Linton M. Traub

Tickets to ride: selecting cargo for clathrin-regulated internalization

Clathrin-mediated endocytosis oversees the constitutive packaging of selected membrane cargoes into transport vesicles that fuse with early endosomes. The process is responsive to activation of signalling receptors and ion channels, promptly clearing post-translationally tagged forms of cargo off the plasma membrane. To accommodate the diverse array of transmembrane proteins that are variably gathered into forming vesicles, a dedicated sorting machinery cooperates to ensure that non-competitive uptake from the cell surface occurs within minutes. Recent structural and functional data reveal remarkable plasticity in how disparate sorting signals are recognized by cargo-selective clathrin adaptors, such as AP-2. Cargo loading also seems to govern whether coats ultimately bud or dismantle abortively at the cell surface.

The trans-Golgi network: a late secretory sorting station

Proteins synthesized on membrane-bound ribosomes are transported through the Golgi apparatus and, on reaching the trans-Golgi network, are sorted for delivery to various cellular destinations. Sorting involves the assembly of cytosol-oriented coat structures which preferentially package cargo into vesicular transport intermediates. Recent studies have shed new light on both the molecular machinery involved and the complexity of the sorting processes.

Disabled-2 exhibits the properties of a cargo-selective endocytic clathrin adaptor

Clathrin‐coated pits at the cell surface select material for transportation into the cell interior. A major mode of cargo selection at the bud site is via the μ2 subunit of the AP‐2 adaptor complex, which recognizes tyrosine‐based internalization signals. Other internalization motifs and signals, including phosphorylation and ubiquitylation, also tag certain proteins for incorporation into a coated vesicle, but the mechanism of selection is unclear. Disabled‐2 (Dab2) recognizes the FXNPXY internalization motif in LDL‐receptor family members via an N‐terminal phosphotyrosine‐binding (PTB) domain. Here, we show that in addition to binding AP‐2, Dab2 also binds directly to phosphoinositides and to clathrin, assembling triskelia into regular polyhedral coats. The FXNPXY motif and phosphoinositides contact different regions of the PTB domain, but can stably anchor Dab2 to the membrane surface, while the distal AP‐2 and clathrin‐binding determinants regulate clathrin lattice assembly. We propose that Dab2 is a typical member of a growing family of cargo‐specific adaptor proteins, including β‐arrestin, AP180, epsin, HIP1 and numb, which regulate clathrin‐coat assembly at the plasma membrane by synchronizing cargo selection and lattice polymerization events.

Sorting it out AP-2 and alternate clathrin adaptors in endocytic cargo selection

The AP-2 adaptor complex is widely viewed as a linchpin molecule in clathrin-mediated endocytosis, simultaneously binding both clathrin and receptors. This dual interaction couples cargo capture with clathrin coat assembly, but it has now been discovered that the association with cargo is tightly regulated. Remarkably, AP-2 is not obligatory for all clathrin-mediated uptake, and several alternate adaptors appear to perform similar sorting and assembly functions at the clathrin bud site.

Sorting in the endosomal system in yeast and animal cells

The endosomal system is a major membrane-sorting apparatus. New evidence reveals that novel coat proteins assist specific sorting steps and docking factors ensure the vectorial nature of trafficking in the endosomal compartment. There is also good evidence for ubiquitin regulating passage of certain proteins into multivesicular late endosomes, which mature by accumulating invaginated membrane. Lipids play a central role in this involution process, as do the class E vacuolar protein-sorting proteins.

Accessory protein recruitment motifs in clathrin-mediated endocytosis.

Clathrin-mediated endocytosis depends upon the interaction of accessory proteins with the alpha-ear of the AP-2 adaptor. We present structural characterization of these regulatory interactions. DPF and DPW motif peptides derived from eps15 and epsin bind in type I beta turn conformations to a conserved pocket on the alpha-ear platform. We show evidence for a second binding site that is DPW motif specific. The structure of a complex with an AP-2 binding segment from amphiphysin reveals a novel binding motif that we term FxDxF, which is engaged in an extended conformation by a unique surface of the platform domain. The FxDxF motif is also used by AP180 and the 170 kDa isoform of synaptojanin and can be found in several potential endocytic proteins, including HIP1, CD2AP, and PLAP. A mechanism of clathrin assembly regulation is suggested by three different AP-2 engagement modes.

