Clathrin Baskets Research Articles

Molecular architecture of synaptic vesicles

Synaptic vesicles (SVs) store and transport neurotransmitters to the presynaptic active zone for release by exocytosis. After release, SV proteins and excess membrane are recycled via endocytosis, and new SVs can be formed in a clathrin-dependent manner. This process maintains complex molecular composition of SVs through multiple recycling rounds. Previous studies explored the molecular composition of SVs through proteomic analysis and fluorescent microscopy, proposing a model for an average SV (1). However, the structural heterogeneity and molecular architecture of individual SVs are not well described. Here, we used cryoelectron tomography to visualize molecular details of SVs isolated from mouse brains and inside cultured neurons. We describe several classes of small proteins on the SV surface and long proteinaceous densities inside SVs. We identified V-ATPases, determined a structure using subtomogram averaging, and showed them forming a complex with the membrane-embedded protein synaptophysin (Syp). Our bioluminescence assay revealed pairwise interactions between vesicle-associated membrane protein 2 and Syp and V-ATPase Voe1 domains. Interestingly, V-ATPases were randomly distributed on the surface of SVs irrespective of vesicle size. A subpopulation of isolated vesicles and vesicles inside neurons contained a partially assembled clathrin coat with an icosahedral symmetry. We observed V-ATPases under clathrin cages in several isolated clathrin-coated vesicles (CCVs). Additionally, from isolated SV preparations and within hippocampal neurons we identified clathrin baskets without vesicles. We determined their and CCVs preferential location in proximity to the cell membrane. Our analysis advances the understanding of individual SVs' diversity and their molecular architecture.

Micellization model for the polymerization of clathrin baskets.

A thermodynamic model is used to investigate the conditions under which clathrin triskelions form polyhedral baskets. The analysis, which is similar to classical methods used to study micelle formation, relates clathrin basket energetics to system parameters linked to triskelial rigidity, the natural curvature of an isolated triskelion, and interactions between triskelial legs in the assembled polyhedra. Mathematical theory predicts that a minimal ("critical") clathrin concentration, C(C), needs to be surpassed in order for basket polymerization to occur, and indicates how C(C), and the amount of polymerized material, depend on the chosen parameters. Analytical expressions are obtained to indicate how changes in the parameters affect the sizes of the polyhedra which arise when the total clathrin concentration exceeds C(C). A continuum analytic approximation then is used to produce numerical results that illustrate the derived dependences.

Clathrin Coat Disassembly by the Yeast Hsc70/Ssa1p and Auxilin/Swa2p Proteins Observed by Single-particle Burst Analysis Spectroscopy

The role of clathrin-coated vesicles in receptor-mediated endocytosis is conserved among eukaryotes, and many of the proteins required for clathrin coat assembly and disassembly have orthologs in yeast and mammals. In yeast, dozens of proteins have been identified as regulators of the multistep reaction required for endocytosis, including those that regulate disassembly of the clathrin coat. In mammalian systems, clathrin coat disassembly has been reconstituted using neuronal clathrin baskets mixed with the purified chaperone ATPase 70-kDa heat shock cognate (Hsc70), plus a clathrin-specific co-chaperone, such as the synaptic protein auxilin. Yet, despite previous characterization of the yeast Hsc70 ortholog, Ssa1p, and the auxilin-like ortholog, Swa2p, testing mechanistic models for disassembly of nonneuronal clathrin coats has been limited by the absence of a functional reconstitution assay. Here we use single-particle burst analysis spectroscopy, in combination with fluorescence correlation spectroscopy, to follow the population dynamics of fluorescently tagged yeast clathrin baskets in the presence of purified Ssa1p and Swa2p. An advantage of this combined approach for mechanistic studies is the ability to measure, as a function of time, changes in the number and size of objects from a starting population to the reaction products. Our results indicate that Ssa1p and Swa2p cooperatively disassemble yeast clathrin baskets into fragments larger than the individual triskelia, suggesting that disassembly of clathrin-coated vesicles may proceed through a partially uncoated intermediate.

