NCBI Bookshelf. A service of the National Library of Medicine, National Institutes of Health.

Madame Curie Bioscience Database [Internet]. Austin (TX): Landes Bioscience; 2000-2013.

Cover of Madame Curie Bioscience Database

Madame Curie Bioscience Database [Internet].

Show details

Clathrin-Mediated Endocytosis

, , and *.

* Corresponding Author: Department of Biology, The Johns Hopkins University, 3400 North Charles Street, Baltimore MD, 21218, USA. Email:ude.uhj@dnaldnewb

Eukaryotic cells use multiple pathways for the endocytic entry of proteins and lipids at the plasma membrane. To date, the best characterized pathway is clathrin-mediated endocytosis. This chapter presents an overview of the mechanisms of clathrin-mediated endocytosis and how it is regulated. We provide a mechanistic description of how a clathrin-coated vesicle (CCV) is formed, from the stages of initiation to scission to uncoating, as well as address important regulation by protein and lipid kinases and phosphatases. Endocytic events are initiated through the concerted action of the clathrin coat and adaptor proteins that select the transmembrane proteins (cargo) that will be carried into the cell in endocytic vesicles. Accessory proteins and the GTPase dynamin work together with forces provided by actin polymerization to complete the formation of the CCV. The ATPase chaperone Hsc70 and the protein auxilin promote CCV uncoating, a necessary step for the vesicle to fuse with endosomes. The synergistic convergence of powerful experimental strategies such as structural, biochemical and genomic approaches, in vitro assays, and real-time imaging in vivo, have combined to allow the new breakthroughs that are discussed.

Introduction

Eukaryotic cells employ numerous portals for the endocytic entry of proteins and lipids at the plasma membrane. These entry pathways include phagocytosis, macropinocytosis, and clathrin- or caveolin-mediated endocytosis1 and of these, clathrin-mediated endocytosis (CME) is the most extensively studied and best understood. Many clathrin-dependent endocytic events mediate cargo transport needed in essentially all cell types. These “housekeeping” forms of CME include the turnover of plasma membrane proteins and lipids, uptake of nutrients such as low-density lipoproteins and iron-saturated transferrin, and endocytosis of a plethora of growth factor receptors following their activation.1-3 Because of its ubiquitous nature, pathogens such as the influenza virus4 and bacterial toxins such as Shiga toxin5 subvert CME to gain entry into cells. The steps required for CME are depicted in Figure 1, and the molecular mechanisms that underlie each of these steps are described in detail in this chapter. Forming a clathrin-coated vesicle (CCV) is a multi-step process that requires the sequential function of more than fifty different proteins. The major protein classes that mediate the formation of a CCV are: (1) the adaptors that select the transmembrane cargo proteins and link the cargo selection/concentration to the polymerization of the clathrin coat, (2) the scission factors such as the GTPase dynamin and its binding partners that couple to force generating events such as actin polymerization, and (3) auxilin and Hsc70 that facilitate the uncoating of the endocytic vesicle.

Figure 1. An overview of the steps in formation of a cargo-laden CCV.

Figure 1

An overview of the steps in formation of a cargo-laden CCV. The major domains of various proteins regulating CCV formation are indicated. Proteins have been assigned to relevant steps in the process (assembly, curvature, or fission), but in some cases (more...)

Clathrin-mediated membrane budding also occurs at the membranes of the trans-Golgi network (TGN), contributing to the generation of carrier vesicles that transport cargo from the TGN to the endosomal system.6 One such set of cargo proteins are the mannose-6-phosphate receptors, which bind to mannose-6-phosphate tagged lysosomal hydrolases in the lumen of the TGN and package these enzymes into CCVs for transport to endosomes/lysosomes.7 Deficiencies in these pathways lead to secretion of lysosomal hydrolases with resultant abnormalities in lysosomal function and the development of lysosomal storage disease.8 Clathrin-mediated trafficking has also been implicated in the retrograde pathway from endosomes to the TGN.9,10

Various systems have been used to study these housekeeping functions of endocytosis, including fibroblasts and the baker's yeast Saccharomyces cerevisiae (Fig. 2). Some tissues and cell types have specialized trafficking needs that are also met through clathrin-mediated mechanisms. For example, in specialized secretory cells, clathrin coats are involved in the formation of secretory granules at the TGN11 and in polarized cells, CCVs are used for the trafficking of certain receptors from the TGN to the basolateral membrane, necessary for the maintenance of polarity.2 Epithelial cells in rat and placental cells in humans use CME for the uptake of maternal immunoglobulins, necessary for the development of maternal derived immunity.12 Perhaps the most striking example of a specialized function for CCVs is seen in neurons (Fig. 2), which communicate by releasing neurotransmitters through fusion of synaptic vesicles (SVs) with the plasma membrane. This leads to the insertion of SV membranes and membrane proteins into the plasma membrane. The endocytic machinery is thus faced with the challenge of the timely and precise retrieval of these components. To overcome this challenge, SV components may retain their unique composition even while embedded in the plasma membrane.13 Alternatively, there may be a large reservoir of SV proteins in the synaptic or axonal plasma membrane that serves as a source for endocytic retrieval.14 Under either circumstance, CCVs are able to selectively retrieve the appropriate protein components and to reform SVs.15 Thus, clathrin-mediated membrane budding contributes to a wide variety of critical cellular processes that are key to the function of essentially all cell types. In this chapter, we will describe the mechanisms involved in the formation of CCVs with a particular emphasis on CCVs that form at the plasma membrane and are involved in CME. The reader is referred to an excellent recent review that summarizes the mechanisms involved in CCV formation at the TGN and compares and contrasts the formation of CCVs at these two cellular sites.16

Figure 2. The three major systems in which endocytosis has been studied include non-polarized cells (fibroblasts), and polarized cells (yeast and neurons).

Figure 2

The three major systems in which endocytosis has been studied include non-polarized cells (fibroblasts), and polarized cells (yeast and neurons). For each system, the major discoveries made, the commonly studied endocytic cargos followed, differences (more...)

Mechanisms of CCV Formation

Initiation of a Clathrin Coated Pit (CCP)

Role of Clathrin and AP-2

Central to the formation of CCPs and CCVs is clathrin itself. The assembly unit of the clathrin coat is the triskelion, composed of three copies of the clathrin-heavy chain (CHC) linked at their C-termini through a trimerization domain.2 The CHCs radiate from this central hub with a characteristic curl that allows the protein to be subdivided into segments referred to as the proximal leg, the knee, the distal leg and the ankle, ending in the N-terminal domain (TD). When triskelia assemble into a clathrin coat, the legs interdigitate to form a lattice of open hexagonal and pentagonal faces with a trimerization domain at each vertex and numerous weak contacts between leg segments stabilizing the lattice.17 Electron cryomicroscopy has revealed that in the assembled coat, the trimerization domain projects inward and makes contacts with the ankle regions of three additional triskelia, each centered two vertices away.18 It is proposed that these contacts are invariant and provide critical stability to the lattice.18,19 Thus, destabilization of the lattice needed for coat disassembly following release of CCVs from the membrane (see below) is likely to be strongly influenced by disruption of this interaction.19

In brain, each CHC is associated in a 1:1 stoichiometry with either of two ˜30 kDa clathrin-light chains (CLCs).20-22 The CLCs lie along the proximal leg segment near the trimerization domain. In vitro, at physiological pH, purified triskelia spontaneously assemble into clathrin cages when they are stripped of CLCs and assembly is inhibited upon readdition of CLCs at molar ratios close to 1:1.23 Thus, the prevailing model is that CLCs regulate assembly in vivo by interfering with contacts between CHCs and preventing unwanted assembly in the cytosol. However, in non-neuronal tissues, CLCs are substoichiometric to CHCs, which calls into question the universal role of CLCs as regulators of clathrin assembly.24 Interestingly, the electron cryomicroscopy analysis of CCVs suggests that CLCs are oriented toward the cytosol, which may better position them to interact with cytosolic regulatory proteins, such as huntingtin-interacting proteins than to regulate CHC assembly.18 Thus, CLCs may function as scaffolding proteins.

As coat assembly begins on the membrane, the triskelia initially form a lattice that functions as a scaffold to recruit a diverse array of clathrin-associated proteins that drive membrane curvature and recruit cargo for subsequent vesicle transport. However, what initiates the formation of CCPs remains elusive. Triskelia do not bind directly to membranes and thus other factors are needed to recruit clathrin and to stabilize its interaction with the membrane. These factors are collectively known as adaptors, and many proteins that fulfill this role have been identified.25 In the case of CME, one key adaptor is adaptor protein 2 (AP-2), a multi-subunit complex composed of two large subunits, α- and β2-adaptin and two smaller subunits, μ2- and σ2-adaptin.26 α- and β2-adaptin are composed of large N-terminal regions that along with μ2 and σ2 form the core of the AP-2 complex (Figs. 1 and 3). The C-terminal regions contain globular, bi-lobed structures referred to as the α- and β2-ears (also termed appendages), which attach to the core via flexible linkers.27 Triskelia bind through the TD of the CHC to a consensus motif in the linker region of β2-adaptin referred to as a clathrin box.28,29 CHC also uses a region outside the TD to target an independent site in the β2-ear and simultanous engagement of both sites is necessary for full triskelia binding efficiency.28,30,31 Two recent studies used mutational analyses to identify the CHC binding site in the β2-ear.30,31 Schmid et al30 assign the CHC binding site to the β2 platform domain while Edeling et al31 attribute binding to the β2 sandwich domain. An independent study by Brodsky and coworkers32 on the interaction of clathrin with the ear of the TGN adaptor GGA1 identified an extended surface on the ear that is targeted by the CHC ankle. As the fold of the GGA1 ear corresponds to the sandwich domain of the β2-ear and as ankle mutants affect the GGA1 ear and the β2-ear in a similar way,32 it seems most likely that the β2-ear sandwich domain harbors the clathrin binding site.

Figure 3. A model of the initiation of clathrin-coated pit formation.

Figure 3

A model of the initiation of clathrin-coated pit formation. A) AP-2 randomly samples the membrane through kinetic action, but with weak affinity. Thus, the equilibrium is predominantly towards the cytosolic pool (equilibrium arrows on left) and the majority (more...)

siRNA-mediated depletion of AP-2 leads to a substantial decrease in membrane association of clathrin,32,33 indicating the importance of this interaction for clathrin recruitment. Therefore, AP-2 is a key component for the nucleation of CCPs at the plasma membrane. Now the question becomes, what recruits AP-2? An early assumption was that AP-2 would be recruited to membranes through interactions with the cytoplasmic tails of receptors that were destined to become cargo of CCVs. Transmembrane proteins require an internalization signal for rapid CME and among the numerous endocytic motifs known, YXXΦ (where Φ represents a bulky hydrophobic residue) and [DE]XXXL[LI] directly bind AP-2.35 To test if sorting signals in receptor cytoplasmic domains are sufficient to initiate the formation of CCPs, Santini and Keen36 overexpressed receptors containing YXXΦ motifs and activated the receptors with an immobilized ligand to prevent receptor endocytosis. No increase in CCP formation or clathrin recruitment to the membrane was observed.36 Thus, it appears that sorting signals in receptor tails are not sufficient to recruit AP-2 and initiate CCP formation.

Role of PtdIns(4,5)P2

There is now abundant evidence that phosphatidylinositol(4,5)bisphosphate [PtdIns(4,5)P2], which is generated primarily on the inner leaflet of the plasma membrane, is crucial for AP-2 recruitment. PtdIns(4,5)P2 binds to α- and μ2-adaptin subunits and some studies have shown that mutation of these binding sites interferes with AP-2 localization.37-39 Overepxression of the PH domain of PLCδ, which binds to and masks PtdIns(4,5)P2, prevents AP-2 from localizing at the surface40 as does decreasing the levels of PtdIns(4,5)P2 through depletion of the PtdIns(4,5)P2 5-kinase Iβ, which converts PtdIns(4)P to PtdIns(4,5)P2.41 Moreover, synaptojanin, a phospholipid phosphatase that converts PtdIns(4,5)P2 to PtdIns(4)P and which was identified based on its role in SV endocytosis42 is implicated in the removal of AP-2 from the membrane.43 In fact, directing synaptojanin to the plasma membrane leads to an acute depletion of PtdIns(4,5)P2 and a rapid loss of endocytic CCPs.44 The formation of PtdIns(4,5)P2 is controlled in large part by lipid kinases that convert PtdIns to PtdIns(4,5)P2. At the TGN, members of the Arf family of small GTPases recruit and activate phospholipid kinases that mediate PtdIns(4,5)P2 synthesis.45 Moreover, Arf6 was shown to facilitate clathrin- and AP-2-mediated CCP nucleation in the synapse via the stimulation of PtdIns(4,5)P2 production by PtdIns kinase Iγ.46 The formation of PtdIns(4,5)P2, triggered by the activation of phospholipid kinases, appears to be key to CCP nucleation.

