Coated Pit Assembly Research Articles

Dynamics of Auxilin 1 and GAK in clathrin-mediated traffic.

Clathrin-coated vesicles lose their clathrin lattice within seconds of pinching off, through the action of the Hsc70 "uncoating ATPase." The J- and PTEN-like domain-containing proteins, auxilin 1 (Aux1) and auxilin 2 (GAK), recruit Hsc70. The PTEN-like domain has no phosphatase activity, but it can recognize phosphatidylinositol phosphate head groups. Aux1 and GAK appear on coated vesicles in successive transient bursts, immediately after dynamin-mediated membrane scission has released the vesicle from the plasma membrane. These bursts contain a very small number of auxilins, and even four to six molecules are sufficient to mediate uncoating. In contrast, we could not detect auxilins in abortive pits or at any time during coated pit assembly. We previously showed that clathrin-coated vesicles have a dynamic phosphoinositide landscape, and we have proposed that lipid head group recognition might determine the timing of Aux1 and GAK appearance. The differential recruitment of Aux1 and GAK correlates with temporal variations in phosphoinositide composition, consistent with a lipid-switch timing mechanism.

Role of the Clathrin Terminal Domain in Regulating Coated Pit Dynamics Revealed by Small Molecule Inhibition

Clathrin-mediated endocytosis (CME) regulates many cell physiological processes such as the internalization of growth factors and receptors, entry of pathogens, and synaptic transmission. Within the endocytic network, clathrin functions as a central organizing platform for coated pit assembly and dissociation via its terminal domain (TD). We report the design and synthesis of two compounds named pitstops that selectively block endocytic ligand association with the clathrin TD as confirmed by X-ray crystallography. Pitstop-induced inhibition of clathrin TD function acutely interferes with receptor-mediated endocytosis, entry of HIV, and synaptic vesicle recycling. Endocytosis inhibition is caused by a dramatic increase in the lifetimes of clathrin coat components, including FCHo, clathrin, and dynamin, suggesting that the clathrin TD regulates coated pit dynamics. Pitstops provide new tools to address clathrin function in cell physiology with potential applications as inhibitors of virus and pathogen entry and as modulators of cell signaling.

Initiator for Coated Pit Assembly

Membrane-sculpting proteins nucleate clathrin-coated pits during endocytosis.

Initiator for Coated Pit Assembly

Initiator for Coated Pit Assembly

Unusual Structural Organization of the Endocytic Proteins AP180 and Epsin 1

Epsin and AP180/CALM are important endocytic accessory proteins that are believed to be involved in the formation of clathrin coats. Both proteins associate with phosphorylated membrane inositol lipids through their epsin N-terminal homology domains and with other components of the endocytic machinery through short peptide motifs in their carboxyl-terminal segments. Using hydrodynamic and spectroscopic methods, we demonstrate that the parts of epsin 1 and AP180 that are involved in protein-protein interactions behave as poorly structured flexible polypeptide chains with little or no conventional secondary structure. The predominant cytosolic forms of both proteins are monomers. Furthermore, we show that recombinant epsin 1, like AP180, drives in vitro assembly of clathrin cages. We conclude that the epsin N-terminal homology domain-containing proteins AP180/CALM and epsin 1 have a very similar molecular architecture that is designed for the rapid and efficient recruitment of the principal coat components clathrin and AP-2 at the sites of coated pit assembly.

Activation of MAPK by TRH requires clathrin-dependent endocytosis and PKC but not receptor interaction with beta-arrestin or receptor endocytosis.

To determine whether the interaction of the TRH receptor with beta-arrestin is necessary for TRH activation of MAPK, cells expressing either intact or truncated, internalization-defective TRH receptors were transfected with a beta-arrestin-green fluorescent protein conjugate. In cells expressing the wild-type pituitary TRH receptor, TRH caused translocation of the beta-arrestin-green fluorescent protein conjugate from the cytosol to the plasma membrane within 30 sec. After 5 min, the beta-arrestin-green fluorescent protein conjugate was visible in vesicles, where it colocalized with rhodamine-labeled TRH. In hypertonic sucrose, the beta-arrestin-green fluorescent protein conjugate translocated to the plasma membrane after TRH addition but did not internalize. In cells expressing the truncated TRH receptor, TRH did not cause translocation of the beta-arrestin-green fluorescent protein conjugate. TRH activated MAPK strongly in cells expressing intact or truncated TRH receptors, indicating that the receptor does not need to bind beta-arrestin or internalize. MAPK activation by TRH, epidermal growth factor, and phorbol ester was strongly inhibited by hypertonic sucrose and concanavalin A, which block movement of proteins into coated pits and coated pit assembly. Hypertonic sucrose did not affect MAPK activation in cells overexpressing MAPK kinase 1. Dominant negative dynamin, which blocks conversion of coated pits to vesicles, also reduced receptor internalization and TRH activation of MAPK. TRH activation of MAPK required PKC but was insensitive to pertussis toxin and did not require ras, epidermal growth factor receptor kinase, or PI3K. These results show that the TRH receptor itself does not need to bind beta-arrestin or undergo sequestration to activate MAPK but that the endocytic pathway must be intact.