Nanomechanics of Clathrin Protein Shells

The nanomechanical properties of protein shells, ranging from virus capsids to clathrin-coated vesicles, are functionally important and of notable recent interest. Using atomic force microscopy (AFM) and, more recently, quartz crystal microbalance-dissipation (QCM-D) instrumentation, we investigate mechanical properties of intact clathrin-coated vesicles (CCVs), reassembled pure clathrin cage baskets, and single triskelia. CCVs are purified subcellular trafficking organelles of about 100 nm diameter, consisting of an outer clathrin lattice shell over a receptor- and adaptor- loaded lipid vesicle. The clathrin cages formed by self-assembly of clathrin triskelia are structurally similar to the outer shell of CCVs. From AFM and complementary QCM-D measurements, we now provide an integrative mechanical model of the clathrin protein shell and of the coupling between the clathrin coat and the membrane vesicle, in biologically relevant fluid environments. Whereas the Young's modulus of the triskelia arm is as high as a few MPa, the effective shear moduli of clathrin triskelia, clathrin baskets, and CCVs are one to two orders of magnitude smaller and vary with the buffer pH and ionic strength, as well as their proximity to a given substrate support. Such modulation of the nanomechanical properties of the assembling/disassembling protein shells may be an essential aspect of cellular function.

A PH domain within OCRL bridges clathrin-mediated membrane trafficking to phosphoinositide metabolism

OCRL, whose mutations are responsible for Lowe syndrome and Dent disease, and INPP5B are two similar proteins comprising a central inositol 5-phosphatase domain followed by an ASH and a RhoGAP-like domain. Their divergent NH2-terminal portions remain uncharacterized. We show that the NH2-terminal region of OCRL, but not of INPP5B, binds clathrin heavy chain. OCRL, which in contrast to INPP5B visits late stage endocytic clathrin-coated pits, was earlier shown to contain another binding site for clathrin in its COOH-terminal region. NMR structure determination further reveals that despite their primary sequence dissimilarity, the NH2-terminal portions of both OCRL and INPP5B contain a PH domain. The novel clathrin-binding site in OCRL maps to an unusual clathrin-box motif located in a loop of the PH domain, whose mutations reduce recruitment efficiency of OCRL to coated pits. These findings suggest an evolutionary pressure for a specialized function of OCRL in bridging phosphoinositide metabolism to clathrin-dependent membrane trafficking.

Clathrin Triskelia Show Evidence of Molecular Flexibility

The clathrin triskelion, which is a three-legged pinwheel-shaped heteropolymer, is a major component in the protein coats of certain post-Golgi and endocytic vesicles. At low pH, or at physiological pH in the presence of assembly proteins, triskelia will self-assemble to form a closed clathrin cage, or “basket”. Recent static light scattering and dynamic light scattering studies of triskelia in solution showed that an individual triskelion has an intrinsic pucker similar to, but differing from, that inferred from a high resolution cryoEM structure of a triskelion in a clathrin basket. We extend the earlier solution studies by performing small-angle neutron scattering (SANS) experiments on isolated triskelia, allowing us to examine a higher q range than that probed by static light scattering. Results of the SANS measurements are consistent with the light scattering measurements, but show a shoulder in the scattering function at intermediate q values (0.016 Å −1), just beyond the Guinier regime. This feature can be accounted for by Brownian dynamics simulations based on flexible bead-spring models of a triskelion, which generate time-averaged scattering functions. Calculated scattering profiles are in good agreement with the experimental SANS profiles when the persistence length of the assumed semiflexible triskelion is close to that previously estimated from the analysis of electron micrographs.

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.

Clathrin exchange during clathrin-mediated endocytosis.

During clathrin-mediated endocytosis, clathrin-coated pits invaginate to form clathrin-coated vesicles (CVs). Since clathrin-coated pits are planar structures, whereas CVs are spherical, there must be a structural rearrangement of clathrin as invagination occurs. This could occur through simple addition of clathrin triskelions to the edges of growing clathrin-coated pits with very little exchange occurring between clathrin in the pits and free clathrin in the cytosol, or it could occur through large scale exchange of free and bound clathrin. In the present study, we investigated this question by studying clathrin exchange both in vitro and in vivo. We found that in vitro clathrin in CVs and clathrin baskets do not exchange with free clathrin even in the presence of Hsc70 and ATP where partial uncoating occurs. However, surprisingly FRAP studies on clathrin-coated pits labeled with green fluorescent protein–clathrin light chains in HeLa cells show that even when endocytosis is blocked by expression of a dynamin mutant or depletion of cholesterol from the membrane, replacement of photobleached clathrin in coated pits on the membrane occurs at almost the same rate and magnitude as when endocytosis is occurring. Furthermore, very little of this replacement is due to dissolution of old pits and reformation of new ones; rather, it is caused by a rapid ATP-dependent exchange of clathrin in the pits with free clathrin in the cytosol. On the other hand, consistent with the in vitro data both potassium depletion and hypertonic sucrose, which have been reported to transform clathrin-coated pits into clathrin cages just below the surface of the plasma membrane, not only block endocytosis but also block exchange of clathrin. Taken together, these data show that ATP-dependent exchange of free and bound clathrin is a fundamental property of clathrin-coated pits, but not clathrin baskets, and may be involved in a structural rearrangement of clathrin as clathrin-coated pits invaginate.