Thus, one could propose a model (Fig. 3) in which cytosolic AP-2 is continuously sampling the plasma membrane through simple kinetic action. Transient increases in PtdIns(4,5)P2 levels through activation of phospholipid kinases would occur in patches on the inner leaflet, generating a transient, low affinity nucleation site that now favors an association of AP-2 with the membrane. The initial interaction likely occurs via PtdIns(4,5)P2 interaction with its binding site on the α-adaptin subunit and is in the 5-10 μM range.47,48 Subsequent interaction of PtdIns(4,5)P2 with its binding site in μ2-adaptin, which occurs following a predicted conformational change in the μ2-subunit, would further increase membrane affinity.48 Both phosphorylation of μ2 and cargo binding stabilize μ2 in an open conformation (Fig. 3 and see below). The α- and β2-ears bind to numerous accessory proteins that typically have multiple ear-binding sites.49,50 This leads to cross-linking of AP-2, creating avidity effects on the interaction of AP-2 with PtdIns(4,5)P2 and further increasing the affinity of the adaptor for the membrane. Adding to this phenomenon is the formation of the clathrin lattice, bound to the β2-ear and linker. The recruitment of cargo provides a final stabilization allowing the CCP to reach maturity.51 Thus, through a series of low affinity interactions, AP-2 coordinates endocytic protein complexes into high affinity and stable endocytic structures. The redundancy that is inherent in this type of multiple, low affinity-based structure can explain why no single mutation in AP-2 that disrupts its interaction with PtdIns(4,5)P2 totally abolishes endocytic function. 47

Contribution of Alternative Adaptors

Also of interest to the early stages of CCP formation is the recognition of the role of alternative cargo adaptors including epsin, assembly protein of 180kDa/clathrin assembly leukemia myeloid protein (AP180/CALM), huntingtin-interacting protein 1 (HIP1), Dab2, autosomal recessive hypercholesteremia (ARH) protein and β-arrestin. Each of these proteins binds to clathrin and PtdIns(4,5)P2, and except for AP180/CALM, are also known to bind directly to specific classes of cargo or endocytic sorting motifs.25 Thus, like AP-2 they recruit clathrin to PtdIns(4,5)P2 nucleation sites and they recruit cargo into nascent CCPs. For example, the low-density lipoprotein receptor (LDLR) contains an internalization sequence FDNPVY, matching the general consensus internalization motif FXNPXY. Structural studies reveal that this motif is incompatible for interaction with AP-252 and knocking down AP-2 by siRNA does not block CME of LDLR.34 Interestingly, ARH protein contains a phosphotyrosine-binding (PTB) domain that binds with high selectivity to non-phosphorylated FXNPXY motifs and several lines of evidence indicate that ARH protein functions as an adaptor to recruit LDLR to nascent CCPs.25 In fact, mutations in ARH protein lead to disruption of LDLR endocytosis and are responsible for the development of ARH in humans.53-55 However, it is worth mentioning that each of the alternative cargo adaptors noted above also bind to the ears of AP-2.56-61 By binding to PtdIns(4,5)P2 and AP-2 simultaneously, they could aid in the development of avidity effects contributing to AP-2 dependent CCP nucleation (Fig. 3). Additionally, by crosslinking the cytoplasmic domains of large cargos such as the LDL receptor, membrane bending forces could be generated by virtue of gathering the large cargo in a small concentrated area. Initiation of membrane curvature has also been attributed to insertion of amphiphathic alpha-helices of various coat-associated proteins into the inner membrane leaflet (discussed below). Thus, depending on the nature of the cargo, the alternative adaptors could either directly or indirectly lead to membrane bending and initiation of CCPs. A key area for the future is to determine when these adaptors function independently of AP-2 and when or if they contribute to AP-2-dependent endocytic function.

Sites of Nucleation (Role of Intersectin)

Another important question relates to the sites where CCPs are nucleated. Do CCPs nucleate at defined sites on the plasma membrane or are nucleation sites determined stochastically? At least in the case of the presynaptic nerve terminal, there appears to be defined endocytic “hot spots” surrounding the active zone, the sites of fusion of neurotransmitter-containing SVs. A strong candidate as an important scaffold protein for these membrane subdomains is dynamin-associated protein of 160 kDa (Dap160).62 Dap160 is the Drosophila homologue of intersectin-short (intersectin-s), an endocytic protein composed of two Eps15 homology (EH) domains, a coiled-coil region, and five tandem Src homology 3 (SH3) domains.63 Intersectin-s localizes to CCPs and interacts with key components of the endocytic machinery including synaptojanin and dynamin.64 In Drosophila neuromuscular synapses, Dap160 is concentrated in a region that precisely surrounds the active zone and that is also enriched in AP-2.62,65 Dap160 loss-of-function mutants are impaired in SV endocytosis and are unable to sustain high-frequency neurotransmitter release.66,67 Moreover, essential endocytic proteins including dynamin and synaptojanin are lost from the synapse, suggesting that Dap160 is a critical scaffold for the organization of endocytic sites. Whether or not intersectin-s plays a similar scaffolding role in mammalian cells has not been extensively studied. Unlike in Drosophila neurons, the major form of intersectin in mammalian neurons is intersectin-long (intersectin-l), a splice variant of intersectin-s that has a C-terminal extension with a Dbl homology (DH), pleckstrin homology (PH) and C2 domain.64 Tandem DH/PH domains are a signature of guanine-nucleotide exchange factors (GEFs) and intersectin-l is a Cdc42-specific GEF that activates actin assembly.68-70 Thus, in mammalian neurons, intersectin-l is ideally situated to act as a scaffolding protein at endocytic active zones and to couple the formation of endocytic vesicles to actin assembly. In yeast, sites of exocytosis found within buds also seem to be spatially coupled to actin patches, which are sites of endocytosis.71 The yeast EH domain protein Pan1, which may be an intersectin homolog, shares some intersectin-l-like functions, including activation of actin assembly.72,73 Interestingly, Pan1 functions as a key regulator during the early steps of actin patch formation.74 Thus, the scaffolding function of intersectin appears to be evolutionarily conserved.

Role of Cargo

Whether or not CCPs are nucleated at defined membrane sites has been more problematic to resolve in non-neuronal cells. In neurons, CME of SVs is compensatory and endocytosis is tightly coupled to exocytosis. Thus, endocytic sites must be spatially constrained near the sites of exocytosis. This is not the case in non-neuronal cells, and in theory, CME could occur at random sites on the plasma membrane. However, early studies did seem to suggest that CME occurs repeatedly at endocytic hot spots. When the formation of CCPs was examined in cultured cells expressing GFP-CLC chimeras, CCPs had a seemingly random distribution but were seen to form repeatedly at defined sites over time, whereas other areas of the membrane were excluded from CCP formation.75 However, a more recent study using fluorescently tagged CLC as well as fluorescently tagged AP-2 and cargo proteins indicated that there are no strongly preferred sites for the nucleation of CCPs.51 Intriguingly, the second study demonstrated that cargo-loaded clathrin clusters grow steadily during their lifetime, with larger cargo particles requiring more time to complete assembly of sufficiently large CCVs. Moreover, clathrin clusters that fail to incorporate cargo are short lived.51 This led to a model in which CCPs initiate randomly, perhaps in response to transient increases in PtdIns(4,5)P2 stimulated by the activation of lipid kinases. The pits begin to grow, but collapse unless they are stabilized by incorporation of cargo (Fig. 3). This model is consistent with in vitro studies48 demonstrating that AP-2 initially interacts with PtdIns(4,5)P2 via the α-adaptin subunit, which causes a conformational change in the μ2 subunit that exposes binding sites for PtdIns(4,5)P2 and the tails of transmembrane receptors bearing YXXΦ motifs (Fig. 3). The combination of interactions strongly increases the affinity of AP-2 for the membrane.48 Moreover, binding of cargo to the μ2 subunit stimulates the activity of μ2-associated lipid kinases providing a further increase in PtdIns(4,5)P2 levels.76 Thus, cargo engagement is critical for CCPs to reach maturation. A key avenue for the future is to determine the spatial and temporal order of these events. It will also be important to understand what triggers the initial formation of PtdIns(4,5)P2 via lipid kinases. Does this occur randomly or are there signaling pathways that determine their activation? Moreover, is there control over when and where PtdIns(4,5)P2 is generated? Are there mechanisms to corral locally synthesized PtdIns(4,5)P2 to prevent its diffusion and dilution? These questions represent interesting avenues for further research.

Role of Phosphorylation in Regulating Coat Assembly

Given the complexity of coat assembly it is perhaps not surprising that the process is highly regulated. A key regulatory mechanism in this regard is protein phosphorylation, which involves the actions of both kinases and phosphatases. Interestingly, phosphorylation has been found to play both positive and negative roles in modulating activities of the endocytic machinery.

Positive Roles

The best example of a positive role of phosphorylation on CME is adaptor-associated kinase 1 (AAK1) modification of the μ2 chain of AP-2. AAK1 was discovered as a CCV-associated protein77 and as a protein kinase that through one domain binds to the appendage of α-adaptin, and through its kinase domain phosphorylates the hinge region of μ2.78 This hinge links the μ2 N-terminal domain, which is embedded within the core of AP-2 to the C-terminal domain that contains the PtdIns(4,5)P2- and cargo-binding sites.37 It is likely that AAK1 corresponds to the long-known CCV-associated activity that phosphorylates μ2 in vivo. Phosphorylated μ2 more readily undergoes the conformational transition that exposes the binding sites on μ2 for PtdIns(4,5)P2 and YXXΦ cargo binding sites. Interestingly, the activity of AAK1 is stimulated greatly by assembled clathrin,78,79 providing a mechanism to link clathrin assembly and cargo loading.51

The site on the μ2 hinge that is modified by AAK1 conforms to the consensus site defined for the related protein kinase in yeast called Prk1p.80,81 Prk1p and its homologue Ark1p have both been implicated in regulating endocytosis in yeast,81,82 although a positive role in yeast similar to AAK1 has not yet been shown. A relatively large number of Prk1p substrates have been characterized that are all implicated in endocytosis; in general, the data are interpreted as Prk1p modifications being inhibitory to endocytosis. Consistent with this, Prk1p and Ark1p are recruited to endocytic sites through interactions with Abp1p, a very late acting factor in endocytosis that is thought to promote disassembly of endocytic complexes.83,84 In the future, it will be interesting to learn if there are positive effects of Ark1p or Prk1p phosphorylation, and/or if there are additional (perhaps inhibited) AAK1 substrates besides μ2-adaptin. However, since AAK1 seems to be controlled by both assembled clathrin and binding to AP-2, its other putative substrates would necessarily need to be in CCPs or CCVs.

An unusual CCV-associated protein called coated vesicle associated kinase of 104kDa (CVAK104) has also been identified.77,85 This protein resembles a kinase, but several key catalytic residues are not conserved relative to bone fide kinases. Nonetheless, a recent report suggests that it could still be catalytically active toward β2-adaptin subunits of AP-2.85 While it is difficult to envision a mechanism of catalysis by CVAK104, it is likely to play a role of some sort in endocytosis given its clear association with CCVs, whether or not it functions as a kinase. Additionally, a recent report found that CVAK104 localizes to endosomes and the TGN, and may thus regulate intracellular CCV-dependent trafficking between these organelles.86

The tyrosine kinase src has been implicated in positively influencing CME through its modification of CHC.87 In response to stimulation of the EGF receptor, src becomes activated and promotes clathrin recruitment to the plasma membrane and EGFR internalization. It remains to be seen if phosphorylation of the conserved Tyr1477, which lies near the CLC binding sites, can directly stimulate assembly of clathrin triskelia into cages, or if the mechanism of enhancing CME is indirect, for example via recruiting a phosphorylation-specific binding partner.

