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Accessory factors in clathrin-dependent synaptic vesicle endocytosis

Clathrin-mediated endocytosis is a special form of vesicle budding important for the internalization of receptors and extracellular ligands, for the recycling of plasma membrane components, and for the retrieval of surface proteins destined for degradation. In nerve terminals, clathrin-mediated endocytosis is crucial for synaptic vesicle recycling. Recent structural studies have provided molecular details of coat assembly. In addition, biochemical and genetic studies have identified numerous accessory proteins that assist the clathrin coat in its function at synapses and in other systems. This review summarizes these advances with a special focus on accessory factors and highlights new aspects of clathrin-mediated endocytosis revealed by the study of these factors.

Key Points

  • Clathrin-mediated endocytosis is a special form of vesicle budding that has a key function in synaptic vesicle recycling at nerve terminals. The fundamental features of this form of endocytosis in nerve terminals are similar to clathrin-mediated endocytosis in other cell types. However, it has some unique characteristics such as its fast kinetics and the existence of several neuron-specific molecules involved in the endocytic process.

  • The main building blocks of the clathrin coats are clathrin and the adaptor protein AP-2. Coat assembly starts with the oligomerization of AP-2 and the subsequent recruitment of clathrin. As the coat expands, the curvature of the membrane becomes more pronounced until it undergoes fission. The newly formed vesicle rapidly sheds its coat.

  • There are many accessory factors that assist the formation of clathrin-coated vesicles. Some of them, such as AP-180, amphiphysin, Eps15, epsin or dynamin are involved in coat assembly and in the regulation of coat dynamics. The protein AP-180, for instance, seems to be important for determining the size of the coat. Similarly, amphiphysin seems to function as a multifunctional adaptor that contributes to the recruitment of coat protein, and dynamin seems to be crucial for the fission event.

  • Other accessory factors coordinate growth of the coat with changes of the lipid bilayer and with modifications of the actin cytoskeleton. For instance, dynamin and some of its binding partners (for example, amphiphysin and syndapin) seem to interact functionally with actin. Similarly, endophilin seems to have lysophosphatidic acid acyl transferase activity that may be important for membrane invagination.

  • Other factors, such as synaptojanin, amphiphysin, intersectin or auxilin, participate in the crosstalk between endocytic mechanisms and intracellular signalling pathways. For instance, synaptojanin is a phosphoinositide phosphatase that regulates a PtdIns(4,5)P2 pool important both for endocytosis and for signalling.

  • One important aspect about the different accessory factors involved in clathrin-mediated endocytosis is that each of them may act at several stages during the endocytic process. This is probably related to the existence in each factor of binding sites for many of the other molecules involved in endocytosis. A current challenge for the field is to establish the hierarchy, the sequence of action, and the precise site of assembly of all of these factors into the clathrin coat.

  • A recurring theme in the study of clathrin-accessory factors is their link to actin. Indeed, the evidence for a role of actin in clathrin-mediated endocytosis is strong in some systems. Its actual involvement is not understood yet but it has been proposed that actin could be involved in the fission step and in the subsequent translocation of vesicles away from the membrane.

  • The fact that clathrin coats form in the synaptic membrane only after exocytosis indicates that the regulation of endocytosis in nerve terminals might be related to the exocytotic process, although the exact mechanism remains to be elucidated. In addition, protein phosphorylation is another mechanism involved in the regulation of the endocytic machinery.

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Figure 1: Domain structure of the coat components and accessory factors of clathrin-mediated endocytosis at the synapse.
Figure 2: Documented interactions of the coat components and accessory factors of clathrin-mediated endocytosis.
Figure 3: Sequential stages in clathrin-mediated endocytosis at the presynaptic terminal.
Figure 4: Disruption of synaptojanin function in the giant reticulospinal synapse of the lamprey.
Figure 5: Amphiphysin, dynamin and clathrin-coat components can transform liposomes into structures similar to the tubules and coated pits observed in situ .

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Acknowledgements

We thank Scott Floyd for discussion and assistance in the preparation of the manuscript. Work carried out in our lab was supported in part by grants from the NIH.

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Correspondence to Pietro De Camilli.