Energetics of Clathrin Basket Assembly

A minimal thermodynamic model is used to study the in vitro equilibrium assembly of reconstituted clathrin baskets. The model contains parameters accounting for i) the combined bending and flexing rigidities of triskelion legs and hubs, ii) the intrinsic curvature of an isolated triskelion, and iii) the free energy changes associated with interactions between legs of neighboring triskelions. Analytical expressions for basket size distributions are derived, and published size distribution data (Zaremba S, Keen JH. J Cell Biol 1983;97: 1339-1347) are then used to provide estimates for net total basket assembly energies. Results suggest that energies involved in adding triskelions to partially formed clathrin lattices are small (of the order of kBT), in accord with the notion that lattice remodeling during basket formation occurs as a result of thermodynamic fluctuations. In addition, analysis of data showing the effects of assembly proteins (APs) on basket size indicates that the binding of APs increases the intrinsic curvature of an elemental triskelial subunit, the stabilizing energy of leg interactions, and the effective leg/hub rigidity. Values of effective triskelial rigidity determined in this investigation are similar to those estimated by previous analysis of shape fluctuations of isolated triskelia.

Complete reconstitution of clathrin basket formation with recombinant protein fragments: adaptor control of clathrin self-assembly.

Clathrin polymerization into a polyhedral basket, surrounding budding membrane vesicles, mediates protein sorting during endocytosis and organelle biogenesis. Adaptor proteins target clathrin assembly to specific membrane sites and sequester receptors into the clathrin coat. We have reconstituted complete clathrin basket formation from recombinantly expressed fragments of clathrin and adaptors. This reconstitution reveals a hierarchy of clathrin self-assembly interactions and demonstrates that adaptors control basket formation by alignment of the distal domains of the clathrin triskelion leg through their binding to the terminal domain.

Role of Cyclin G-associated Kinase in Uncoating Clathrin-coated Vesicles from Non-neuronal Cells

Auxilin is a brain-specific DnaJ homolog that is required for Hsc70 to dissociate clathrin from bovine brain clathrin-coated vesicles. However, Hsc70 is also involved in uncoating clathrin-coated vesicles formed at the plasma membrane of non-neuronal cells suggesting that an auxilin homolog may be required for uncoating in these cells. One candidate is cyclin G-associated kinase (GAK), a 150-kDa protein expressed ubiquitously in various tissues. GAK has a C-terminal domain with high sequence similarity to auxilin; like auxilin this C-terminal domain consists of three subdomains, an N-terminal tensin-like domain, a clathrin-binding domain, and a C-terminal J-domain. Western blot analysis shows that GAK is present in rat liver, bovine testes, and bovine brain clathrin-coated vesicles. More importantly, liver clathrin-coated vesicles, which contain GAK but not auxilin, are uncoated by Hsc70, suggesting that GAK acts as an auxilin homolog in non-neuronal cells. In support of this view, the clathrin-binding domain of GAK alone induces clathrin polymerization into baskets and the combined clathrin-binding domain and J-domain of GAK supports uncoating of AP180-clathrin baskets by Hsc70 at pH 7 and induces Hsc70 binding to clathrin baskets at pH 6. Immunolocalization studies suggest that GAK is a cytosolic protein that is concentrated in the perinuclear region; it appears to be highly associated with the trans-Golgi where the budding of clathrin-coated vesicles occurs. We propose that GAK is a required cofactor for the uncoating of clathrin-coated vesicles by Hsc70 in non-neuronal cells.