Negative Roles

In general, phosphorylation of the endocytic machinery has more commonly been associated with inhibiting protein interactions that underlie the formation of functional protein complexes. For example, the dephosphins, which include CLCs, α- and β2-adaptin subunits of AP-2, amphiphysin, dynamin, AP180 and synaptojanin, correspond to a wide range of neuronal endocytic proteins that are phosphorylated and inactive in resting neurons, and coordinately activated by calcineurin-dependent dephosphorylation upon stimulation of SV exocytosis. 88,89 Recent evidence has focused on casein kinase II (CKII) and Cdk5/p35 as the best candidates for CCV-associated inhibitory kinases. Numerous substrates have been identified for each, both in vitro and in vivo.

CKII has long been implicated in inhibitory effects on its endocytic substrates, perhaps as the enzyme that acts on the dephosphins. Interestingly, CKII is inactive when it is associated with CCVs, but it is activated by CCV uncoating.90 The inhibitory activity of the CCVs was found to be PtdIns(4,5)P2, possibly via competitive binding of the lipid in the kinase active site.91 This sets up an interesting scenario, in which PtdIns(4,5)P2, an early component of CCV assembly, recruits CKII but suppresses its activity until the lipid is degraded by synaptojanin, presumably to facilitate the uncoating of CCVs for the recycling and reuse of endocytic components.

For Cdk5/p35, which is a neuronal-specific protein, controversy exists with regard to the relevant in vivo substrate(s) and the consequences of phosphorylation. A recent report suggests that dynamin 1 is the key Cdk5/p35 substrate in neurons, and that CME is inhibited when syndapin 1, a partner of multiple endocytic components, has reduced binding to phosphorylated dynamin 1.92 Other key Cdk5/p35 substrates with potential impact on CME include synaptojanin, phosphorylation of which inhibits lipid phosphatase activity, thus maintaining high levels of PtdIns(4,5)P2 and impeding uncoating; and amphiphysin, phosphorylation of which inhibits binding to endophilin, thus diminishing dynamin recruitment.93-95 By analogy, yeast has a related kinase called Pho85p that phosphorylates the SH3 domain of Rvs167p, a yeast homologue of amphiphysin, and inhibits its binding to the yeast WASp homologous protein Las17p.96

The existence of ubiquitously expressed Cdk5 homologues has not been reported in metazoans, but related activities may exist. For example, the substrate consensus site for Cdk5/p35 includes Ser/Thr-Pro; the ubiquitously expressed mitotic kinase Cdc2 has a similar substrate consensus site. Mitotic cell extracts have been shown to phosphorylate epsins at Cdc2 consensus sites to inhibit their interactions with AP-2 in vitro.97 Another kinase with a similar substrate consensus site is the Drosophila minibrain kinase and its mammalian homologue Dyrk1A. Dyrk1A phosphorylates dynamin and amphiphysin, which in most situations inhibits binding to their partners.98 Interestingly though, phosphorylation of dynamin also seems to enhance binding to Grb2, suggesting a potential effect of this kinase on signal transduction pathways.99 An important caveat to keep in mind when interpreting many phosphorylation studies is the need for a correspondence of in vitro results to in vivo events.

An siRNA-based genome-wide study of all known or predicted human kinases in the genome (referred to as the kinome) was used to study the roles of various kinases in endocytosis. 100 This study monitored the infectivity of two viruses, SV40 and VSV, which are internalized via caveolar- and clathrin-dependent pathways, respectively. Thus, these viruses were used as indicators of internalization via caveolae or CCPs. Based on the clathrin-dependent entry of VSV as a prerequisite for infectivity, this study provided independent evidence that AAK1 and CKII alpha 1 are important for CME. Additionally, numerous other kinases that regulate endocytosis (that use both protein and lipid substrates) were identified in or confirmed by this study. For instance, siRNA depletion of Rho-kinase reduces CME, consistent with another study that showed Rho-kinase phosphorylation of endophilin and commensurate inhibition of EGFR internalization.101 It is extremely intriguing that various subsets of kinases were classified as either affecting both pathways, or having reciprocal effects on each pathway. This suggests that there are many levels of regulation by kinases, and communication between distinct endocytic pathways that remain to be understood.

Phosphatases

In spite of the clearly important regulatory roles played by protein kinases, surprisingly little is known about the phosphatases that counteract the effects of the kinases. As mentioned above, calcineurin is a major Ca2+-activated protein phosphatase that regulates SV recycling.89 In yeast, the type I protein phosphatase Glc7 is recruited to endocytic sites through its binding partner Scd5, where it may counteract the effects of the Prk1 and Ark1 kinases.102,103 Besides these two specific examples of phosphatases that regulate the endocytic machinery, there are some suggestions in the literature that tyrosine phosphatases may influence the endocytosis of cell surface proteins and the signaling pathways regulated by activated signaling receptors. For example, RTKs can control their own endocytosis and trafficking itineraries through tyrosine phosphorylation of their cytoplasmic tails and of downstream signaling components, and these events would need to be reversed by tyrosine phosphatases.104 In the case of angiotensin II signaling, the dual-specificity phosphatase MKP7 is recruited to activated receptors by the adaptor β-arrestin, where it dephosphorylates components of signaling cascade.105 In another case, protein tyrosine phosphatases have been implicated in regulating dynamin-dependent endocytosis of the renal outer medullary potassium channel 1.106 A more focused effort toward identifying and characterizing protein phosphatases that contribute to endocytic regulation will be necessary for a complete understanding of endocytosis.

Membrane curvature

ENTH Domains

Once nucleation has occurred and the clathrin machinery begins to assemble on the membrane, it needs to carry out one of its major functions, namely to generate highly curved membrane vesicles from essentially flat bilayers. It has long been known that purified clathrin triskelia, under appropriate non-physiological buffer conditions, spontaneously assemble in vitro into clathrin cages with a diameter similar to CCVs seen in cells. This led to models in which the assembly of clathrin was sufficient to drive membrane budding and curvature. However, other components of the clathrin machinery are needed to promote clathrin assembly under physiological conditions. It has also become apparent that the generation of membrane curvature involves multiple proteins acting at the protein-lipid interface.107

One recurring theme is that proteins use amphipathic helices to promote membrane curvature. Insertion of the helices into the cytosolic leaflet displaces lipid headgroups and creates an asymmetry between the leaflets of the bilayer. The bilayer couple hypothesis proposes that such leaflet asymmetry causes spontaneous membrane curvature toward the cytosolic side.108 Proteins bearing epsin N-terminal homology (ENTH) domains provide one example of this mechanism. ENTH domains were originally identified by sequence alignment, which revealed that the N-terminus of epsin contained an ˜150 residue region conserved in proteins from a diverse range of species,109,110 while the more divergent C-terminal region contained motifs that bind to clathrin and AP-2.56,64 The ENTH domain of epsin has a compact globular structure composed of 8 α-helices111 and binds to PtdIns(4,5)P2 through basic residues in helices 1, 3 and 4 and the loop between helix 1 and 2.112,113 Upon PtdIns(4,5)P2 binding, unstructured residues at the N-terminus of the ENTH domain form a new α-helix that has a series of hydrophobic residues on its outer surface.114 The new helix inserts into PtdIns(4,5)P2-containing monolayers, which can be measured based on changes in surface pressure.115 Mutation of key hydrophobic residues in the helix prevents both membrane insertion and the development of curvature, even though a flat clathrin lattice forms on the membrane.114,115 This suggests that clathrin assembly is not the primary mechanism for curvature generation but may instead form a mold with which the deformed membrane can be structured into a highly curved vesicle. However, clathrin triskelia have been found to exchange between the membrane and the cytosol;116 if this exchange can be coupled with transitions of the membrane pool from flat hexagonal lattices to rounded hexagon/pentagon lattices, then an alternate explanation may hold. This alternate proposal is that thermal fluctuations may account for the initial membrane curvature, which in turn is stabilized by clathrin assembly around the curved membrane.117

In addition to epsin, which functions in CCV formation at the plasma membrane, enthoprotin/CLINT/epsinR contains an ENTH domain, binds to clathrin adaptor protein 1 (AP-1) and functions in clathrin trafficking at the TGN and endosomes.77,118-122 Therefore, ENTH domain-based amphipathic helix-mediated membrane deformation may be a widely utilized phenomenon occurring on multiple cellular membranes. The ENTH-domain mechanism of lipid binding and bending has been contrasted to the AP180 N-terminal homology (ANTH)-domain mechanism of lipid binding mediated by AP180/CALM proteins. ANTH domains bind phosphoinositides via a basic patch on the surface of the folded structure, have a lower affinity for the membrane, and lack the membrane bending effect seen with ENTH domains.112,115

Curvature of COPII Vesicles

Similar principles regarding the membrane insertion of amphipathic helices operate in the formation of COPII-coated vesicles at the endoplasmic reticulum (ER).123,124 Sar1p is a small GTPase that guides the recruitment of the COPII adaptor and coat proteins, Sec23/24p and Sec13/31p onto ER membranes during COPII vesicle formation. Exchange of GDP for GTP allows Sar1p-GTP to translocate from the cytosol to the ER membrane and also leads to the disruption of a hydrophobic pocket normally occupied by an N-terminal amphipathic helix, resulting in membrane insertion of the helix.125 In vitro, the helix can deform synthetic liposomes into narrow tubules; in cells, mutation of hydrophobic residues in the helix yields Sar1p mutants that are unable to form highly curved membranes despite normal recruitment of coat proteins.123,124 Interestingly, the Sec23/24p dimer forms a structure with a concave surface enriched in basic residues and with a shape consistent with the curvature of a COPII vesicle.126 It is an appealing hypothesis that membrane deformation induced by Sar1p could be stabilized or promoted by electrostatic interactions between the bilayer and Sec23/24p.

BAR and N-BAR Domains

Another protein module participating in the formation of membrane curvature is the BAR (BIN1/amphiphysin/Rvs167p) domain. The acronym originated based on the similarity between the N-terminal regions of amphiphysin I and II, nerve terminal-enriched proteins participating in CME; BIN1, an amphiphysin II splice variant; and Rvs167p, a yeast amphiphysin homologue.127-129 The function of the BAR domain was first suggested by the observation that the N-terminus of amphiphysin I, which mediates amphiphysin I homodimerization as well as heterodimerization with amphiphysin II,130,131 could bind and tubulate liposomes.132 Determination of the crystal structure of the BAR domain from Drosophila amphiphysin revealed a dimer in which each monomer is composed of three long, kinked α-helices forming a six helix bundle around the dimer interface.133 The overall structure of the dimer is an elongated crescent with extensive positive charge along a concave surface. This allows the BAR domain to form electrostatic interactions with negatively charged membranes such that they can sense curvature. Thus, BAR domain proteins have been proposed to act as molecular switches that induce protein recruitment or activity after a certain degree of membrane curvature is achieved during vesicle formation.107 Moreover, it is possible that BAR domains may in fact contribute to the induction of curvature by forcing membranes to conform to the concave face of the module.107

In some cases, BAR domains are found in combination with an N-terminal amphipathic helix (N-BAR domains). One such N-BAR domain protein is endophilin, which was originally identified and implicated in CME based on its interaction with dynamin and synaptojanin.134,135 The crystal structure of the N-BAR domain of a brain-enriched form of endophilin, endophilin A1 has recently been solved.136-138 The structure is similar to that of amphiphysin with the exception of the N-terminal helix and the presence of two largely disordered 30-residue domains inserted into helix 1 and projecting from the concave face of the dimer. The N-terminal helix, which was previously demonstrated to be important for liposome binding and tubulation,139 folds upon membrane binding and is peripherally bound in the plane of the membrane at the phosphate level of the headgroups, ideally situated to effect membrane curvature similarly to ENTH domains.136,137 The insert at the membrane interface forms a ridge that penetrates into the bilayer, further enhancing liposome tubulation.136,137 Thus, similar to Sar1/Sec23/Sec24p, N-BAR domains appear capable of both inducing and stabilizing membrane curvature. BAR and N-BAR domains are now recognized in a variety of proteins, many of which participate in some way in membrane remodeling. It is worth noting that the structure of the endophilin N-BAR domain is not consistent with a proposed lysophosphatidic acyltransferase (LPAAT) activity that has been ascribed to this protein and in fact, recent studies have eliminated the notion of an LPAAT activity in endophilin.140

F-BAR Domains, Coupling Actin to Endocytosis

The FCH (Fes/CIP4 homology) domain is a protein module found in a wide variety of proteins, many of which have been linked through genetics or biochemical studies to the regulation of the actin cytoskeleton ( see refs. 141,142 and refs. therein). One FCH domain-bearing protein is syndapin/PACSIN, which functions in CME via interactions with dynamin and synaptojanin and also contributes to regulation of actin via interactions with N-WASP, a key regulator of actin assembly.143,144 A careful reevaluation of many FCH domain proteins has revealed an additional C-terminal coiled-coil domain that, when considered as part of the FCH domain, predicts a larger protein module that is homologous throughout its length with BAR domains.141,142 Moreover, like the BAR domain, the extended module is predicted to have three distinct α-helices. This module has been named the extended FC (EFC) domain and alternatively, the FCH and BAR (F-BAR) domain. EFC/F-BAR domains bind to lipids; their addition to liposomes causes tubulation much like that seen with BAR domains, with the exception that the tubules formed in the presence of EFC/F-BAR domains are larger in diameter. Binding to both phosphatidylserine and PtdIns(4,5)P2 has been reported.141,142 Structural analysis of the EFC/F-BAR domain should reveal much information about its lipid specificity, relationship to the BAR domain, and its mechanism of action.

