles by the uncoating ATPase

The uncoating ATPase has been shown to dissociate clathrin from both clathrin-coated vesicles and synthetic clathrin baskets (Rothman, J. E., and Schmid, S. L. (1986) Cell 46, 5-9). In the present study, we investigated the mechanism of action of the uncoating ATPase using intact coated vesicles isolated from bovine brain. We observed an initial burst of uncoating followed by much slower steady-state uncoating. The initial burst of uncoating was essentially stoichiometric with each molecule of uncoating ATPase apparently binding to one leg of the clathrin triskelion. When the enzyme was preincubated with equimolar ADP, Pi, and ATP, rather than just ATP alone, both the initial burst and the slow steady-state uncoating were markedly inhibited, suggesting that the combination of ADP and Pi is a strong competitive inhibitor of ATP binding. However, kinetic studies suggested that ADP and Pi dissociates from the enzyme relatively rapidly unless clathrin is also bound to the enzyme. These results suggest that, after the uncoating ATPase rapidly removes a stoichiometric amount of clathrin while ATP is hydrolyzed at the active site, slow release of ADP and Pi from the resulting enzyme.clathrin.ADP.Pi complex limits the rate at which further uncoating occurs.     

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.     

Trimeric binding of the 70-kD uncoating ATPase to the vertices of clathrin triskelia: a candidate intermediate in the vesicle uncoating reaction

Clathrin-coated vesicles were uncoated with the 70-kD "uncoating ATPase" from bovine brain, and the molecular products were visualized by freeze-etch electron microscopy. This yielded images of released clathrin triskelia with up to three 70-kD uncoating ATPase molecules bound to their vertices. Likewise, incubation of soluble clathrin triskelia with purified uncoating ATPase also led to trimeric binding of the ATPase to the vertices of clathrin triskelia. However, this occurred only when either EDTA or nonhydrolyzable analogues of ATP were present, in which case the ATPase also appeared to self-associate. When ATP was present instead, no 70-kD ATPases could be found on clathrin triskelia and all ATPases remained monomeric. These observations support the notion that ATP controls an allosteric conversion of the 70- kD uncoating ATPase between two different molecular conformations, an ATP-charged state in which the molecule has relatively low affinity for itself as well as low affinity for clathrin, and an ATP-discharged state in which both of these affinities are high. We presume that in vivo, the latter condition is brought about by ATP hydrolysis and product release, at which point the ATPase will bind tightly to clathrin and/or self-associate. We further propose that these reactions, when occurring in concert within a clathrin lattice, will tend to destabilize it by a mechanism we call "protein polymer competition". We stress the analogies between such a mechanism of uncoating and the ATP-driven events in muscle contraction. Finally, we show that under experimental conditions in which the uncoating ATPase fully removes the coats from brain coated vesicles, identical aliquots of the enzyme do not affect plasmalemmal coated pits in situ. This remarkable selectivity, the mechanism of which remains a complete mystery, is at least consistent with the idea that the 70-kD ATPase indeed plays a role in uncoating coated vesicles after they have formed in vivo.     

Assembly and packing of clathrin into coats

We present a model for the packing of clathrin molecules into the characteristic hexagons and pentagons covering coated pits and vesicles. The assembly unit is a symmetrical trimer with three extended legs. Polymerization of these units occurs in seconds under suitable conditions, giving empty polyhedral cages resembling the structures around coated vesicles. Images of small, negatively stained fragments of cages, assembled directly on electron microscope grids, reveal details of the structure, which correlate well with the predicted features of the model. There is one clathrin trimer at each polyhedral vertex, and each leg of the trimer extends along two neighboring polyhedral edges. Quasi-equivalent packing in pentagons and hexagons in polyhedra of different sizes requires a variable joint at the vertex of the molecule and a hinge in each leg. The construction of clathrin coats is remarkable for the extended fibrous contacts that each molecule makes with many others. Such contacts may confer mechanical strength combined with flexibility needed when a vesicle is pinched off from the membrane.     