Characterization of D10S and K71E mutants of human cytosolic hsp70.

To determine the effect of mutations at the nucleotide-binding site of recombinant Hsp70 on its interaction with protein and peptide substrates, point mutations were made at D10 and K71, two residues at the active site. The D10S mutation weakened both ATP and ADP binding, while the K71E mutation weakened only ATP binding. In binding experiments using Hsp70 with no bound nucleotide, the mutated Hsp70s interacted with clathrin and peptide just like the wild-type Hsp70. However, the D10 mutation completely abolished the effects of both ATP and ADP on peptide and clathrin binding. The K71 mutation also abolished the effect of ATP on substrate binding, but ADP, which still bound tightly, had its normal effect on substrate binding. In addition, the D10S and K71E mutants had greatly reduced ability to uncoat clathrin-coated vesicles at pH 7.0, bind to clathrin baskets at pH 6.0, and undergo polymerization induced by YDJ1 in the presence of ATP. We conclude, first, that nucleotides must bind strongly to Hsp70 to affect substrate binding and, second, that interaction of Hsp70 with DnaJ homologues may also require a strongly bound ATP.

Clathrin self-assembly is regulated by three light-chain residues controlling the formation of critical salt bridges.

Clathrin self-assembly into a polyhedral lattice mediates membrane protein sorting during endocytosis and organelle biogenesis. Lattice formation occurs spontaneously in vitro at low pH and, intracellularly, is triggered by adaptors at physiological pH. To begin to understand the cellular regulation of clathrin polymerization, we analyzed molecular interactions during the spontaneous assembly of recombinant hub fragments of the clathrin heavy chain, which bind clathrin light-chain subunits and mimic the self-assembly of intact clathrin. Reconstitution of hubs using deletion and substitution mutants of the light-chain subunits revealed that the pH dependence of clathrin self-assembly is controlled by only three acidic residues in the clathrin light-chain subunits. Salt inhibition of hub assembly identified two classes of salt bridges which are involved and deletion analysis mapped the clathrin heavy-chain regions participating in their formation. These combined observations indicated that the negatively charged regulatory residues, identified in the light-chain subunits, inhibit the formation of high-affinity salt bridges which would otherwise induce clathrin heavy chains to assemble at physiological pH. In the presence of light chains, clathrin self-assembly depends on salt bridges that form only at low pH, but is exquisitely sensitive to regulation. We propose that cellular clathrin assembly is controlled via the simple biochemical mechanism of reversing the inhibitory effect of the light-chain regulatory sequence, thereby promoting high-affinity salt bridge formation.

Auxilin-induced interaction of the molecular chaperone Hsc70 with clathrin baskets.

We previously reported that a 100-kDa cofactor, recently identified as auxilin, is a DnaJ homolog which is required for Hsc70 to uncoat clathrin baskets. In the present study we investigated the effect of auxilin on the interaction of Hsc70 with pure clathrin baskets at pH 6, where no uncoating occurs. In a reaction which required auxilin, the baskets activated the Hsc70 ATPase activity more than 100-fold with an apparent dissociation constant of about 0.2 microM. Maximal ATPase activity occurred at a 1 to 1 molar ratio of auxilin to clathrin triskelion independent of the Hsc70 concentration suggesting that auxilin is primarily complexed with the clathrin baskets. The binding of Hsc70 to baskets also required auxilin, but less auxilin was needed for maximum binding than for maximum ATPase activity showing that auxilin can catalytically induce binding of Hsc70. The binding also required ATP; Hsc70 dissociated from baskets with a 6 min half-life when ATP was hydrolyzed to ADP. In contrast to auxilin, the assembly proteins, AP-2 and AP180, did not support activation of the Hsc70 ATPase activity by clathrin baskets nor did soluble clathrin triskelions at pH 7 significantly activate the ATPase activity with auxilin present. Therefore, the interaction of auxilin, clathrin baskets, and Hsc70-ATP is highly specific with auxilin first binding to a clathrin triskelion in the baskets and then Hsc70-ATP strongly binding to the auxilin-clathrin complex; the auxilin can then migrate to another clathrin triskelion before the ATPase cycle is complete.

Effect of yeast and human DnaJ homologs on clathrin uncoating by 70 kilodalton heat shock protein.