In addition to causing membrane tubulation in vitro, EFC/F-BAR domains also induce tubular invaginations of the plasma membrane when expressed in cells.141,142 Interestingly, this phenomenon is enhanced by disruption of the actin cytoskeleton. Many of the known EFC/ F-BAR proteins bind to N-WASP (e.g., syndapin/PACSIN), which could contribute to N-WASP recruitment to the plasma membrane. N-WASP activates actin assembly, which is known to cooperate with dynamin to drive vesicle fission from the membrane (see below). Disruption of actin assembly could give EFC/F-BAR domains more time to cause membrane tubules to form before they are pinched off the membrane. Moreover, dynamin itself is another major binding partner for several EFC/F-BAR, BAR and N-BAR domain proteins, and dynamin overexpression antagonizes tubule formation.141,142 Thus, budding of CCVs can be initiated and sustained by protein modules such as ENTH, BAR, N-BAR and F-BAR/EFC domains. Proteins bearing these modules can also both recruit dynamin and recruit directly or indirectly proteins that stimulate actin assembly. Dynamin and actin cooperate to drive membrane fission. By the formation of such protein networks that initiate, stabilize and resolve membrane curvaturedependent processes, early events and late events in the formation of vesicles can be cooperatively linked.

Scission

After clathrin and AP-2, dynamin was one of the first proteins found to participate in clathrin-mediated membrane budding. Dynamin contains an N-terminal GTPase domain, a PH domain that binds predominantly to PtdIns(4,5)P2,145 a GTPase effector domain (GED) that acts as an intramolecular GAP,146 and a proline-rich domain (PRD) that binds to a large number of SH3 domain-bearing proteins.147 Its role in CME was first suggested by its identification as the product of the shibire gene in Drosophila.148 Conditional mutations in shibire cause rapid paralysis with the appearance of endocytic vesicles that remain attached to the plasma membrane by narrow membrane stalks, each decorated with an electron dense collar. 149 It was subsequently demonstrated that purified dynamin oligomerizes to form rings with an inner diameter similar to the diameter of the membrane stalks seen in shibire flies.150 Moreover, in synaptosomes (pinched off and purified presynaptic nerve terminals), the addition of GTPγS, which locks dynamin into a GTP-bound state, led to the presence of clathrin-coated structures that remained attached to the plasma membrane, often with highly extended membrane necks.151 The necks were coated with dynamin, which appeared to wrap around the neck in a spiral. Together, these data led to the hypothesis that dynamin functions in vesicle fission as a mechanochemical enzyme that utilizes GTP hydrolysis to provide a twisting force that severs the membrane tube.

Various models have been subsequently proposed for dynamin function. When PtdIns(4,5)P2-containing lipid nanotubes were used as a substrate for dynamin self-assembly, the GTP-bound protein was seen to form tightly packed dynamin rings.152 Upon GTP hydrolysis, the spacing between the rings was increased suggesting that a lengthwise conformational change (spring-like) could allow for vesicle fission.152 Thus, dynamin would function as a mechanochemical enzyme but in a unique conformational manner. An alternative model suggested that dynamin functions analogously to other GTPases, as a time-limited recruitment factor.146 This model came from the analysis of dynamin mutants that inhibit either dynamin self-assembly or the intramolecular GAP activity (which requires self-assembly to place the GED domain of one member of a dynamin oligomer in proximity to the GTPase domain of another). In both cases, dynamin remains in an active, GTP bound state and this was in fact found to stimulate CME, suggesting that GTP-bound dynamin recruits in effectors required for fission.146 Thus, the exact mechanism by which dynamin mediates membrane fission has remained elusive. However, a recent paper has provided new evidence for the old model, that GTP hydrolysis-dependent twisting of dynamin provides constriction and tension in membrane fission.153 When observed in real time in vitro, it was seen that addition of GTP, but not GDP or GTPγS to dynamin-coated lipid tubules resulted in twisting of the tubules and supercoiling. This twisting motion was confirmed using streptavidin beads conjugated to biotinylated dynamin, in which the beads were seen to swing around the tubules. This swinging/twisting action created a longitudinal tension that was released when the tubules underwent fission. Thus, dynamin appears most likely to function, at least in part, as a mechanochemical GTPase using GTP-dependent twisting forces coupled with tension to allow for membrane fission late in vesicle budding.

Uncoating

Once the CCVs have been liberated from the membrane by dynamin-dependent scission, the vesicles uncoat before transport to subsequent stations in the endocytic pathway. Since clathrin assembly can proceed spontaneously, an energy-dependent step is necessary to dissociate the clathrin cage. In fact, in vitro uncoating assays have demonstrated that the heat shock cognate 70 (Hsc70), an ATPase, is a critical factor in CCV uncoating.154 A key cofactor in the uncoating reaction is auxilin, which comes in two forms. Auxilin 1 is brain specific and contains an N-terminal region with sequence similarity to the phosphatase and C2 domains of PTEN (a PtdIns-3-phosphatase), a central region with motifs for binding to clathrin and AP-2, and a C-terminal DNAJ domain. Auxilin 2 (also known as cyclin G-associated kinase, GAK) has a ubiquitous tissue distribution and a similar domain structure but with an additional N-terminal ser/thr kinase domain. Auxilins bind directly clathrin and AP-2, and through the DNAJ domain, they also bind directly to Hsc70, thus recruiting the ATP-bound ATPase to CCVs. Since partially assembled lattices should be able to bind auxilins, an important question is what prevents Hsc70 from mediating premature uncoating. For example, auxilin may only be recruited at specific stages of the CCV life cycle or the ATPase activity of Hsc70 may be regulated to only activate uncoating at specific times. Two recent studies have examined the recruitment of auxilin over the life cycle of CCVs.155,156 Interestingly, they found that while auxilin is present at low levels during CCV formation, there is a major burst of recruitment following the peak of dynamin recruitment late in the CCV life cycle. Using a combination of epifluorescence and evanescent field microscopy, the investigators determined that auxilin and clathrin first leave the evanescent field as the vesicles move from the plasma membrane and then disappear from the epifluorescence field as the CCVs uncoat. Interestingly, the late recruitment of auxilin is dependent on the PTEN-like domain, which binds to specific inostiol phospholipids, most notable, PtdIns(3)P.155,156 Thus, a switch in inositol phospholipid levels late in CCV formation may act as a trigger to recruit auxilin/Hsc70 to mediate CCV uncoating. Consistent with such a model, knock out of synaptojanin leads to an accumulation of coated CCVs,43 suggesting that this lipid phosphatase is a critical switch for uncoating.

Interestingly, a recent study in neurons has revealed a potentially novel mechanism for the recruitment of Hsc70 in the uncoating of SVs. The adhesion molecule, CHL1, a member of the immunoglobulin superfamily was found to bind to Hsc70 in nerve terminals157 CHL1 was found to be targeted to SVs by endocytosis; deficiency of CHL1 leads to abnormally high numbers of CCVs and the inability to release clathrin. Thus, CHL1 may represent an additional mechanism to recruit Hsc70 for CCV uncoating during SV recycling.

Actin

One of binding partners for the PRD of dynamin is the SH3 domain protein cortactin.158 Cortactin also binds to actin and activates the Arp2/3 complex, which stimulates the formation of branched filamentous actin. Interestingly, live cell imaging has revealed that late stages in the invagination of CCPs are accompanied by recruitment of dynamin and a burst of actin assembly at CCPs.159 A more recent study has in fact revealed that cortactin recruitment and actin assembly coincides precisely with the fission reaction, and that blocking actin assembly with latrunculin-B inhibits vesicle scission.160 Thus, it appears that actin assembly works in cooperation with dynamin to drive the fission reaction. In addition to a role in the late stages of endocytosis, actin has also been implicated in early stages of endocytic internalization.161

Resolution of the Order of Endocytic Events in Yeast by Time-Lapse Microscopy

Two early studies pointed to roles for actin in endocytosis. An initial hint was provided at the apical domain of polarized epithelial cells, where CME was sensitive to the actin depolymerizing agent cytochalasin.162 A major role for actin as a functional participant in endocytosis was indicated by studies from the Riezman lab that found endocytosis defects in yeast with mutations in actin or in the fimbrin homologue Sac6p, a protein that bundles actin filaments.163 Further linking actin to endocytosis in yeast were the observations that most endocytosis mutants exhibited perturbations in the organization and localization of actin, and that most endocytosis proteins colocalized with actin and actin-associated proteins in small peripheral spots known as actin patches. Interestingly, careful analysis of cells double labeled for certain pairs of endocytic proteins showed that the colocalization was often not exact, but that a percentage of patches had only one of the two proteins while other patches had both proteins.164 Did this mean that yeast has multiple patches with distinct compositions for distinct purposes? Or, did the distinct compositions reflect patches at different stages of maturation? Other important questions were raised, such as what is the role for actin in yeast endocytosis, and does yeast endocytosis have any relationship to the mechanisms used by mammalian cells?

In a landmark paper by Drubin and colleagues, real-time two-color microscopy studies have begun to place the network of endocytic proteins in yeast into an ordered pathway.165 Yeast strains were created that had either GFP or CFP/RFP fused at the C-terminus of various proteins; importantly, these fusions were made at the chromosomal locus for each protein. Thus, the chimeric proteins were expressed as the sole source of the protein and at endogenous levels from the normal gene promoter. Additionally, the chimeras could be tested to ensure that the normal function is unaffected by the C-terminal GFP fusion. The initial study assessed six proteins (Las17p/WASp, Pan1p/intersectin, Sla1p, Sla2p/HIP1, Arc15p - an Arp2/3 subunit and Abp1p/mABP1), and found three characteristic dynamic behaviors: stationary, initially stationary followed by a phase of brief motility, and a brief motility phase followed by a more extensive motility phase.165 These behaviors were modeled as corresponding to CCP formation, CCP invagination, and CCP scission to release a CCV. A subsequent study examined the dynamic behavior of about sixty proteins implicated in endocytosis or actin dynamics relative to the initial set that was observed; this showed that some proteins exhibited yet other behaviors, primarily around the transitions between the brief motility (invagination) and the extensive motility phase (scission).74 These studies have had two major impacts: first, the network of protein interactions can now be understood in the context of the sequence of events that underlies endocytic internalization; second, a more restricted subset of potential functions for each protein can be predicted based on its dynamic behavior.