Epsin 1 is a Polyubiquitin-Selective Clathrin-Associated Sorting Protein

Epsin 1 engages several core components of the endocytic clathrin coat, yet the precise mode of operation of the protein remains controversial. The occurrence of tandem ubiquitin‐interacting motifs (UIMs) suggests that epsin could recognize a ubiquitin internalization tag, but the association of epsin with clathrin‐coat components or monoubiquitin is reported to be mutually exclusive. Here, we show that endogenous epsin 1 is clearly an integral component of clathrin coats forming at the cell surface and is essentially absent from caveolin‐1‐containing structures under normal conditions. The UIM region of epsin 1 associates directly with polyubiquitin chains but has extremely poor affinity for monoubiquitin. Polyubiquitin binding is retained when epsin synchronously associates with phosphoinositides, the AP‐2 adaptor complex and clathrin. The enrichment of epsin within clathrin‐coated vesicles purified from different tissue sources varies and correlates with sorting of multiubiquitinated cargo, and in cultured cells, polyubiquitin, rather than non‐conjugable monoubiquitin, promotes rapid internalization. As epsin interacts with eps15, which also contains a UIM region that binds to polyubiquitin, epsin and eps15 appear to be central components of the vertebrate poly/multiubiquitin‐sorting endocytic clathrin machinery.

The autosomal recessive hypercholesterolemia (ARH) protein interfaces directly with the clathrin-coat machinery

The low density lipoprotein (LDL) receptor plays a pivotal role in cholesterol metabolism. Inherited mutations that disturb the activity of the receptor lead to elevations in plasma cholesterol levels and early-onset coronary atherosclerosis. Defects in either the LDL receptor or apolipoprotein B, the proteinaceous component of LDL particles that binds the LDL receptor, elevate circulating LDL-cholesterol levels in an autosomal-dominant fashion, with heterozygotes displaying values between homozygous and normal individuals. Rarely, similar clinical phenotypes occur with a recessive pattern of inheritance, and several genetic lesions in the autosomal recessive hypercholesterolemia (ARH) gene on chromosome 1 have been mapped in this class of patients. ARH has an N-terminal phosphotyrosine-binding (PTB) domain evolutionarily related to that found in Disabled-2 and numb, two endocytic proteins. PTB domains bind to the consensus sequence FXNPXY, corresponding to the internalization motif of the LDL receptor. We show here that in addition to the FXNPXY sequence, ARH binds directly to soluble clathrin trimers and to clathrin adaptors by a mode involving the independently folded appendage domain of the β subunit. At steady state, ARH colocalizes with endocytic proteins in HeLa cells, and the LDL receptor fluxes through peripheral ARH-positive sites before delivery to early endosomes. Because ARH also binds directly to phosphoinositides, which regulate clathrin bud assembly at the cell surface, our data suggest that in ARH patients, defective sorting adaptor function in hepatocytes leads to faulty LDL receptor traffic and hypercholesterolemia.

Syp1 is a conserved endocytic adaptor that contains domains involved in cargo selection and membrane tubulation.

Internalization of diverse transmembrane cargos from the plasma membrane requires a similarly diverse array of specialized adaptors, yet only a few adaptors have been characterized. We report the identification of the muniscin family of endocytic adaptors that is conserved from yeast to human beings. Solving the structures of yeast muniscin domains confirmed the unique combination of an N‐terminal domain homologous to the crescent‐shaped membrane‐tubulating EFC/F‐BAR domains and a C‐terminal domain homologous to cargo‐binding μ homology domains (μHDs). In vitro and in vivo assays confirmed membrane‐tubulation activity for muniscin EFC/F‐BAR domains. The μHD domain has conserved interactions with the endocytic adaptor/scaffold Ede1/eps15, which influences muniscin localization. The transmembrane protein Mid2, earlier implicated in polarized Rho1 signalling, was identified as a cargo of the yeast adaptor protein. These and other data suggest a model in which the muniscins provide a combined adaptor/membrane‐tubulation activity that is important for regulating endocytosis.

Cargo Recognition in Clathrin-Mediated Endocytosis

The endosomal system is expansive and complex, characterized by swift morphological transitions, dynamic remodeling of membrane constituents, and intracellular positioning changes. To properly navigate this ever-altering membrane labyrinth, transmembrane protein cargoes typically require specific sorting signals that are decoded by components of protein coats. The best-characterized sorting process within the endosomal system is the rapid internalization of select transmembrane proteins within clathrin-coated vesicles. Endocytic signals consist of linear motifs, conformational determinants, or covalent modifications in the cytosolic domains of transmembrane cargo. These signals are interpreted by a diverse set of clathrin-associated sorting proteins (CLASPs) that translocate from the cytosol to the inner face of the plasma membrane. Signal recognition by CLASPs is highly cooperative, involving additional interactions with phospholipids, Arf GTPases, other CLASPs, and clathrin, and is regulated by large conformational changes and covalent modifications. Related sorting events occur at other endosomal sorting stations.