The role of dynamin and its binding partners in coated pit invagination and scission.

Plasma membrane clathrin-coated vesicles form after the directed assembly of clathrin and the adaptor complex, AP2, from the cytosol onto the membrane. In addition to these structural components, several other proteins have been implicated in clathrin-coated vesicle formation. These include the large molecular weight GTPase, dynamin, and several Src homology 3 (SH3) domain-containing proteins which bind to dynamin via interactions with its COOH-terminal proline/arginine-rich domain (PRD). To understand the mechanism of coated vesicle formation, it is essential to determine the hierarchy by which individual components are targeted to and act in coated pit assembly, invagination, and scission. To address the role of dynamin and its binding partners in the early stages of endocytosis, we have used well-established in vitro assays for the late stages of coated pit invagination and coated vesicle scission. Dynamin has previously been shown to have a role in scission of coated vesicles. We show that dynamin is also required for the late stages of invagination of clathrin-coated pits. Furthermore, dynamin must bind and hydrolyze GTP for its role in sequestering ligand into deeply invaginated coated pits. We also demonstrate that the SH3 domain of endophilin, which binds both synaptojanin and dynamin, inhibits both late stages of invagination and also scission in vitro. This inhibition results from a reduction in phosphoinositide 4,5-bisphosphate levels which causes dissociation of AP2, clathrin, and dynamin from the plasma membrane. The dramatic effects of the SH3 domain of endophilin led us to propose a model for the temporal order of addition of endophilin and its binding partner synaptojanin in the coated vesicle cycle.

Synaptotagmin Regulation of Coated Pit Assembly

Synaptotagmins bind clathrin AP-2 with high affinity via their second C(2) domain, which indicates they are involved in coated pit function. We now report that expression of synaptotagmins lacking either the second C(2) domain or the entire cytoplasmic region potently inhibit endocytosis. Inhibition was dependent on two intramembrane cysteine residues that were found to be essential for synaptotagmin oligomerization. Cells expressing the wild-type, but not the mutant, truncated synaptotagmin fragment had a reduced number of clathrin-coated pits. These results suggest that the formation of synaptotagmin multimers is an important step in the regulation of coated pit assembly.

Inhibition of clathrin-coated pit assembly by an Eps15 mutant.

Recent data have shown that Eps15, a newly identified component of clathrin-coated pits constitutively associated with the AP-2 complex, is required for receptor-mediated endocytosis. However, its precise function remains unknown. Interestingly, Eps15 contains three EH (Eps15-Homology) domains also found in proteins required for the internalization step of endocytosis in yeast. Results presented here show that EH domains are required for correct coated pit targeting of Eps15. Furthermore, when cells expressed an Eps15 mutant lacking EH domains, the plasma membrane punctate distribution of both AP-2 and clathrin was lost, implying the absence of coated pits. This was further confirmed by the fact that dynamin, a GTPase found in coated pits, was homogeneously redistributed on the plasma membrane and that endocytosis of transferrin, a specific marker of clathrin-dependent endocytosis, was strongly inhibited. Altogether, these results strongly suggest a role for Eps15 in coated pit assembly and more precisely a role for Eps15 in the docking of AP-2 onto the plasma membrane. This hypothesis is supported by the fact that a GFP fusion protein encoding the ear domain of (alpha)-adaptin, the AP-2 binding site for Eps15, was efficiently targeted to plasma membrane coated pits.