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DATABASE LINKS

Clathrin

α-adaptin

β2 adaptin

LAP

Unc-11

dynamin

shibire

profilin

synaptojanin

Unc-26

amphiphysin

endophilin

intersectin

syndapin

N-WASP

EPS15

epsin

liquid facets

HSC70

tensin

PTEN

HIP1

HIP1R

stoned

Glossary

ENDOSOME

A membrane-bound organelle that carries materials ingested by endocytosis and commonly passes them to lysosomes for degradation or recycles them to the cell surface.

QUANTAL UPTAKE

The content of a single synaptic vesicle taken up by the cell after endocytosis.

PHOSPHOINOSITIDE

A generic term used to define any inositol-containing glycerophospholipid.

TRANS-ACTING FACTOR

An agent whose effects are independent of its direct interaction with the element being regulated.

DOMINANT-NEGATIVE INTERFERENCE

The interference of a mutant protein on the activity of the wild type following the formation of a heteromeric complex.

LIPOSOME

A lipid vesicle artificially formed by sonicating lipids in an aqueous solution.

PROFILIN

Multifunctional actin-binding protein that regulates actin dynamics. It can bind and sequester actin monomers, catalyse nucleotide exchange on actin, and promote monomer addition at the barbed end of a growing filament.

SH3 DOMAINS

Src-homology region 3 domains. Protein modules of about 50 amino acids that recognize and bind proline-containing sequences.

PLEIOTROPISM

The ability of a single protein to have several, seemingly unrelated biological actions.

ALTERNATIVE SPLICING

During splicing, introns are excised from RNA following transcription and the cut ends are rejoined to form a continuous message. Alternative splicing gives rise to different messages from the same DNA molecule.

LYSOPHOSPHATIDIC ACID ACYL TRANSFERASE ACTIVITY

Enzymatic activity that leads to the generation of phosphatidic acid from lysophosphatidic acid and acyl-coenzyme A.

STERIC HINDRANCE

Prevention of a reaction between different molecules as a result of their sizes or spatial disposition.

EH DOMAINS

Protein–protein interaction modules that recognize the consensus motifs NPF or other aromatic and hydrophobic di- and tripeptides in target proteins.

RAS PROTEINS

A group of proteins involved in growth, differentiation and signalling that require the binding of GTP to enter into their active state.

DISABLED

A non-receptor tyrosine kinase that has SH2 and SH3 protein-interaction domains.

PLECKSTRIN

A protein of unknown function originally identified in platelets. It is a principal substrate of protein kinase C. Pleckstrin-homology domains are sequences of about 100 amino acids present in many signalling molecules.

WISKOTT–ALDRICH SYNDROME

X-linked recessive immunodeficiency caused by mutations in the WASP gene. This gene encodes a protein capable of interacting with actin, the Arp2/3 complex, PtdIns(4,5)P2 and members of the Rho family of GTPases, molecules that regulate the polymerization of actin.

ARP2/3 COMPLEX

Actin-related protein complex. A complex of seven proteins that participates in the nucleation of crosslinked actin networks.

FILOPODIA

Thin protrusions from a cell that usually contain microfilaments.

LAMELLIPODIA

Flattened projections of the plasma membrane of eukaryotic cells.

VHS DOMAIN

Protein–protein and protein–membrane interaction module present in various proteins. Derives its name from the first three proteins in which it was found: Vps27p, Hrs and STAM.

ARMADILLO REPEAT

Protein motif of 42 amino acids described in the Drosophila melanogaster Armadillo protein. It mediates protein–protein interactions with molecules such as the cadherins.

HRB PROTEIN

HIV-1 Rev-binding protein. Hrb is related to the nucleoporins, a class of proteins that mediate nucleocytoplasmic transport.

REV PROTEIN

A component of the human immunodeficiency virus (HIV).

UBIQUITINATION

The attachment of the protein ubiquitin to lysine residues of other proteins.

BICISTRONIC SEQUENCE

A single DNA or RNA sequence that encodes for two proteins.

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Slepnev, V., De Camilli, P. Accessory factors in clathrin-dependent synaptic vesicle endocytosis. Nat Rev Neurosci 1, 161–172 (2000). https://doi.org/10.1038/35044540

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