Studies in yeast have also highlighted the importance of the Arp2/3 complex in endocytic internalization. Arp2/3 subunits localize at actin patches, and mutation of these subunits leads to endocytosis defects (e.g., see ref. 166). The seven-subunit Arp2/3 complex requires an activating factor for it to efficiently and maximally stimulate the polymerization of actin monomers into filaments, although a recent in vitro study found that Arp2/3 could partially stimulate polymerization of yeast actin in the absence of activators.167 All five characterized Arp2/3 activators in yeast are implicated in endocytosis: Las17p (WASp homologue), Pan1p (intersectin homologue), Myo3p and Myo5p (type I myosins) and Abp1p (mABP1 homologue) (reviewed in ref. 168). Interestingly, these proteins evidently have functions at each stage of internalization. Las17p is an early acting factor; unlike its homologue WASp which requires a combination of Cdc42-GTP and PtdIns(4,5)P2 to activate Arp2/3, Las17p instead appears to be constitutively active and is regulated by transient association with the inhibitor proteins Sla1p and Bbc1p, via SH3 domains found in both regulatory partners.169 The Arp2/3 activation properties of Pan1p may be important at the transition to invagination or scission, and also requires Pan1p binding to F-actin.170 The type I myosins, which have both motor domains and Arp2/ 3 activation domains, are also important at the transition to invagination or scission.171 Recent work suggests that the Myo5 Arp2/3 activation domain helps produce actin filaments that the Myo5 motor domain then moves perpendicular to the plasma membrane in the direction of the budding vesicle.172 The analogy of this mechanism to mammalian endocytosis may not be perfectly parallel, as only fungal type I myosins have Arp2/3 stimulatory activity in addition to motor activity. Finally, Abp1p acts at the latest stages of internalization.165 While Abp1p was initially characterized as a weak Arp2/3 stimulator, more recent data suggests that it may be a master inhibitor of endocytosis that shuts off the process by inhibiting Arp2/3 activity and by recruiting negative regulatory factors.172,173 For example, Abp1p binds to both Prk1p and Ark1p, the protein kinases that phosphorylate many endocytic machinery components, and to the synaptojanin-related inositol phosphatase Inp52p.83,84

For actin polymerization to provide a driving force for vesicle formation, it must be connected to the nascent endocytic vesicle to transmit the forces. A candidate to fulfill this connecting function is one of the first endocytosis proteins isolated in yeast, the Sla2/End4p protein. 174,175 The yeast Sla2p protein is homologous to the mammalian HIP1R (Huntingtin-interacting protein-1 related) protein.176 Both proteins bind PtdIns(4,5)P2, F-actin, clathrin light chain and other components of the endocytic vesicle, and thus have the properties to suggest they could link polymerizing F-actin to the vesicle membrane or coat.59,176,177 Consistent with acting as F-actin/vesicle connecting proteins, cells lacking Sla2p or HIP1R have uncontrolled actin polymerization at nascent endocytic sites with no corresponding vesicle invagination.177,178

There are several fundamental differences in the machinery necessary for endocytosis in yeast versus animal cells. Most notably is the relatively greater importance of actin in supporting endocytosis in yeast. Conversely, several key factors that are critical for endocytosis in animal cells are apparently less important in yeast. For instance, while yeast has an AP-2 complex that localizes to the plasma membrane, there are no known endocytic cargos that require AP-2 for uptake, and no known phenotypes of an AP-2 deletion yeast cell.179-182 Likewise, Vps1p, the closest yeast homologue to dynamin, has only thus far been implicated in trafficking at the Golgi.183 However, Vps1p has been found to associate with the endocytic protein Sla1p and to affect the organization of the cortical actin patches at the plasma membrane, suggesting the potential for Vps1p playing at least a supporting role for endocytosis in yeast.184

Role of Actin in Mammalian Systems

The first indication of a role for actin in endocytosis in animal cells was discovered for endocytosis from the apical side of polarized epithelial cells, which was sensitive to the commonly used actin inhibitor cytochalasin.162 More recently, new methods of observing endocytosis, and the use of more potent inhibitors such as the latrunculins have revealed a more widespread sensitivity of endocytosis in mammalian cells to these inhibitors.159,160,185,186 However, this is still not as clear-cut as in yeast cells, as it depends on the cell type, the inhibitor being used, the cargo being monitored, and whether the dorsal vs. ventral side of the cell is being observed.

Many homologous actin-binding and actin-polymerizing factors involved in endocytosis in yeast and mammalian cells are known (reviewed in refs. 71,168). The established and proposed functions for these factors have been extensively reviewed recently,168,187,188 and thus the reader is referred to these reviews for a comprehensive treatment of this topic. To highlight an example of an actin-associated endocytic protein specific to animal cells, the Type VI myosin is proposed to help mediate the movements of newly formed clathrin-coated vesicles as they leave the peri-cortical actin filament-rich region.189,190 Localized vesicle-associated actin polymerization is also suggested to provide forces for propulsion of vesicles (reviewed in ref. 191). It seems that there are many potential ways in which actin filaments can be used to generate forces to support varied aspects of membrane trafficking and protein sorting. Thus, while there are some differences in the details of endocytic mechanisms in yeast and animal cells, there nonetheless are many shared features, and perhaps more to be discovered in the future.

Major Unresolved Questions

In this rapidly moving field, in spite of the amazing progress that has been made in the past decade, many important fundamental questions remain, and new ones have emerged. The key question of what determines when and where an endocytic pit will form is still open; it seems likely that the answer will involve an integration of exocytosis, signaling events, and cell polarity cues. Likewise, uncovering the mechanistic roles that actin plays both at multiple stages of internalization and in post-internalization CCV fate will be difficult to untangle, but will be rewarded by revealing functions for components of the endocytic machinery. In addition, the idea that cells regulate overall endocytic flux by balancing the flow through clathrin-dependent and clathrin-independent pathways has been suggested for many years; the intriguing findings stemming from the kinome analysis will be an excellent starting point to define the mechanisms by which such regulation could be mediated. Another long-term goal for the future will be to determine the temporal and spatial relationship between all of the protein components of the complex web of proteins involved in clathrin-mediated membrane budding. In this regard, important strides are being made in live cell imaging using different combinations of proteins tagged with fluorescent proteins. Finally, a key goal will be the in vitro reconstitution of CCV formation using synthetic lipid membranes and purified proteins. This will be an extreme challenge given the complex nature of the protein machinery and the lipid regulation. The dizzying rate of new discoveries in the endocytosis field in the past few years suggests that exciting solutions to these enigmas will be forthcoming.

Acknowledgements

PSM acknowledges support from the Canadian Institutes of Health Research (CIHR). BR is supported by a CIHR fellowship. PSM is a Fonds de la recherché en santé Québec Senior Scholar and holds the James McGill Chair of McGill University. BW acknowledges support from the National Institutes of Health and The Johns Hopkins University, and thanks Lymarie Maldonado-Báez for helpful comments.