Synaptotagmin I is a high affinity receptor for clathrin AP-2: Implications for membrane recycling

In nerve terminals, Ca(2+)-stimulated synaptic vesicle exocytosis is rapidly followed by endocytosis. Synaptic vesicle endocytosis requires clathrin-coated pits similar to receptor-mediated endocytosis in fibroblasts. Binding of clathrin AP-2 (adaptor complex) to an unidentified high affinity membrane receptor appears to be necessary for coated pit assembly in fibroblasts. We now show that synaptic vesicles have a high affinity AP-2 site (KD, approximately 1 x 10(-10) M) similar to the one observed in fibroblasts. Using a combination of competition and direct binding assays, we demonstrate that synaptotagmin I, an intrinsic membrane protein of synaptic vesicles, has all of the properties of the AP-2 receptor and that AP-2 binds to the second C2 domain in the molecule. Thus, synaptotagmin I may be a multifunctional protein with a function in endocytosis in addition to the previously proposed role in exocytosis.

Multiple GTP-binding proteins participate in clathrin-coated vesicle-mediated endocytosis.

We have examined the effects of various agonists and antagonists of GTP-binding proteins on receptor-mediated endocytosis in vitro. Stage-specific assays which distinguish coated pit assembly, invagination, and coat vesicle budding have been used to demonstrate requirements for GTP-binding protein(s) in each of these events. Coated pit invagination and coated vesicle budding are both stimulated by addition of GTP and inhibited by GDP beta S. Although coated pit invagination is resistant to GTP gamma S, A1F4-, and mastoparan, late events involved in coated vesicle budding are inhibited by these antagonists of G protein function. Earlier events involved in coated pit assembly are also inhibited by GTP gamma S, A1F4-, and mastoparan. These results demonstrate that multiple GTP-binding proteins, including heterotrimeric G proteins, participate at discrete stages in receptor-mediated endocytosis via clathrin-coated pits.

The appendage domain of the AP-2 subunit is not required for assembly or invagination of clathrin-coated pits.

Coated pits contain a resident membrane molecule(s) that binds clathrin AP-2 with high affinity. AP-2 binding to this site is likely to be the first step in coated pit assembly because this subunit functions as a template for the polymerization of clathrin into flat polygonal lattices. Integral membrane proteins involved in receptor mediated endocytosis cluster in the newly assembled pits as they invaginate and bud from the membrane. The AP-2 subunit is a multi-domain, molecular complex that can be separated by proteolysis into a brick-shaped core and ear-like appendage domains. We have used this property to identify the domain involved in the various stages of coated pit assembly and budding. We found that the core of AP-2 is the domain that binds both to membranes and to triskelions during assembly. Triskelions are perfectly capable of forming lattices on the membrane bound cores. Clathrin lattices bound only to core domains were also able to invaginate normally. Limited proteolysis was also useful for further characterizing the AP-2 binding site. Elastase treatment of the inside membrane surface released a peptide fraction that is able to bind AP-2 in solution and prevent it from interacting with membranes. Affinity purification of binding activity yielded a collection of peptides that was dominated by a 45-kD species. This is the candidate peptide for containing the AP-2-binding site. Therefore, the appendage domain does not directly participate in any of the assembly or invagination events required for coated pit function.

Cytosol- and clathrin-dependent stimulation of endocytosis in vitro by purified adaptors.

Using stage-specific assays for receptor-mediated endocytosis of transferrin (Tfn) into perforated A431 cells we show that purified adaptors stimulate coated pit assembly and ligand sequestration into deeply invaginated coated pits. Late events in endocytosis involving membrane fission and coated vesicle budding which lead to the internalization of Tfn are unaffected. AP2, plasma membrane adaptors, are active at physiological concentrations, whereas AP1, Golgi adaptors, are inactive. Adaptor-dependent stimulation of Tfn sequestration requires cytosolic clathrin, but is unaffected by clathrin purified from coated vesicles suggesting that soluble and assembled clathrin pools are functionally distinct. In addition to adaptors and cytosolic clathrin other, as yet unidentified, cytosolic factors are also required for efficient coated pit invagination. These results provide new insight into the mechanisms and regulation of coated pit assembly and invagination.

The mechanism of receptor-mediated endocytosis: more questions than answers.

Receptor-mediated endocytosis occurs via clathrin-coated pits and is therefore coupled to the dynamic cycle of assembly and disassembly of the coat constituents. These coat proteins comprise part, but certainly not all, of the machinery involved in the recognition of membrane receptors and their selective packaging into transport vesicles for internalization. Despite considerable knowledge about the biochemistry of coated vesicles and purified coat proteins, little is known about the mechanisms of coated pit assembly, receptor-sorting and coated vesicle formation. Cell-free assays which faithfully reconstitute these events provide powerful new tools with which to elucidate the overall mechanism of receptor-mediated endocytosis.