Beta-internexin is a microtubule-associated protein identical to the 70-kDa heat-shock cognate protein and the clathrin uncoating ATPase

We have examined the relationship of the ubiquitous 68-70-kDa cytoskeletal-associated protein beta-internexin (Napolitano, E. W., Pachter, J. S., Chin, S. S. M., and Liem, R. K. H. (1985) J. Cell Biol. 101, 1323-1331) to heat-shock cognate 70 (hsc70), the major constitutive member of the mammalian heat-shock protein 70 (hsp70) family of stress proteins. We purify beta-internexin from rat brain microtubules and confirm its identity with hsc70 and the clathrin-uncoating ATPase by the following criteria: 1) The partial sequence of a cyanogen bromide-derived peptide from beta-internexin matches the inferred amino acid sequence of the cDNA clone pRC62 encoding hsc70 from rat brain (O'Malley, K., Mauron, A., Barchas, J. D., and Kedes, L. (1985) Mol. Cell. Biol. 5, 3476-3483). 2) Mixing experiments followed by two-dimensional gel analyses reveal the precise co-migration of beta-internexin, the clathrin-uncoating ATPase, and the in vitro translation product of cDNA clone pHSP-4 encoding rat brain hsc70. 3) beta-Internexin is recognized by a monoclonal antibody reactive against the class of hsp70 proteins. 4) beta-Internexin purified from a microtubule-associated protein-enriched fraction of rat brain by virtue of high affinity binding to ATP-agarose possesses clathrin cage-specific ATPase activity.     

ATP catalyzes the sequestration of clathrin during enzymatic uncoating

ATP facilitates the sequestration of displaced triskelions by uncoating protein. In so doing, ATP is not hydrolyzed; nor does the concentration of ATP affect the equilibrium of this binding. However, the rates of both the binding of uncoating protein to clathrin and of their dissociation are greatly accelerated by ATP. These properties suggest that ATP acts catalytically to speed the capture of displaced triskelions by uncoating protein, as well as stoichiometrically in its hydrolysis to drive the displacement of triskelions from cages. The nucleotide specificity of this "catalytic" site for ATP on the uncoating protein is much less strict than that of the distinct "hydrolytic" site that drives the ATP-dependent displacement of triskelions from cages.     

Two classes of binding sites for uncoating protein in clathrin triskelions

Clathrin released from coated vesicles or empty cages by the ATP-dependent action of uncoating protein exists as a complex with the uncoating protein. Despite its apparent consumption during a round of uncoating, we have found that uncoating protein functions as an enzyme in that it rapidly and spontaneously recycles from its product (triskelions) to its substrate (cages). The binding of uncoating protein to clathrin triskelions is a complex equilibrium that involves the interaction of uncoating protein with at least two distinct sites on the clathrin molecule. Limited proteolysis dissected clathrin into two domains, each of which contained distinct binding sites. Binding to one of these sites, located on the proximal leg of a triskelion, was dependent upon the presence of light chains and was unstable to gel filtration. Binding to the second kind of site, located on the distal portion of a triskelion leg, was stable to gel filtration and was independent of the presence of light chains.     

The generation of curved clathrin coats from flat plaques

Flat clathrin lattices or 'plaques' are commonly believed to be the precursors to clathrin-coated buds and vesicles. The sequence of steps carrying the flat hexagonal lattice into a highly curved polyhedral cage with exactly 12 pentagons remains elusive, however, and the large numbers of disrupted interclathrin connections in previously proposed conversion pathways make these scenarios rather unlikely. The recent notion that clathrin can make controlled small conformational transitions opens new avenues. Simulations with a self-assembling clathrin model suggest that localized conformational changes in a plaque can create sufficiently strong stresses for a dome-like fragment to break apart. The released fragment, which is strongly curved but still hexagonal, may subsequently grow into a cage by recruiting free triskelia from the cytoplasm, thus building all 12 pentagonal faces without recourse to complex topological changes. The critical assembly concentration in a slightly acidic in vitro solution is used to estimate the binding energy of a cage at 25-40 k(B) T/clathrin.     