We recently found that the DnaJ homolog auxilin is required for Hsc70 to uncoat clathrin baskets. In the present study, we investigated the effect of two other DnaJ homologs, YDJ1 from yeast and HDJ1 from humans, on the uncoating activity of Hsc70. Neither YDJ1 nor HDJ1 substituted for auxilin in supporting uncoating. Rather, in the presence of auxilin, both YDJ1 and HDJ1 strongly inhibited uncoating at pH 7 and also prevented the binding of Hsc70 to clathrin baskets at pH 6. Both YDJ1, as shown previously, and HDJ1 catalytically induce polymerization of Hsc70 into large polymers in ATP, and the YDJ1 concentration required to inhibit uncoating was similar to the concentration required for polymerization. However, uncoating was almost completely inhibited even at low concentrations of Hsc70 where only partial polymerization occurs, suggesting that YDJ1 inhibits uncoating not only by polymerizing the Hsc70 but also by some other mechanism as well. The effects of YDJ1 and HDJ1 were completely reversible; when they were removed, the Hsc70 regained full activity. Since both YDJ1 and HDJ1 inhibited the uncoating of clathrin baskets by brain cytosol as well as by purified Hsc70, this could be a physiological phenomenon which could affect other activities of Hsc70 in addition to uncoating.

Mechanism of clathrin basket dissociation: separate functions of protein domains of the DnaJ homologue auxilin.

Auxilin was recently identified as cofactor for hsc70 in the uncoating of clathrin-coated vesicles (Ungewickell, E., H. Ungewickell, S.E. Holstein, R. Lindner, K. Prasad, W. Barouch, B. Martin, L.E. Greene, and E. Eisenberg. 1995. Nature (Lond.). 378: 632-635). By constructing different glutathione-S-transferase (GST)-auxilin fragments, we show here that cooperation of auxilin's J domain (segment 813-910) with an adjoining clathrin binding domain (segment 547-814) suffices to dissociate clathrin baskets in the presence of hsc70 and ATP. When the two domains are expressed as separate GST fusion proteins, the cofactor activity is lost, even though both retain their respective functions. The clathrin binding domain binds to triskelia like intact auxilin with a maximum stoichiometry of 3 and concomitantly promotes their assembly into regular baskets. A fragment containing auxilin's J domain associates in an ATP-dependent reaction with hsc70 to form a complex with a half-life of 8 min at 25 degrees C. When the clathrin binding domain and the J domain are recombined via dimerization of their GST moieties, cofactor activity is partially recovered. The interaction between auxilin's J domain and hsc70 causes rapid hydrolysis of bound ATP. Release of inorganic phosphate appears to be correlated with the disintegration of the complex between auxilin's J domain and hsc70. We infer that the metastable complex composed of auxilin, hsc70, ADP, and P(i) contains an activated form of hsc70, primed to engage clathrin that is brought into apposition with it by the DnaJ homologue auxilin.

Purification of a New Clathrin Assembly Protein from Bovine Brain Coated Vesicles and Its Identification as Myelin Basic Protein

The multimeric clathrin assembly proteins AP-1 and AP-2 with molecular masses of approximately 270 kDa and the monomeric clathrin assembly proteins AP180 and auxilin with molecular masses of approximately 90 kDa catalyze the assembly of clathrin into artificial clathrin baskets under physiological conditions. We have now identified a much smaller approximately 20-kDa clathrin assembly protein in 0.5 M Tris, pH 7.0, extracts of bovine-brain coated vesicles and purified it to near homogeneity. A polyclonal antibody against this protein did not cross-react with any of the other assembly proteins, and sequencing data suggest that this new protein is similar or identical to myelin basic protein (MBP). At a molar ratio of 3 molecules per clathrin triskelion, MBP catalyzes polymerization of clathrin into artificial baskets that appear structurally similar to the baskets assembled by the other assembly proteins. In addition, like the other baskets, the clathrin-MBP baskets are uncoated by hsp70. MBP represents a significant fraction of the total assembly protein activity present in 0.5 M Tris, pH 7.0, extracts of coated vesicles. It is not clear if it acts as an assembly protein in vivo, but because it is well characterized and easily available, MBP will be a useful protein to investigate the mechanism of clathrin assembly and disassembly in vitro.