22] Receptor-mediated endocytosis in semiintact cells

Publisher Summary This chapter discusses receptor-mediated endocytosis in semiintact cells. Many biologically important macromolecules are internalized into cells with the complex, multistep process of receptor-mediated endocytosis. Bound ligands and their receptors are first clustered into specialized coated pit regions of the cell surface. Cell surface receptors internalized via coated pits fall into two classes. One class, including, transferrin and low-density lipoprotein (LDL) receptors, are constitutively localized in coated pits and efficiently internalized at rates independent of bound ligand. In contrast, unoccupied receptors such as epidermal growth factor (EGF) and insulin receptors are internalized at low basal rates. These events not only require the addition of coat constituents but are accompanied by morphological changes in the coat, which progresses from an initially planar structure to a highly curved, invaginated pit. Finally, a membrane fission event occurs and the coat is sealed around the newly budded coated vesicle. Molecular dissection of these biochemically diverse events is facilitated by the use of cell-free assays that allow distinct stages to be assayed independently. In the chapter, the assay described was used to examine receptor-mediated endocytosis in A431 cells. These assays allow independent measurement of coated pit assembly, invagination, and coated vesicle budding.

Clathrin-coated pits contain an integral membrane protein that binds the AP-2 subunit with high affinity.

Coated pits will assemble onto purified plasma membranes that are attached to a poly-L-lysine coated substratum (Moore, M. S., Mahaffey, D. T., Brodsky, F. M., and Anderson, R. G. W. (1987) Science 236, 558-563; Mahaffey, D. T., Moore, M. S., Brodsky, F. M., and Anderson, R. G. W. (1989) J. Cell Biol. 108, 1615-1624). To better understand the assembly reaction, we have purified both clathrin triskelion and AP-2 subunits from bovine brain and assayed for their ability to bind to the cytoplasmic surface of attached membranes. Two types of membranes were analyzed: those washed with a high pH buffer that selectively removes triskelions and those washed with a high salt buffer that removes both the AP-2 and the triskelion subunits. We found that purified AP-2 subunits bind with high affinity (Kd approximately 3 x 10(-8) M) to salt stripped membranes. Binding is saturable and abolished by treating membranes with less than 20 micrograms/ml of elastase. When membranes were treated with elastase before the salt wash and then salt washed and assayed for AP-2 binding, normal binding was seen, which indicates that the presence of clathrin-coated pits protects the binding site from the protease. Membranes that had rebound AP-2 did not bind purified triskelions, even though high pH buffer-washed membranes that bear endogenous AP-2 bound triskelions with high affinity (Kd approximately 3 x 10(-9) M) and supported lattice assembly. We conclude that coated pit assembly is initiated by the binding of AP-2 to an integral membrane protein but that the AP-2 complex must be activated by an unknown process before the coated pit lattice will assemble.

Towards an in vitro system for studying clathrin-coated pit function.

To elucidate the molecular basis of coated pit function, we are developing a method for reconstituting the endocytic cycle in a cell-free system. Using purified membranes attached to a solid substratum, we have successfully reconstituted coated pit assembly. We now show that the endogenous coated pits on these membranes spontaneously round up and leave the membrane at 37 degrees C. As measured by radioimmunoassay, clathrin loss is both pH- and temperature-dependent. The loss is also inhibited by pretreating the membranes with apyrase at 37 degrees C, which suggests that nucleotide triphosphates are required.

Assembly of clathrin-coated pits onto purified plasma membranes.

During receptor-mediated endocytosis, coated pits invaginate to form coated vesicles, clathrin and associated proteins dissociate from the vesicle membrane, and these proteins form new coated pits at the cell surface. As a means of elucidating molecular mechanisms that govern the function of coated pits, the assembly phase of this cycle was reconstituted by incubating purified membranes that were treated to remove endogenous coated pits with cytoplasm extracted from cultured cells. The in vitro assembly of coated pits on these membranes satisfactorily mimics many features of coated pit formation in the intact cell. These studies indicate that: the membranes contain a limited number of coated pit assembly sites that bind clathrin with high affinity; the half-time for assembly is 5 minutes both at 4 degrees C and 37 degrees C; during assembly, proteins with molecular sizes of 180, 110, and 36 kilodaltons are recruited to the plasma membrane; and assembly is not dependent on adenosine triphosphate, but this nucleotide triggers a temperature-dependent loss of coated pits that are assembled in the absence of adenosine triphosphate.