An outer membrane enzyme encoded by Salmonella typhimurium lpxR that removes the 3'-acyloxyacyl moiety of lipid A

The Salmonella and related bacteria modify the structure of the lipid A portion of their lipopolysaccharide in response to environmental stimuli. Some lipid A modifications are required for virulence and resistance to cationic antimicrobial peptides. We now demonstrate that membranes of Salmonella typhimurium contain a novel hydrolase that removes the 3'-acyloxyacyl residue of lipid A in the presence of 5 mM Ca2+. We have identified the gene encoding the S. typhimurium lipid A 3'-O-deacylase, designated lpxR, by screening an ordered S. typhimurium genomic DNA library, harbored in Escherichia coli K-12, for expression of Ca2+-dependent 3'-O-deacylase activity in membranes. LpxR is synthesized with an N-terminal type I signal peptide and is localized to the outer membrane. Mass spectrometry was used to confirm the position of lipid A deacylation in vitro and the release of the intact 3'-acyloxyacyl group. Heterologous expression of lpxR in the E. coli K-12 W3110, which lacks lpxR, resulted in production of significant amounts of 3'-O-deacylated lipid A in growing cultures. Orthologues of LpxR are present in the genomes of E. coli O157:H7, Yersinia enterocolitica, Helicobacter pylori, and Vibrio cholerae. The function of LpxR is unknown, but it could play a role in pathogenesis because it might modulate the cytokine response of an infected animal.     

Assembly polypeptides from coated vesicles mediate reassembly of unique clathrin coats

A protein activity has been identified in extracts of coated vesicles that enables purified clathrin triskelions to reassemble in vitro into coat structures of uniform size. Coats formed in the presence of this preparation, regardless of the buffer system employed, are uniform in size with a mean diameter of 78 nm (+/- 5 nm SD) and a sedimentation coefficient (S20,w) of approximately 250S. Analysis of the reassembled coats on dodecyl sulfate acrylamide gels reveals that they have specifically incorporated three polypeptides from the preparation: those of Mr congruent to 52,000, 100,000, and 110,000. The 52,000-, 100,000-, and 110,000-mol-wt polypeptides are incorporated in molar ratios of 0.85, 1.11, and 0.26, respectively, per three clathrin monomers (equivalent to one triskelion). We therefore designate these as assembly polypeptides (AP). In contrast, coats formed from clathrin alone, under permissive buffer conditions, are larger (400S), more heterogeneous in size (101 nm +/- 15 nm SD), and are composed only of clathrin and its associated light chains. These biochemical and biophysical characteristics distinguish AP-reassembled coats from coats formed by triskelions alone. AP-reassembled coats can be isolated, dissociated, then reassembled in the absence of any other factors. This recycling indicates that all the information needed for reassembly is present in the coat-incorporated polypeptides themselves. Reassembly is stoichiometric and saturable with respect to both clathrin and AP concentration. In the presence of AP, significant coat reassembly occurs at clathrin concentrations as low as 0.06 mg/ml. AP-mediated reassembly proceeds at 4 degrees, 22 degrees, and 37 degrees C. Coat formation also proceeds efficiently at intracellular pH values (7.2- 7.5) in the presence of AP. In its absence, reassembly does not occur at all above pH 6.7. In summary, AP promotes clathrin reassembly into coat structures of uniform size and distinctive composition under physiologically relevant salt, temperature, and pH conditions. In addition, the close similarity in size between AP-reassembled coats in vitro and coated membranes in the Golgi region in vivo raises the possibility that AP in the cell may be associated with this subpopulation of coat structures.     

Single-stranded-DNA-dependent ATPase from HeLa cells that stimulates DNA polymerase alpha-primase activity: purification and characterization of the ATPase

A single-stranded DNA-dependent ATPase that cofractionates during the early stages of purification of a multiprotein DNA polymerase alpha complex from HeLa cells has been purified to homogeneity. The ATPase is part of a 16S multienzyme DNA polymerase alpha complex that is fully active in SV40 DNA replication in vitro. The ATPase hydrolyzes ATP to ADP in a reaction that is completely dependent on the presence of DNA. DNA in single-stranded form is strongly preferred as a cofactor, and polydeoxynucleotides with adenine or thymidine residues are highly effective. Glycerol gradient sedimentation showed that the purified ATPase sedimented at an s20,w of 7 S, and polyacrylamide gel electrophoresis under denaturing conditions reveals two polypeptides with relative molecular weights of 83,000 and 68,000. Both of these polypeptides have purine nucleotide binding sites as revealed by photoaffinity cross-linking experiments. ATP binds to the two subunits more efficiently than GTP, and CTP or UTP does not cross-link with the two polypeptides. DNA synthesis catalyzed by purified HeLa cell DNA polymerase alpha-primase is stimulated in the presence of ATPase and ATP at an optimum concentration of 2 mM. Analysis of the DNA product by gel electrophoresis indicates that with poly(dT) but not phage M13 DNA as template the ATPase overcomes a lag and decreases the length of nascent DNA chains synthesized by the DNA polymerase alpha-primase complex.     