References

1.
Conner SD, Schmid SL. Regulated portals of entry into the cell. Nature. 2003;422(6927):37–44. [PubMed: 12621426]
2.
Brodsky FM. et al. Biological basket weaving: Formation and function of clathrin-coated vesicles. Annu Rev Cell Dev Biol. 2001;17:517–568. [PubMed: 11687498]
3.
McPherson PS, Kay BK, Hussain NK. Signaling on the endocytic pathway. Traffic. 2001;2(6):375–384. [PubMed: 11389765]
4.
Rust MJ. et al. Assembly of endocytic machinery around individual influenza viruses during viral entry. Nat Struct Mol Biol. 2004;11(6):567–573. [PMC free article: PMC2748740] [PubMed: 15122347]
5.
Sandvig K, van Deurs B. Endocytosis, intracellular transport, and cytotoxic action of Shiga toxin and ricin. Physiol Rev. 1996;76(4):949–966. [PubMed: 8874490]
6.
Robinson MS. Adaptable adaptors for coated vesicles. Trends Cell Biol. 2004;14(4):167–174. [PubMed: 15066634]
7.
Griffiths G. et al. The mannose 6-phosphate receptor and the biogenesis of lysosomes. Cell. 1988;52(3):329–341. [PubMed: 2964276]
8.
Ludwig T, Le Borgne R, Hoflack B. Roles for mannose-6-phosphate receptors in lysosomal enzyme sorting, IGF-II binding and clathrin-coat assembly. Trends Cell Biol. 1995;5(5):202–206. [PubMed: 14731450]
9.
Meyer C. et al. mu1A-adaptin-deficient mice: Lethality, loss of AP-1 binding and rerouting of mannose 6-phosphate receptors. Embo J. 2000;19(10):2193–2203. [PMC free article: PMC384363] [PubMed: 10811610]
10.
Hinners I, Tooze SA. Changing directions: Clathrin-mediated transport between the Golgi and endosomes. J Cell Sci. 2003;116(Pt 5):763–771. [PubMed: 12571274]
11.
Austin C, Hinners I, Tooze SA. Direct and GTP-dependent interaction of ADP-ribosylation factor 1 with clathrin adaptor protein AP-1 on immature secretory granules. J Biol Chem. 2000;275(29):21862–21869. [PubMed: 10807927]
12.
Pearse BM, Bretscher MS. Membrane recycling by coated vesicles. Annu Rev Biochem. 1981;50:85–101. [PubMed: 7023370]
13.
Willig KI. et al. STED microscopy reveals that synaptotagmin remains clustered after synaptic vesicle exocytosis. Nature. 2006;440(7086):935–939. [PubMed: 16612384]
14.
Fernandez-Alfonso T, Kwan R, Ryan TA. Synaptic vesicles interchange their membrane proteins with a large surface reservoir during recycling. Neuron. 2006;51(2):179–186. [PubMed: 16846853]
15.
Murthy VN, De Camilli P. Cell biology of the presynaptic terminal. Annu Rev Neurosci. 2003;26:701–728. [PubMed: 14527272]
16.
McNiven MA, Thompson HM. Vesicle formation at the plasma membrane and trans-Golgi network: The same but different. Science. 2006;313(5793):1591–1594. [PubMed: 16973870]
17.
Wakeham DE. et al. Clathrin self-assembly involves coordinated weak interactions favorable for cellular regulation. Embo J. 2003;22(19):4980–4990. [PMC free article: PMC204494] [PubMed: 14517237]
18.
Fotin A. et al. Molecular model for a complete clathrin lattice from electron cryomicroscopy. Nature. 2004;432(7017):573–579. [PubMed: 15502812]
19.
Fotin A. et al. Structure of an auxilin-bound clathrin coat and its implications for the mechanism of uncoating. Nature. 2004;432(7017):649–653. [PubMed: 15502813]
20.
Blondeau F. et al. Tandem MS analysis of brain clathrin-coated vesicles reveals their critical involvement in synaptic vesicle recycling. Proc Natl Acad Sci USA. 2004;101(11):3833–3838. [PMC free article: PMC374330] [PubMed: 15007177]
21.
Kirchhausen T, Harrison SC. Protein organization in clathrin trimers. Cell. 1981;23(3):755–761. [PubMed: 7226229]
22.
Ungewickell E, Branton D. Assembly units of clathrin coats. Nature. 1981;289(5796):420–422. [PubMed: 7464911]
23.
Ungewickell E, Ungewickell H. Bovine brain clathrin light chains impede heavy chain assembly in vitro. J Biol Chem. 1991;266(19):12710–12714. [PubMed: 2061336]
24.
Girard M. et al. Non-stoichiometric relationship between clathrin heavy and light chains revealed by quantitative comparative proteomics of clathrin-coated vesicles from brain and liver. Mol Cell Proteomics. 2005;4(8):1145–1154. [PubMed: 15933375]
25.
Traub LM. Sorting it out: AP-2 and alternate clathrin adaptors in endocytic cargo selection. J Cell Biol. 2003;163(2):203–208. [PMC free article: PMC2173531] [PubMed: 14581447]
26.
Ritter B. et al. Molecular mechanisms in clathrin-mediated membrane budding revealed through subcellular proteomics. Biochem Soc Trans. 2004;32(Pt 5):769–773. [PubMed: 15494011]
27.
Owen DJ, Collins BM, Evans PR. Adaptors for clathrin coats: Structure and function. Annu Rev Cell Dev Biol. 2004;20:153–191. [PubMed: 15473838]
28.
Owen DJ. et al. The structure and function of the beta 2-adaptin appendage domain. Embo J. 2000;19(16):4216–4227. [PMC free article: PMC302036] [PubMed: 10944104]
29.
ter Haar E, Harrison SC, Kirchhausen T. Peptide-in-groove interactions link target proteins to the beta-propeller of clathrin. Proc Natl Acad Sci USA. 2000;97(3):1096–1100. [PMC free article: PMC15533] [PubMed: 10655490]
30.
Schmid EM. et al. Role of the AP2 beta-appendage hub in recruiting partners for clathrin-coated vesicle assembly. PLoS Biol. 2006;4(9):e262. [PMC free article: PMC1540706] [PubMed: 16903783]
31.
Edeling MA. et al. Molecular switches involving the AP-2 beta2 appendage regulate endocytic cargo selection and clathrin coat assembly. Dev Cell. 2006;10(3):329–342. [PubMed: 16516836]
32.
Knuehl C. et al. Novel binding sites on clathrin and adaptors regulate distinct aspects of coat assembly. Traffic. 2006;7(12):1688–1700. [PubMed: 17052248]
33.
Hinrichsen L. et al. Effect of clathrin heavy chain- and alpha-adaptin-specific small inhibitory RNAs on endocytic accessory proteins and receptor trafficking in HeLa cells. J Biol Chem. 2003;278(46):45160–45170. [PubMed: 12960147]
34.
Motley A. et al. Clathrin-mediated endocytosis in AP-2-depleted cells. J Cell Biol. 2003;162(5):909–918. [PMC free article: PMC2172830] [PubMed: 12952941]
35.
Bonifacino JS, Traub LM. Signals for sorting of transmembrane proteins to endosomes and lysosomes. Annu Rev Biochem. 2003;72:395–447. [PubMed: 12651740]
36.
Santini F, Keen JH. Endocytosis of activated receptors and clathrin-coated pit formation: Deciphering the chicken or egg relationship. J Cell Biol. 1996;132(6):1025–1036. [PMC free article: PMC2120763] [PubMed: 8601582]
37.
Collins BM. et al. Molecular architecture and functional model of the endocytic AP2 complex. Cell. 2002;109(4):523–535. [PubMed: 12086608]
38.
Gaidarov I, Keen JH. Phosphoinositide-AP-2 interactions required for targeting to plasma membrane clathrin-coated pits. J Cell Biol. 1999;146(4):755–764. [PMC free article: PMC2156139] [PubMed: 10459011]
39.
Rohde G, Wenzel D, Haucke V. A phosphatidylinositol (4,5)-bisphosphate binding site within mu2-adaptin regulates clathrin-mediated endocytosis. J Cell Biol. 2002;158(2):209–214. [PMC free article: PMC2173125] [PubMed: 12119359]
40.
Jost M. et al. Phosphatidylinositol-4,5-bisphosphate is required for endocytic coated vesicle formation. Curr Biol. 1998;8(25):1399–1402. [PubMed: 9889104]
41.
Padron D. et al. Phosphatidylinositol phosphate 5-kinase Ibeta recruits AP-2 to the plasma membrane and regulates rates of constitutive endocytosis. J Cell Biol. 2003;162(4):693–701. [PMC free article: PMC2173809] [PubMed: 12913109]
42.
McPherson PS. et al. A presynaptic inositol-5-phosphatase. Nature. 1996;379(6563):353–357. [PubMed: 8552192]
43.
Cremona O. et al. Essential role of phosphoinositide metabolism in synaptic vesicle recycling. Cell. 1999;99(2):179–188. [PubMed: 10535736]
44.
Zoncu R. et al. Loss of endocytic clathrin-coated pits upon acute depletion of phosphatidylinositol 4,5-bisphosphate. Proc Natl Acad Sci USA. 2007;104(10):3793–3798. [PMC free article: PMC1805489] [PubMed: 17360432]
45.
Godi A. et al. ARF mediates recruitment of PtdIns-4-OH kinase-beta and stimulates synthesis of PtdIns(4,5)P2 on the Golgi complex. Nat Cell Biol. 1999;1(5):280–287. [PubMed: 10559940]
46.
Krauss M. et al. ARF6 stimulates clathrin/AP-2 recruitment to synaptic membranes by activating phosphatidylinositol phosphate kinase type Igamma. J Cell Biol. 2003;162(1):113–124. [PMC free article: PMC2172713] [PubMed: 12847086]
47.
Motley AM. et al. Functional analysis of AP-2 alpha and mu2 subunits. Mol Biol Cell. 2006;17(12):5298–5308. [PMC free article: PMC1679692] [PubMed: 17035630]
48.
Honing S. et al. Phosphatidylinositol-(4,5)-bisphosphate regulates sorting signal recognition by the clathrin-associated adaptor complex AP2. Mol Cell. 2005;18(5):519–531. [PubMed: 15916959]
49.
McPherson PS, Ritter B. Peptide motifs: Building the clathrin machinery. Mol Neurobiol. 2005;32(1):73–87. [PubMed: 16077185]
50.
Traub LM. Common principles in clathrin-mediated sorting at the Golgi and the plasma membrane. Biochim Biophys Acta. 2005;1744(3):415–437. [PubMed: 15922462]
51.
Ehrlich M. et al. Endocytosis by random initiation and stabilization of clathrin-coated pits. Cell. 2004;118(5):591–605. [PubMed: 15339664]
52.
Owen DJ, Evans PR. A structural explanation for the recognition of tyrosine-based endocytotic signals. Science. 1998;282(5392):1327–1332. [PubMed: 9812899]
53.
Eden ER. et al. Use of homozygosity mapping to identify a region on chromosome 1 bearing a defective gene that causes autosomal recessive homozygous hypercholesterolemia in two unrelated families. Am J Hum Genet. 2001;68(3):653–660. [PMC free article: PMC1274478] [PubMed: 11179013]
54.
Eden ER. et al. Restoration of LDL receptor function in cells from patients with autosomal recessive hypercholesterolemia by retroviral expression of ARH1. J Clin Invest. 2002;110(11):1695–1702. [PMC free article: PMC151635] [PubMed: 12464675]
55.
Garcia CK. et al. Autosomal recessive hypercholesterolemia caused by mutations in a putative LDL receptor adaptor protein. Science. 2001;292(5520):1394–1398. [PubMed: 11326085]
56.
Chen H. et al. Epsin is an EH-domain-binding protein implicated in clathrin-mediated endocytosis. Nature. 1998;394(6695):793–797. [PubMed: 9723620]
57.
He G. et al. ARH is a modular adaptor protein that interacts with the LDL receptor, clathrin, and AP-2. J Biol Chem. 2002;277(46):44044–44049. [PubMed: 12221107]
58.
Laporte SA. et al. The beta2-adrenergic receptor/betaarrestin complex recruits the clathrin adaptor AP-2 during endocytosis. Proc Natl Acad Sci USA. 1999;96(7):3712–3717. [PMC free article: PMC22359] [PubMed: 10097102]
59.
Legendre-Guillemin V. et al. HIP1 and HIP12 display differential binding to F-actin, AP2, and clathrin: Identification of a novel interaction with clathrin light chain. J Biol Chem. 2002;277(22):19897–19904. [PubMed: 11889126]
60.
Metzler M. et al. HIP1 functions in clathrin-mediated endocytosis through binding to clathrin and adaptor protein 2. J Biol Chem. 2001;276(42):39271–39276. [PubMed: 11517213]
61.
Morris SM, Cooper JA. Disabled-2 colocalizes with the LDLR in clathrin-coated pits and interacts with AP-2. Traffic. 2001;2(2):111–123. [PubMed: 11247302]
62.
Roos J, Kelly RB. Dap160, a neural-specific Eps15 homology and multiple SH3 domain-containing protein that interacts with Drosophila dynamin. J Biol Chem. 1998;273(30):19108–19119. [PubMed: 9668096]
63.
Yamabhai M. et al. Intersectin, a novel adaptor protein with two Eps15 homology and five Src homology 3 domains. J Biol Chem. 1998;273(47):31401–31407. [PubMed: 9813051]
64.
Hussain NK. et al. Splice variants of intersectin are components of the endocytic machinery in neurons and non-neuronal cells. J Biol Chem. 1999;274(22):15671–15677. [PubMed: 10336464]
65.
Gonzalez-Gaitan M, Jackle H. Role of Drosophila alpha-adaptin in presynaptic vesicle recycling. Cell. 1997;88(6):767–776. [PubMed: 9118220]
66.
Koh TW, Verstreken P, Bellen HJ. Dap160/intersectin acts as a stabilizing scaffold required for synaptic development and vesicle endocytosis. Neuron. 2004;43(2):193–205. [PubMed: 15260956]
67.