ATPase activity associated with the uncoating of clathrin baskets by Hsp70.

In the presence of ATP, bovine brain hsp70 has been shown to remove clathrin from bovine brain clathrin-coated vesicles in a rapid stoichiometric initial burst followed by slow steady-state uncoating. In addition, it has been found recently that a 100-kDa cofactor is required for hsp70 to uncoat clathrin baskets prepared with the assembly protein AP-2. In this study the ATPase activity associated with uncoating was investigated, with baskets formed from clathrin and assembly proteins. Mixed assembly proteins or assembly protein AP-2 could not be used in ATPase studies because they activated the hsp70 ATPase activity even in the absence of clathrin. However, this was not the case with assembly protein AP180. A stoichiometric initial burst of ATP hydrolysis was found to accompany the initial burst of uncoating of AP180-clathrin baskets by hsp70, with 1 mol of hydrolyzed ATP/mol of released clathrin heavy chain. Furthermore, the presence of a 100-kDa cofactor was needed for both processes. These results suggest that an initial burst of uncoating occurs with all clathrin baskets, that an initial burst of ATP hydrolysis accompanies this initial burst of uncoating, and that a 100-kDa cofactor is required for both.

A protein cofactor is required for uncoating of clathrin baskets by uncoating ATPase.

Immediately after clathrin-coated pits pinch off from the cell membrane to form clathrin-coated vesicles, clathrin dissociates from the vesicles. In vitro studies suggest that this dissociation is carried out by the uncoating ATPase, a constitutive member of the 70-kDa heat shock family. Aside from the requirement for ATP, nothing is known about the regulation of the uncoating process. We now show that clathrin baskets prepared from highly purified clathrin and AP2, the assembly protein associated with plasma membrane coated vesicles, cannot be uncoated by the bovine brain uncoating ATPase alone. A 100-kDa protein cofactor, which was isolated from coated vesicles, is essential for uncoating by the uncoating ATPase. This cofactor restores normal uncoating when present at a molar ratio of about 1 to 10 to clathrin and uncoating ATPase.

Localization of anti-clathrin antibody in the sarcomere and sensitivity of myofibril structure to chloroquine suggest a role for clathrin in myofibril assembly

Immunofluorescence microscopy has been used to demonstrate that X22, a monoclonal antibody specific for clathrin heavy chain, localizes in repetitive bands that appear soon after the fusion of skeletal myoblasts into multinucleate fibers. This organization has been found in cultures containing myotubes that develop in vitro from explants of newborn rat hindlimb cells and in myotubes derived from the L8E63 myogenic line. Bands were also prominent in skinned fibers prepared from adult rat soleus muscle and in cardiac myocytes grown in vitro from 4-day heart ventricles. Immunofluorescence banding was localized in the sarcomere as a doublet, with one element on either side of the Z line. Evidence that supports the conclusion that the reaction with X22 antibody is specific and indicative of the localization of clathrin in the sarcomere includes: (1) Identical titration of X22 antibody reactivity with the determinant in coated vesicles and in the sarcomere. (2) Conditions (eg., pH and Tris) that disrupt clathrin baskets or prevent its assembly likewise disrupt the localization of X22 in bands. (3) Chloroquine inhibits both the normal trafficking of clathrin in the cell and X22 banding in the sarcomere. (4) Immunoblot analysis of myotube lysates reveals a single band with an electrophoretic mobility identical to the 180,000-Da clathrin heavy chain. (5) The assembly of clathrin into sarcomeric bands occurs early in the development of the myofibrillar apparatus. Quantitation of the appearance of X22 banding in primary cultures of myotubes indicates that it precedes that of other myofibrillar proteins and that assembly takes place in the following order: X22, titin, myosin heavy chain, actin, and desmin. The assembly of myosin, titin, and actin into sarcomeric bands, as well as X22, is inhibited by chloroquine. Upon prolonged exposure to chloroquine previously assembled proteins are drastically reduced or no longer evident in the sarcomere. On the basis of these results and considering the role of clathrin in intracellular transport and its capacity to interact with actin and α-actinin, we suggest that clathrin may have diverse roles in the assembly, integrity, and functioning of the sarcomere and its integration with the sarcolemma. The early organization of X22 into bands further suggests that clathrin may also function early in the assembly of the contractile system.