Functional organization of clathrin in coats: combining electron cryomicroscopy and X-ray crystallography.

The sorting of specific proteins into clathrin-coated pits and the mechanics of membrane invagination are determined by assembly of the clathrin lattice. Recent structures of a six-fold barrel clathrin coat at 21 A resolution by electron cryomicroscopy and of the clathrin terminal domain and linker at 2.6 A by X-ray crystallography together show how domains of clathrin interact and orient within the coat and reveal the strongly puckered shape and conformational variability of individual triskelions. The beta propeller of the terminal domain faces the membrane so that recognition segments from adaptor proteins can extend along its lateral grooves. Clathrin legs adapt to different coat environments in the barrel by flexing along a segment at the knee that is free of contacts with other molecules.     

Assembled and unassembled pools of clathrin: a quantitative study using an enzyme immunoassay

Using polyclonal antibodies raised against clathrin, we have developed an enzyme-linked immunoassay that can specifically measure the quantity of clathrin in crude cell extracts. We found that the quantity (weight percent of total protein) of clathrin was similar in cell types that exhibit large differences in their levels of endocytosis and exocytosis (lymphoid cells, 0.11%; liver cells, 0.07%, fibroblasts, 0.18%; myeloma cells, 0.16%). However, the quantity of clathrin was found to be significantly higher in brain cortex (0.75%). Cellular clathrin was separated by high-speed centrifugation into two fractions: an unassembled form present in high-speed supernatants and an assembled form (clathrin coats) present in the pellets. We show that the fraction of clathrin in the unassembled state varies considerably depending on the cell type studied (14% in brain cortex to 70% in lymphocytes). Our data support the view that the amount of clathrin (relative to total cell protein) in eucaryotic cells is not related to the extent of receptor-mediated endocytosis and intracellular membrane traffic. However, the fraction of assembled clathrin seems to be higher in endocytically and/or exocytically active cells.     

TrimerDimer: an oligonucleotide-based saturation mutagenesis approach that removes redundant and stop codons

9-fluorenylmethoxycarbonyl (Fmoc) and 4,4′-dimethoxytrityl (DMTr) are orthogonal hydroxyl protecting groups that have been used in conjunction to assemble oligonucleotide libraries whose variants contain wild-type and mutant codons randomly interspersed throughout a focused DNA region. Fmoc is labile to organic bases and stable to weak acids, whereas DMTr behaves oppositely. Based on these chemical characteristics, we have now devised TrimerDimer, a novel codon-based saturation mutagenesis approach that removes redundant and stop codons during the assembly of degenerate oligonucleotides. In this approach, five DMTr-protected trinucleotide phosphoramidites (dTGG, dATG, dTTT, dTAT and dTGC) and five Fmoc-protected dinucleotide phosphoramidites (dAA, dTT, dAT, dGC and dCG) react simultaneously with a starting oligonucleotide growing on a solid support. The Fmoc group is then removed and the incorporated dimers react with a mixture of three DMTr-protected monomer phosphoramidites (dC, dA and dG) to produce 15 trinucleotides: dCAA, dAAA, dGAA, dCTT, dATT, dGTT, dCAT, dAAT, dGAT, dCGC, dAGC, dGGC, dCCG, dACG and dGCG. After one mutagenic cycle, 20 codons are generated encoding the 20 natural amino acids. TrimerDimer was tested by randomizing the four contiguous codons that encode amino acids L64–G67 of an engineered, nonfluorescent GFP protein. Sequencing of 89 nonfluorescent mutant clones and isolation of two fluorescent mutants confirmed the principle.     

Assembly of the mannose-6-phosphate receptor into reconstituted clathrin coats.

In ionic conditions in which clathrin coats are stable, the mannose-6-phosphate receptor associates with the 100-kd/50-kd coat complexes purified from bullock brain coated vesicles. These aggregates exist as striking spherical structures of 300-1000 A diameter. When clathrin is included in the assembly mixture, cages are formed which apparently encapsulate these aggregates, giving, in the absence of lipid, structures resembling full coated vesicles.