Marie B. et al. Dap160/intersectin scaffolds the periactive zone to achieve high-fidelity endocytosis and normal synaptic growth. Neuron. 2004;43(2):207–219. [PubMed: 15260957]
68.
Hussain NK. et al. Endocytic protein intersectin-l regulates actin assembly via Cdc42 and N-WASP. Nat Cell Biol. 2001;3(10):927–932. [PubMed: 11584276]
69.
Karnoub AE. et al. Molecular basis for Rac1 recognition by guanine nucleotide exchange factors. Nat Struct Biol. 2001;8(12):1037–1041. [PubMed: 11685227]
70.
Zamanian JL, Kelly RB. Intersectin 1L guanine nucleotide exchange activity is regulated by adjacent src homology 3 domains that are also involved in endocytosis. Mol Biol Cell. 2003;14(4):1624–1637. [PMC free article: PMC153127] [PubMed: 12686614]
71.
Engqvist-Goldstein AE, Drubin DG. Actin assembly and endocytosis: From yeast to mammals. Annu Rev Cell Dev Biol. 2003;19:287–332. [PubMed: 14570572]
72.
Miliaras NB, Wendland B. EH Proteins: Multivalent regulators of endocytosis (and other pathways) Cell Biochem Biophys. 2004;41(2):295–318. [PubMed: 15475615]
73.
Duncan MC. et al. Yeast Eps15-like endocytic protein, Pan1p, activates the Arp2/3 complex. Nat Cell Biol. 2001;3(7):687–690. [PubMed: 11433303]
74.
Kaksonen M, Toret CP, Drubin DG. A modular design for the clathrin- and actin-mediated endocytosis machinery. Cell. 2005;123(2):305–320. [PubMed: 16239147]
75.
Gaidarov I. et al. Spatial control of coated-pit dynamics in living cells. Nat Cell Biol. 1999;1(1):1–7. [PubMed: 10559856]
76.
Krauss M. et al. Stimulation of phosphatidylinositol kinase type I-mediated phosphatidylinositol (4,5)-bisphosphate synthesis by AP-2mu-cargo complexes. Proc Natl Acad Sci USA. 2006;103(32):11934–11939. [PMC free article: PMC1567676] [PubMed: 16880396]
77.
Wasiak S. et al. Enthoprotin: A novel clathrin-associated protein identified through subcellular proteomics. J Cell Biol. 2002;158(5):855–862. [PMC free article: PMC2173151] [PubMed: 12213833]
78.
Conner SD, Schroter T, Schmid SL. AAK1-mediated micro2 phosphorylation is stimulated by assembled clathrin. Traffic. 2003;4(12):885–890. [PubMed: 14617351]
79.
Jackson AP. et al. Clathrin promotes incorporation of cargo into coated pits by activation of the AP2 adaptor micro2 kinase. J Cell Biol. 2003;163(2):231–236. [PMC free article: PMC2173513] [PubMed: 14581451]
80.
Huang B. et al. Identification of novel recognition motifs and regulatory targets for the yeast actin-regulating kinase Prk1p. Mol Biol Cell. 2003;14(12):4871–4884. [PMC free article: PMC284791] [PubMed: 13679512]
81.
Zeng G, Cai M. Regulation of the actin cytoskeleton organization in yeast by a novel serine/ threonine kinase Prk1p. J Cell Biol. 1999;144(1):71–82. [PMC free article: PMC2148122] [PubMed: 9885245]
82.
Cope MJ. et al. Novel protein kinases Ark1p and Prk1p associate with and regulate the cortical actin cytoskeleton in budding yeast. J Cell Biol. 1999;144(6):1203–1218. [PMC free article: PMC2150571] [PubMed: 10087264]
83.
Stefan CJ. et al. The phosphoinositide phosphatase Sjl2 is recruited to cortical actin patches in the control of vesicle formation and fission during endocytosis. Mol Cell Biol. 2005;25(8):2910–2923. [PMC free article: PMC1069591] [PubMed: 15798181]
84.
Fazi B. et al. Unusual binding properties of the SH3 domain of the yeast actin-binding protein Abp1: Structural and functional analysis. J Biol Chem. 2002;277(7):5290–5298. [PubMed: 11668184]
85.
Conner SD, Schmid SL. CVAK104 is a novel poly-L-lysine-stimulated kinase that targets the beta2-subunit of AP2. J Biol Chem. 2005;280(22):21539–21544. [PubMed: 15809293]
86.
Duwel M, Ungewickell EJ. Clathrin-dependent association of CVAK104 with endosomes and the trans-Golgi network. Mol Biol Cell. 2006;17(10):4513–4525. [PMC free article: PMC1635376] [PubMed: 16914521]
87.
Wilde A. et al. EGF receptor signaling stimulates SRC kinase phosphorylation of clathrin, influencing clathrin redistribution and EGF uptake. Cell. 1999;96(5):677–687. [PubMed: 10089883]
88.
Slepnev VI. et al. Role of phosphorylation in regulation of the assembly of endocytic coat complexes. Science. 1998;281(5378):821–824. [PubMed: 9694653]
89.
Cousin MA, Robinson PJ. The dephosphins: Dephosphorylation by calcineurin triggers synaptic vesicle endocytosis. Trends Neurosci. 2001;24(11):659–665. [PubMed: 11672811]
90.
Korolchuk VI, Banting G. CK2 and GAK/auxilin2 are major protein kinases in clathrin-coated vesicles. Traffic. 2002;3(6):428–439. [PubMed: 12010461]
91.
Korolchuk VI, Cozier G, Banting G. Regulation of CK2 activity by phosphatidylinositol phosphates. J Biol Chem. 2005;280(49):40796–40801. [PubMed: 16157582]
92.
Anggono V. et al. Syndapin I is the phosphorylation-regulated dynamin I partner in synaptic vesicle endocytosis. Nat Neurosci. 2006;9(6):752–760. [PMC free article: PMC2082060] [PubMed: 16648848]
93.
Lee SY. et al. Regulation of synaptojanin 1 by cyclin-dependent kinase 5 at synapses. Proc Natl Acad Sci USA. 2004;101(2):546–551. [PMC free article: PMC327184] [PubMed: 14704270]
94.
Tomizawa K. et al. Cophosphorylation of amphiphysin I and dynamin I by Cdk5 regulates clathrin-mediated endocytosis of synaptic vesicles. J Cell Biol. 2003;163(4):813–824. [PMC free article: PMC2173686] [PubMed: 14623869]
95.
Floyd SR. et al. Amphiphysin 1 binds the cyclin-dependent kinase (cdk) 5 regulatory subunit p35 and is phosphorylated by cdk5 and cdc2. J Biol Chem. 2001;276(11):8104–8110. [PubMed: 11113134]
96.
Friesen H. et al. Regulation of the yeast amphiphysin homologue Rvs167p by phosphorylation. Mol Biol Cell. 2003;14(7):3027–3040. [PMC free article: PMC165695] [PubMed: 12857883]
97.
Chen H. et al. The interaction of epsin and Eps15 with the clathrin adaptor AP-2 is inhibited by mitotic phosphorylation and enhanced by stimulation-dependent dephosphorylation in nerve terminals. J Biol Chem. 1999;274(6):3257–3260. [PubMed: 9920862]
98.
Murakami N. et al. Phosphorylation of amphiphysin I by minibrain kinase/dual-specificity tyrosine phosphorylation-regulated kinase, a kinase implicated in Down syndrome. J Biol Chem. 2006;281(33):23712–23724. [PubMed: 16733250]
99.
Chen-Hwang MC. et al. Dynamin is a minibrain kinase/dual specificity Yak1-related kinase 1A substrate. J Biol Chem. 2002;277(20):17597–17604. [PubMed: 11877424]
100.
Pelkmans L. et al. Genome-wide analysis of human kinases in clathrin- and caveolae/raft-mediated endocytosis. Nature. 2005;436(7047):78–86. [PubMed: 15889048]
101.
Kaneko T. et al. Rho mediates endocytosis of epidermal growth factor receptor through phosphorylation of endophilin A1 by Rho-kinase. Genes Cells. 2005;10(10):973–987. [PubMed: 16164598]
102.
Chang JS. et al. Protein phosphatase-1 binding to scd5p is important for regulation of actin organization and endocytosis in yeast. J Biol Chem. 2002;277(50):48002–48008. [PubMed: 12356757]
103.
Chang JS. et al. Cortical recruitment and nuclear-cytoplasmic shuttling of Scd5p, a protein phosphatase-1-targeting protein involved in actin organization and endocytosis. Mol Biol Cell. 2006;17(1):251–262. [PMC free article: PMC1345663] [PubMed: 16251346]
104.
Sorkin A, Von Zastrow M. Signal transduction and endocytosis: Close encounters of many kinds. Nat Rev Mol Cell Biol. 2002;3(8):600–614. [PubMed: 12154371]
105.
Willoughby EA, Collins MK. Dynamic interaction between the dual specificity phosphatase MKP7 and the JNK3 scaffold protein beta-arrestin 2. J Biol Chem. 2005;280(27):25651–25658. [PubMed: 15888437]
106.
Sterling H. et al. Inhibition of protein-tyrosine phosphatase stimulates the dynamin-dependent endocytosis of ROMK1. J Biol Chem. 2002;277(6):4317–4323. [PMC free article: PMC2822458] [PubMed: 11719519]
107.
McMahon HT, Gallop JL. Membrane curvature and mechanisms of dynamic cell membrane remodelling. Nature. 2005;438(7068):590–596. [PubMed: 16319878]
108.
Sheetz MP, Singer SJ. Biological membranes as bilayer couples: A molecular mechanism of drug-erythrocyte interactions. Proc Natl Acad Sci USA. 1974;71(11):4457–4461. [PMC free article: PMC433905] [PubMed: 4530994]
109.
Rosenthal JA. et al. The epsins define a family of proteins that interact with components of the clathrin coat and contain a new protein module. J Biol Chem. 1999;274(48):33959–33965. [PubMed: 10567358]
110.
Kay BK. et al. Identification of a novel domain shared by putative components of the endocytic and cytoskeletal machinery. Protein Sci. 1999;8(2):435–438. [PMC free article: PMC2144257] [PubMed: 10048338]
111.
Hyman J. et al. Epsin 1 undergoes nucleocytosolic shuttling and its eps15 interactor NH(2)-terminal homology (ENTH) domain, structurally similar to Armadillo and HEAT repeats, interacts with the transcription factor promyelocytic leukemia Zn(2)+ finger protein (PLZF) J Cell Biol. 2000;149(3):537–546. [PMC free article: PMC2174850] [PubMed: 10791968]
112.
Ford MG. et al. Simultaneous binding of PtdIns(4,5)P2 and clathrin by AP180 in the nucleation of clathrin lattices on membranes. Science. 2001;291(5506):1051–1055. [PubMed: 11161218]
113.
Itoh T. et al. Role of the ENTH domain in phosphatidylinositol-4,5-bisphosphate binding and endocytosis. Science. 2001;291(5506):1047–1051. [PubMed: 11161217]
114.
Ford MG. et al. Curvature of clathrin-coated pits driven by epsin. Nature. 2002;419(6905):361–366. [PubMed: 12353027]
115.
Stahelin RV. et al. Contrasting membrane interaction mechanisms of AP180 N-terminal homology (ANTH) and epsin N-terminal homology (ENTH) domains. J Biol Chem. 2003;278(31):28993–28999. [PubMed: 12740367]
116.
Yim YI. et al. Exchange of clathrin, AP2 and epsin on clathrin-coated pits in permeabilized tissue culture cells. J Cell Sci. 2005;118(Pt 11):2405–2413. [PubMed: 15923653]
117.
Hinrichsen L. et al. Bending a membrane: How clathrin affects budding. Proc Natl Acad Sci USA. 2006;103(23):8715–8720. [PMC free article: PMC1482644] [PubMed: 16735469]
118.
Duncan MC, Payne GS. ENTH/ANTH domains expand to the Golgi. Trends Cell Biol. 2003;13(5):211–215. [PubMed: 12742163]
119.
Duncan MC, Costaguta G, Payne GS. Yeast epsin-related proteins required for Golgi-endosome traffic define a gamma-adaptin ear-binding motif. Nat Cell Biol. 2003;5(1):77–81. [PubMed: 12483220]
120.
Hirst J. et al. EpsinR: An ENTH domain-containing protein that interacts with AP-1. Mol Biol Cell. 2003;14(2):625–641. [PMC free article: PMC149997] [PubMed: 12589059]
121.
Kalthoff C. et al. Clint: A novel clathrin-binding ENTH-domain protein at the Golgi. Mol Biol Cell. 2002;13(11):4060–4073. [PMC free article: PMC133614] [PubMed: 12429846]
122.
Mills IG. et al. EpsinR: An AP1/clathrin interacting protein involved in vesicle trafficking. J Cell Biol. 2003;160(2):213–222. [PMC free article: PMC2172650] [PubMed: 12538641]
123.
Bielli A. et al. Regulation of Sar1 NH2 terminus by GTP binding and hydrolysis promotes membrane deformation to control COPII vesicle fission. J Cell Biol. 2005;171(6):919–924. [PMC free article: PMC2171319] [PubMed: 16344311]
124.
Lee MC. et al. Sar1p N-terminal helix initiates membrane curvature and completes the fission of a COPII vesicle. Cell. 2005;122(4):605–617. [PubMed: 16122427]
125.
Huang M. et al. Crystal structure of Sar1-GDP at 1.7 A resolution and the role of the NH2 terminus in ER export. J Cell Biol. 2001;155(6):937–948. [PMC free article: PMC2150902] [PubMed: 11739406]
126.
Bi X, Corpina RA, Goldberg J. Structure of the Sec23/24-Sar1 prebudding complex of the COPII vesicle coat. Nature. 2002;419(6904):271–277. [PubMed: 12239560]
127.
David C, Solimena M, De Camilli P. Autoimmunity in stiff-Man syndrome with breast cancer is targeted to the C-terminal region of human amphiphysin, a protein similar to the yeast proteins, Rvs167 and Rvs161. FEBS Lett. 1994;351(1):73–79. [PubMed: 8076697]
128.
Ramjaun AR. et al. Identification and characterization of a nerve terminal-enriched amphiphysin isoform. J Biol Chem. 1997;272(26):16700–16706. [PubMed: 9195986]
129.
Sakamuro D. et al. BIN1 is a novel MYC-interacting protein with features of a tumour suppressor. Nat Genet. 1996;14(1):69–77. [PubMed: 8782822]
130.
Ramjaun AR. et al. The N terminus of amphiphysin II mediates dimerization and plasma membrane targeting. J Biol Chem. 1999;274(28):19785–19791. [PubMed: 10391921]
131.
Wigge P. et al. Amphiphysin heterodimers: Potential role in clathrin-mediated endocytosis. Mol Biol Cell. 1997;8(10):2003–2015. [PMC free article: PMC25662] [PubMed: 9348539]
132.
Takei K. et al. Functional partnership between amphiphysin and dynamin in clathrin-mediated endocytosis. Nat Cell Biol. 1999;1(1):33–39. [PubMed: 10559861]
133.
Peter BJ. et al. BAR domains as sensors of membrane curvature: The amphiphysin BAR structure. Science. 2004;303(5657):495–499. [PubMed: 14645856]
134.
de Heuvel E. et al. Identification of the major synaptojanin-binding proteins in brain. J Biol Chem. 1997;272(13):8710–8716. [PubMed: 9079704]
135.
Ringstad N, Nemoto Y, De Camilli P. The SH3p4/Sh3p8/SH3p13 protein family: Binding partners for synaptojanin and dynamin via a Grb2-like Src homology 3 domain. Proc Natl Acad Sci USA. 1997;94(16):8569–8574. [PMC free article: PMC23017] [PubMed: 9238017]
136.
Gallop JL. et al. Mechanism of endophilin N-BAR domain-mediated membrane curvature. Embo J. 2006;25(12):2898–2910. [PMC free article: PMC1500843] [PubMed: 16763559]
137.
Masuda M. et al. Endophilin BAR domain drives membrane curvature by two newly identified structure-based mechanisms. Embo J. 2006;25(12):2889–2897. [PMC free article: PMC1500852] [PubMed: 16763557]
138.
Weissenhorn W. Crystal structure of the endophilin-A1 BAR domain. J Mol Biol. 2005;351(3):653–661. [PubMed: 16023669]
139.
Farsad K. et al. Generation of high curvature membranes mediated by direct endophilin bilayer interactions. J Cell Biol. 2001;155(2):193–200. [PMC free article: PMC2198845] [PubMed: 11604418]
140.
Gallop JL, Butler PJ, McMahon HT. Endophilin and CtBP/BARS are not acyl transferases in endocytosis or Golgi fission. Nature. 2005;438(7068):675–678. [PubMed: 16319893]
141.
Itoh T. et al. Dynamin and the actin cytoskeleton cooperatively regulate plasma membrane invagination by BAR and F-BAR proteins. Dev Cell. 2005;9(6):791–804. [PubMed: 16326391]
142.
Tsujita K. et al. Coordination between the actin cytoskeleton and membrane deformation by a novel membrane tubulation domain of PCH proteins is involved in endocytosis. J Cell Biol. 2006;172(2):269–279. [PMC free article: PMC2063556] [PubMed: 16418535]
143.
Modregger J. et al. All three PACSIN isoforms bind to endocytic proteins and inhibit endocytosis. J Cell Sci. 2000;113(Pt 24):4511–4521. [PubMed: 11082044]
144.
Qualmann B. et al. Syndapin I, a synaptic dynamin-binding protein that associates with the neural Wiskott-Aldrich syndrome protein. Mol Biol Cell. 1999;10(2):501–513. [PMC free article: PMC25183] [PubMed: 9950691]
145.
Salim K. et al. Distinct specificity in the recognition of phosphoinositides by the pleckstrin homology domains of dynamin and Bruton's tyrosine kinase. Embo J. 1996;15(22):6241–6250. [PMC free article: PMC452447] [PubMed: 8947047]
146.
Sever S, Muhlberg AB, Schmid SL. Impairment of dynamin's GAP domain stimulates receptor-mediated endocytosis. Nature. 1999;398(6727):481–486. [PubMed: 10206643]
147.
Gout I. et al. The GTPase dynamin binds to and is activated by a subset of SH3 domains. Cell. 1993;75(1):25–36. [PubMed: 8402898]
148.
van der Bliek AM, Meyerowitz EM. Dynamin-like protein encoded by the Drosophila shibire gene associated with vesicular traffic. Nature. 1991;351(6325):411–414. [PubMed: 1674590]
149.
Kosaka T, Ikeda K. Possible temperature-dependent blockage of synaptic vesicle recycling induced by a single gene mutation in Drosophila. J Neurobiol. 1983;14(3):207–225. [PubMed: 6304244]
150.
Hinshaw JE, Schmid SL. Dynamin self-assembles into rings suggesting a mechanism for coated vesicle budding. Nature. 1995;374(6518):190–192. [PubMed: 7877694]
151.
Takei K. et al. Tubular membrane invaginations coated by dynamin rings are induced by GTP-gamma S in nerve terminals. Nature. 1995;374(6518):186–190. [PubMed: 7877693]
152.
Stowell MH. et al. Nucleotide-dependent conformational changes in dynamin: Evidence for a mechanochemical molecular spring. Nat Cell Biol. 1999;1(1):27–32. [PubMed: 10559860]
153.
Roux A. et al. GTP-dependent twisting of dynamin implicates constriction and tension in membrane fission. Nature. 2006;441(7092):528–531. [PubMed: 16648839]
154.
Kirchhausen T. Clathrin. Annu Rev Biochem. 2000;69:699–727. [PubMed: 10966473]
155.
Lee DW. et al. Recruitment dynamics of GAK and auxilin to clathrin-coated pits during endocytosis. J Cell Sci. 2006;119(Pt 17):3502–3512. [PubMed: 16895969]
156.
Massol RH. et al. A burst of auxilin recruitment determines the onset of clathrin-coated vesicle uncoating. Proc Natl Acad Sci USA. 2006;103(27):10265–10270. [PMC free article: PMC1502446] [PubMed: 16798879]
157.
Leshchyns'ka I. et al. The adhesion molecule CHL1 regulates uncoating of clathrin-coated synaptic vesicles. Neuron. 2006;52(6):1011–1025. [PubMed: 17178404]
158.
McNiven MA. et al. Regulated interactions between dynamin and the actin-binding protein cortactin modulate cell shape. J Cell Biol. 2000;151(1):187–198. [PMC free article: PMC2189798] [PubMed: 11018064]
159.
Merrifield CJ. et al. Imaging actin and dynamin recruitment during invagination of single clathrin-coated pits. Nat Cell Biol. 2002;4(9):691–698. [PubMed: 12198492]
160.
Merrifield CJ, Perrais D, Zenisek D. Coupling between clathrin-coated-pit invagination, cortactin recruitment, and membrane scission observed in live cells. Cell. 2005;121(4):593–606. [PubMed: 15907472]
161.
Yarar D, Waterman-Storer CM, Schmid SL. A dynamic actin cytoskeleton functions at multiple stages of clathrin-mediated endocytosis. Mol Biol Cell. 2005;16(2):964–975. [PMC free article: PMC545926] [PubMed: 15601897]
162.
Gottlieb TA. et al. Actin microfilaments play a critical role in endocytosis at the apical but not the basolateral surface of polarized epithelial cells. J Cell Biol. 1993;120(3):695–710. [PMC free article: PMC2119548] [PubMed: 8381123]
163.
Kubler E, Riezman H. Actin and fimbrin are required for the internalization step of endocytosis in yeast. Embo J. 1993;12(7):2855–2862. [PMC free article: PMC413538] [PubMed: 8335001]
164.
Warren DT. et al. Sla1p couples the yeast endocytic machinery to proteins regulating actin dynamics. J Cell Sci. 2002;115(Pt 8):1703–1715. [PubMed: 11950888]
165.
Kaksonen M, Sun Y, Drubin DG. A pathway for association of receptors, adaptors, and actin during endocytic internalization. Cell. 2003;115(4):475–487. [PubMed: 14622601]
166.
Moreau V. et al. The yeast actin-related protein Arp2p is required for the internalization step of endocytosis. Mol Biol Cell. 1997;8(7):1361–1375. [PMC free article: PMC276158] [PubMed: 9243513]
167.
Wen KK, Rubenstein PA. Acceleration of yeast actin polymerization by yeast Arp2/3 complex does not require an Arp2/3-activating protein. J Biol Chem. 2005;280(25):24168–24174. [PubMed: 15857833]
168.
Kaksonen M, Toret CP, Drubin DG. Harnessing actin dynamics for clathrin-mediated endocytosis. Nat Rev Mol Cell Biol. 2006;7(6):404–414. [PubMed: 16723976]
169.
Rodal AA. et al. Negative regulation of yeast WASp by two SH3 domain-containing proteins. Curr Biol. 2003;13(12):1000–1008. [PubMed: 12814545]
170.
Toshima J. et al. Phosphoregulation of Arp2/3-dependent actin assembly during receptor-mediated endocytosis. Nat Cell Biol. 2005;7(3):246–254. [PubMed: 15711538]
171.
Jonsdottir GA, Li R. Dynamics of yeast Myosin I: Evidence for a possible role in scission of endocytic vesicles. Curr Biol. 2004;14(17):1604–1609. [PubMed: 15341750]
172.
Sun Y, Martin AC, Drubin DG. Endocytic internalization in budding yeast requires coordinated actin nucleation and myosin motor activity. Dev Cell. 2006;11(1):33–46. [PubMed: 16824951]
173.
D'Agostino JL, Goode BL. Dissection of Arp2/3 complex actin nucleation mechanism and distinct roles for its nucleation-promoting factors in Saccharomyces cerevisiae. Genetics. 2005;171(1):35–47. [PMC free article: PMC1456526] [PubMed: 16183906]
174.
Holtzman DA, Yang S, Drubin DG. Synthetic-lethal interactions identify two novel genes, SLA1 and SLA2, that control membrane cytoskeleton assembly in Saccharomyces cerevisiae. J Cell Biol. 1993;122(3):635–644. [PMC free article: PMC2119656] [PubMed: 8335689]
175.
Raths S. et al. End3 and end4: Two mutants defective in receptor-mediated and fluid-phase endocytosis in Saccharomyces cerevisiae. J Cell Biol. 1993;120(1):55–65. [PMC free article: PMC2119492] [PubMed: 8380177]
176.
Kalchman MA. et al. HIP1, a human homologue of S. cerevisiae Sla2p, interacts with membrane-associated huntingtin in the brain. Nat Genet. 1997;16(1):44–53. [PubMed: 9140394]
177.
Engqvist-Goldstein AE. et al. The actin-binding protein Hip1R associates with clathrin during early stages of endocytosis and promotes clathrin assembly in vitro. J Cell Biol. 2001;154(6):1209–1223. [PMC free article: PMC2150824] [PubMed: 11564758]
178.
Wesp A. et al. End4p/Sla2p interacts with actin-associated proteins for endocytosis in Saccharomyces cerevisiae. Mol Biol Cell. 1997;8(11):2291–2306. [PMC free article: PMC25709] [PubMed: 9362070]
179.
Huh WK. et al. Global analysis of protein localization in budding yeast. Nature. 2003;425(6959):686–691. [PubMed: 14562095]
180.
Stepp JD. et al. A late Golgi sorting function for Saccharomyces cerevisiae Apm1p, but not for Apm2p, a second yeast clathrin AP medium chain-related protein. Mol Biol Cell. 1995;6(1):41–58. [PMC free article: PMC275813] [PubMed: 7749194]
181.
Huang KM. et al. Clathrin functions in the absence of heterotetrameric adaptors and AP180-related proteins in yeast. Embo J. 1999;18(14):3897–3908. [PMC free article: PMC1171466] [PubMed: 10406795]
182.
Yeung BG, Phan HL, Payne GS. Adaptor complex-independent clathrin function in yeast. Mol Biol Cell. 1999;10(11):3643–3659. [PMC free article: PMC25654] [PubMed: 10564262]
183.
Vater CA. et al. The VPS1 protein, a homolog of dynamin required for vacuolar protein sorting in Saccharomyces cerevisiae, is a GTPase with two functionally separable domains. J Cell Biol. 1992;119(4):773–786. [PMC free article: PMC2289700] [PubMed: 1429836]
184.
Yu X, Cai M. The yeast dynamin-related GTPase Vps1p functions in the organization of the actin cytoskeleton via interaction with Sla1p. J Cell Sci. 2004;117(Pt 17):3839–3853. [PubMed: 15265985]
185.
Fujimoto LM. et al. Actin assembly plays a variable, but not obligatory role in receptor-mediated endocytosis in mammalian cells. Traffic. 2000;1(2):161–171. [PubMed: 11208096]
186.
Lamaze C. et al. The actin cytoskeleton is required for receptor-mediated endocytosis in mammalian cells. J Biol Chem. 1997;272(33):20332–20335. [PubMed: 9252336]
187.
Moseley JB, Goode BL. The yeast actin cytoskeleton: From cellular function to biochemical mechanism. Microbiol Mol Biol Rev. 2006;70(3):605–645. [PMC free article: PMC1594590] [PubMed: 16959963]
188.
Smythe E, Ayscough KR. Actin regulation in endocytosis. J Cell Sci. 2006;119(Pt 22):4589–4598. [PubMed: 17093263]
189.
Buss F. et al. Myosin VI isoform localized to clathrin-coated vesicles with a role in clathrin-mediated endocytosis. Embo J. 2001;20(14):3676–3684. [PMC free article: PMC125554] [PubMed: 11447109]
190.
Hasson T. Myosin VI: Two distinct roles in endocytosis. J Cell Sci. 2003;116(Pt 17):3453–3461. [PubMed: 12893809]
191.
Taunton J. Actin filament nucleation by endosomes, lysosomes and secretory vesicles. Curr Opin Cell Biol. 2001;13(1):85–91. [PubMed: 11163138]
Copyright © 2000-2013, Landes Bioscience.
Bookshelf ID: NBK6479

Views

  • PubReader
  • Print View
  • Cite this Page

Related information

  • PMC
    PubMed Central citations
  • PubMed
    Links to PubMed

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...